Advances in Tissue Banking, Vol. 7

Rduances in

Tissue Banking Vol.7

Editor G l y n O P h i l l i p s

The Chinese saying on the cover has been used as a motivation for the International Atomic Energy Agency radiation and tissue banking programme.

Rduances in

Tissue Banking Vol.7

SERIES IN ADVANCES IN TISSUE BANKING Editor-in-Chief: G. 0. Phillips

Published Vol. 1

Advances in Tissue Banking edited by G. O. Phillips et al.

Vol. 2

Advances in Tissue Banking edited by G. O. Phillips et al.

Vol. 3

Advances in Tissue Banking edited by G. O. Phillips et al.

Vol. 4

Advances in Tissue Banking edited by G. O. Phillips et al.

Vol. 5

The Scientific Basis of Tissue Transplantation edited by A. Nather

Vol. 6

Advances in Tissue Banking edited by G. O. Phillips

Vol. 7

Advances in Tissue Banking edited by G. O. Phillips

Hduances in

Tissue Banking Vol.7

Editor-in-Chief

Glyn O Phillips Research Transfer Ltd, Cardiff, Wales, UK

This is a special volume associated with the World Congress of Tissue Banking held in Boston, USA, to which all international Tissue Banking Associations contributed. The International Atomic Energy Agency provided from their extensive tissue banking programme. Thanks, in particular, are due to Dr Sam Doppelt and Jorge Morales for their support. Regional Editors

A Nather

(Asia Pacific) National University Hospital Tissue Bank, Singapore

D

M S t r o n g (North America) Puget Sound Blood Center, USA

R von Versen

(Europe) German Institute for Cell and Tissue Banking, Germany

YJ? World Scientific NEW JERSEY

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ADVANCES IN TISSUE BANKING (Vol. 7) Copyright © 2004 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

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ADVANCES IN TISSUE BANKING

International Advisory Board H. Burchardt, LISA A. Gross, Canada M. Itoman, Japan J. Kearney, UK J. Komender, Poland B. Loty, France P. Mericka, Czech Republic D.A.F. Morgan, Australia D. Pegg, UK M. Salai, Israel W.W. Tomford, USA Y. Vajaradul, Thailand H. Winkler, Austria N. Yusof, Malaysia N. Triantafyllou, Greece R. Capanna, Italy W.W. Boeckx, Belgium C.J. Yim, Korea

V

LIST OF CONTRIBUTORS

JORGE MORALES IAEA Department of Technical Cooperation Vienna, Austria GLYN O. PHILLIPS Phillips Hydrocolloids Research Ltd. 2 Plymouth Drive Radyr, Cardiff CF15 8BL, UK TED EASTLUND Division of Transfusion Medicine Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455, USA D. MICHAEL STRONG Puget Sound Blood Center/Northwest Tissue Centre 921 Terry Ave, Seattle, Washington 98104, USA JEROEN VAN BAARE, STEPHAN VEHMEIJER and ROLF BLOEM Netherlands Bone bank Foundation, Portgebouw Noord, Rijrisburgerweg 10 2333 AA Leiden, The Netherlands OCTAVIO V MARTINEZ University of Miami Tissue Bank Department of Orthopaedics (R-12) University of Miami, PO Box 016960 Miami, Florida, US 33101 Vll

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List of Contributors

AXEL PRUSS Institute for Transfusion Medicine, Tissue Bank University Hospital Charite Schumannstr. 20/21, D-10117, Berlin MOUJAHED KAO and GEORG PAULI Robert Koch-Institut, Retrovirology Norduter 20, D-13353, Berlin, Germany MARTELL WINTERS Bioburden Section Leader, Nelson Laboratories, Inc. 6280 South Redwood Road Salt Lake City, UT 82123 Y. YU, J.B. CHEN, J.-L. YANG and W.R. WALSH Orthopaedic Research Laboratories Prince of Wales Hospital University of New South Wales Sydney 2031 NSW, Australia R. VERHEUL, N. JOHNSON and D.A.F. MORGAN Queensland Bone Bank Princess Alexandra Hospital Health Service District Wooloongabba 4102 QLD, Australia LARS FROMMELT Institut fur Infektiologie klinische Mikrobiologie und Krankenhaushygiene ENDO-KLINIK Holstenstr. 2, D-22607 Hamburg LUTZ GURTLER Friedrich Loeffler Institut fur Medizinische Mikrobiologie Ernst Moritz Arndt Universitat, Matin-Luther-Strasse D-17487 Greifswald

List of Contributors

THOMAS VON GARREL Klinik fur Unfall-, Wiederherstellungs- und Handchirurgie Philipps Universitat, Baldingerstrasse, D-35043 Marburg PAUL L. ROMAIN Department of Rheumatology The Cambridge Hospital 1493 Cambridge Street, Cambridge, MA 02139 JEROEN VAN BAARE Netherlands Bone bank Foundation Portgebouw Noord, Rijnsburgerweg 10 2333 AA Leiden, The Netherlands GER KROPMAN Euro Skin bank, Beverwijk, The Netherlands MARCO ANTONIO GARCES MORALES and CESER ALEJANDRO REYNAGA LUNA Plastic and Burns Department Hospital Nacional Arzobispo Loayza Av. Alfonso Ugarte s / n Lima, Peru HENRY J. MANKIN Orthopaedic Oncology Service Massachusetts General Hospital Harvard Medical School, Boston, MA 02114 WONG YONG SHON, CHANG YONG HUR and SOONG HYUN JUNG Department of Orthopaedic Surgery Guro Hospital, Korea University #80, Guro-Dong, Guro-Ku, Seoul, Korea 152-703

IX

X

List of Contributors

PETR VISNA Traumatological Hospital Ponavka 6, 602 00 Brno, Czech Republic JIRI ADLER Tissue Bank, University Hospital Brno Czech Republic L. PASA and R. HART Traumatological Hospital Brno Czech Republic J. FOLVARSKY University Hospital, Hradec Kralove Czech Republic V.I. SAVELIEV, LA. KUZNETSOV, A.V. KALININ, A.A. BULATOV and LA. SOLODOV Russian Research Institute of Traumatology and Orthopaedics named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia A.V. KALININ, V.I. SAVELIEV and A.A. BULATOV Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia LUCA DAINESE, GIANLUCA POLVANI, ANNA GUARINO and PAOLO BIGLIOLI Department of Cardiac Surgery University of Milan-Italian Homograft Bank (BIO) Centro Cardiologico Monzino, IRCCS Via Parea 4, 20138 Milan, Italy

List of Contributors

MARILENA FORMATO Department of Physiological, Biochemical and Cellular Sciences, University of Sassari Via Muroni 25, 07100 Sassari, Italy HAN-KI PARK, YOUNG-HWAN PARK, SANG-HYUN LIM, JONG-HOON KIM, and BUM-KOO CHO Yonsei Cardiovascular Research Institute Cardiovascular Hospital Yonsei University College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea SANG-HO CHO Department of Pathology Yonsei University, College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea JONG-CHUL PARK and DONG-WOOK HAN Department of Medical Engineering Yonsei University, College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea CHEE-SOON YOON Department of Thoracic and Cardiovascular Surgery College of Medicine, Konyang University 685 Gasuwondong, Seogu, Daejun, Korea SHI-HO KIM Department of Thoracic and Cardiovascular Surgery College of Medicine, Donga University 3-1 Dongdaesindong, Seogu, Pusan, Korea SAM-YOON LEE Department of Thoracic and Cardiovascular Surgery College of Medicine, Wonkwang University 344-2 Shinyongdong, Iksanshi, Junrabukdo, Korea

XI

Xll

List of Contributors

DORIS A. TAYLOR Center for Cardiovascular Repair University of Minnesota BSBE 7-105 312 Church Street 5E Minneapolis, MN 55455 SITARAM EMANI, MATTHEW ELLIS and RICHARD B. THOMPSON Departments of Medicine and Surgery Duke University Medical Center Box 3345, DUMC Durham, NC 27710 DAGMAR HAVRANOVA, JIRI ADLER, JANA KOMARKOVA, ANNA TEJKALOVA and EVA HLAVACKOVA Tissue Bank, University Hospital Brno Jihlavska 20, 625 00 Brno, Czech Republic EVA VLKOVA, HANA HRUBA and MONIKA HORACKOVA Ophthalmology Department University Hospital Brno Jihlavska 20, 625 00 Brno, Czech Republic MAHMOOD FARAZDAGHI Tissue Banks International, 815 Park Avenue, Baltimore, Maryland 21201, USA SONJA GRUNEWALD, UWE PAASCH and HANS-JUERGEN GLANDER Department of Dermatology/Division of Andrology University of Leipzig, Stephanstrasse 11 D-04103 Leipzig, Germany

List of Contributors

CORNELIA THIEME, UWE PAASCH and HANS-JUERGEN GLANDER Department of Andrology, University of Leipzig Stephanstrasse 11 04103 Leipzig, Germany YOUNG-HWAN PARK, DONG-WOOK HAN and JONGCHUL PARK Department of Thoracic and Cardiovascular Surgery Yonsei Cardiovascular Research Institute Cardiovascular Hospital Yonsei University College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea SHI-HO KIM Department of Thoracic and Cardiovascular Surgery, Donga University College of Medicine 3-1 Dongdaesindong, Seogu, Pusan, Korea SCOTT A. BARBOUR and WARREN KING Palo Alto Medical Foundation Palo Alto, California

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PREFACE

With confidence I can say that this is the most comprehensive volume dealing with tissue banking presently available. There are 27 full chapter contributions from the most distinguished and experienced practitioners in the subject: surgeons, microbiologists, and tissue bankers. The volume deals with all the major facets of tissue banking and utilisation: procurement processing, and utilisation. Safety of Tissues (Section II) Safety of allografts is now a major concern due to microbial and viral contamination of tissues even in the most sophisticated centres. Thus publication here of the International Atomic Energy Agency's Code of Practice for the Radiation Sterilisation of Tissues is an important event, as is their guidance on Standards and Public Awareness of this often misunderstood technology. There is now convincing evidence that safety has been compromised by certain regimes used to prepare human tissues for transplantation. Drs Eastland and Strong document fully the diseases which have been transmitted though tissues, in some instances, leading to fatalities. Thereafter, Section 2 continues to provide an in-depth investigation of this problem, particularly in bone and a consideration of safe and effective methods to use such tissues. The University of Miami Tissue Bank has been a leader in methods of microbiological screening of donors, and the final tissues. Dr Martinez (Chapter 5) delivers this experience concisely to us. The potential presence of viruses poses a formidable challenge to the tissue banker, and even when introducing an end-sterilisation radiation step, the outcome is XV

XVI

Preface

not clear-cut. Dr Axel Pruss and colleagues (Chapter 6) have taken us forward in a giant step in their careful controlled study of this problem. All roads seem now to lead to the desirability of adopting an end-sterilisation process if safe tissue are to be guaranteed (Chapter 7). When ionising radiation is used as part of a standardised working system of a tissue bank, then a degree of sterility assurance can be achieved. The IAEA programme has devoted considerable resources to define such procedures and the Code of Practice (Chapter 8) and Standards (Chapter 9) within which such a Code should be employed. There are limits to such use of radiation for bone, depending on the effects of radiation on mechanical strength and osteoinductivity (Chapter 10) of the final product. It is necessary to evaluate this aspect as Dr Yu and colleagues have done. The Marburg Bone Sterilisation Process for femoral heads may not have a universal application, but its effectiveness when properly used cannot be denied, as demonstrated by Dr von Garrel and his colleagues (Chapter 11). Ethical and Social Attitudes (Section III) The social and legal problems which arose in the UK, as a result of the unlawful procurement of tissues, have adversely affected public attitudes towards the procurement of tissues for cadaveric donors. Dr Paul L. Romain (Chapter 12) sets out in stark terms the ethical challenges which this field now poses. The justification for the practice remains the health benefits which such donation permits. In particular, the availability of such tissues in the wake of disasters such as has been experienced first in the Netherlands (Chapter 13) and then in Peru (Chapter 14) is evaluated. These contributions provide the perfect justification for having a ready supply of safe tissue grafts for immediate treatment of burns or traumatic damage.

Preface

xvii

Tissue Grafts in Orthopaedics (Section IV) The main customers of tissue banks continue to be orthopaedic surgeons. In this volume we are privileged to have an outstanding contribution from the doyen of this valuable technology — Dr Henry Mankin (Chapter 15). What a contribution he has made, and how glad we all are that he continues to be active and inspire further his one-time students, who are now distinguished exponents in their own right. This section demonstrates again the value of using both fresh and processed allografts in revision arthroplasty, and knee defects (Chapters 16 and 17). For the first time in this series we are able to learn of the long Russian experience in this field (Chapter 18 and 19). Other Areas Covered (Sections V-VIII) Cardiovascular grafts sperm banking and controlled process for the cryopreservation of tissues (Chapters 24-27) are other subjects covered in this volume. The motivation for such a comprehensive volume came in the Congress held in Boston which drew together all the international associations of tissue banking: American, Asia Pacific, Latin American and European. The whole world has been harnessed to construct this outstanding and historic volume.

Glyn O. Phillips Editor-in-Chief

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CONTENTS

List of Contributors

vii

Preface

xv

Section I:

Chapter 1

Chapter 2

The Contribution of the International Atomic Energy Agency (IAEA) to Tissue Banking

1

The International Atomic Energy Agency (IAEA) Programme in Radiation and Tissue Banking: Past and Present

3

IAEA Public Awareness Strategies for Tissue Banks

13

Section II:

Safety of Tissue Allografts

49

Chapter 3

Infectious Disease Transmission through Tissue Transplantation

51

Chapter 4 Chapter 5 Chapter 6 Chapter 7

Bacterial Contamination of Bone Allografts in The Netherlands

133

Microbiological Screening of Cadaver Donors and Tissues for Transplantation

143

Safety of Virus Inactivation Methods for Allogeneic Avital Bone Tissue Transplants

157

Methods of Culturing, Problems Associated with Bacteriostasis, and Radiation Sterilisation Options

193

Contents

XX

Chapter 8

IAEA Code of Practice for the Radiation Sterilisation of Tissue Allografts: Requirements for Validation and Routine Control

211

IAEA International Standards for Tissue Banks

267

In Vivo Assessment of Gamma Irradiated Bone: Osteoconductivity and Osteoinductivity

321

Disinfection of Femoral Heads for Bone Grafting Using the Marburg Bone Bank System (Lobator Sd 1) — A Retrospective Evaluation of Quality Control in the Endo-Klinik Bone Bank

339

Ethical and Social Aspects of Tissue Banking

353

Tissue Banking and Transplantation: The Ethical Challenges

355

The Volendam Burn Disaster and the Importance of International Collaboration in Tissue Banking

369

The Need for a Tissue Bank in a Disaster: Experience in Arzobispo Loayza National Hospital after the Tragedy in "Mesa Redonda", Lima, Peru

375

Section IV:

Tissue Grafts in Orthopaedics

387

Chapter 15

Major Limb Reconstruction Using Massive Cadaveric Allografts

389

Revision Arthroplasty Using Fresh Frozen Allograft with Cemented Cup for Acetabular Bone Deficiency

417

Chapter 9 Chapter 10

Chapter 11

Section III: Chapter 12 Chapter 13

Chapter 14

Chapter 16

Contents

Chapter 17

Present Ways of Treating Chondral and Osteochondral Knee Defects

431

Knee Joint Ligament Alloplasty with Tendon Grafts Sterilised with Gaseous Ethylene Oxide

455

New Approaches to Comparative Evaluation of Allogenic and Autologous Bone Transplants Procured in Various Ways

467

Section V:

Cardiovascular Grafts

483

Chapter 20

Cryopreservation of Porcine Aortic Valve: Open Status of the Aortic Leaflets Results in Increased Matrix Glycosaminoglycans Structural Maintenance

485

Pathologic Changes of the Cryopreserved Carotid Artery and Jugular Vein Implanted at the Canine Carotid Artery

501

Cellular Therapy for Heart Failure: A Review of Skeletal Myoblast Transplantation into Infarcted Myocardium

519

Section VI:

Cornea Grafts

547

Chapter 23

Cornea Transplantation Strategy — Organ Culture versus Cold Storage

549

Chapter 18

Chapter 19

Chapter 21

Chapter 22

Section VII: Sperm Banking Chapter 24

Characterisation and Depletion of Membrane Deteriorated Human Spermatozoa After Cryopreservation

559

561

Contents

xxu

Chatper 25

A Repository System for Cryopreserved Semen Samples and Testicular Biopsies Embedded in a Workflow Management System

Section VIII: Cryopreservation Chapter 26

Chapter 27

575 591

Finding the Ideal Freezing Curve for Tissues through Indirect Themophysical Calculation

593

The Safe and Effective Use of Allograft Tissue: An update

611

SECTION I: THE CONTRIBUTION OF THE INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) TO TISSUE BANKING

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

1 THE INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) PROGRAMME IN RADIATION AND TISSUE BANKING: PAST AND PRESENT

JORGE MORALES IAEA D e p a r t m e n t of Technical Cooperation, Vienna, Austria GLYN O. PHILLIPS Phillips Hydrocolloids Research Ltd, Cardiff, Wales, UK

1. The Early Period The IAEA has gained more experience and success than any other international organisation in establishing Tissue Banks in developing countries and applying ionizing radiation for sterilising tissue grafts used in transplant surgery (in orthopaedic reconstruction, treatment for cancer, trauma and high velocity impact damage), the treatment of burns, leprosy and intractable skin wounds, and pressure sore ulcers. This extensive programme has its origins in an IAEA Expert group meeting in the Joliot Curie Radiobiology Institute in Budapest around 1971, when it was resolved that there were advantages to be gained by using ionizing radiations to sterilise human and animal tissue. Yet for the early years, these interests 3

4

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were pursued under a broad cover of the sterilisation of medical products. The first declaration of the IAEA's official move into this field came with the 1974 Symposium in Bombay on "Sterilisation of Medical Products and Biological Tissues". The first vehicle used by IAEA was the Research Coordination Programme. Interested parties were encouraged to collaborate to study the effects of radiation on tissues. The outcome of this work was the IAEA Advisory Group Meeting held in Athens in 1976 on "The Effects of Sterilising Radiation Doses upon the Antigenic Properties of Proteins and Biological Tissues". The Proceedings of the meeting have been published (Phillips et al., 1978). In retrospect, it can now be realized how historic this meeting was; with pioneers in the broader subject of Tissue Banking participating: Gary E. Friedlander, Kenneth W. Sell, D. Michael Strong, K. Ostrowski, Sandor Pellet, Rudolph Klen, A. Dziedic-Goclawska, Pe Khin and Nicholas Triantafyllou. Thereafter, the programme gained momentum. Meetings under the auspices of IAEA were held in the Republic of Korea (1978) and Czechoslovakia (1981). The experience of the Eastern European countries was particularly helpful over this early period. The first truly Tissue-Banking contract (BUR/7/004) provided by IAEA emerged shortly afterwards, and in March 1983 a Tissue Bank was established in Burma (now Myanmar). From the mid-1980's the name "Tissue Banking" began to appear in official IAEA titles of programmes. Among the first was Sri Lanka where the late Dr. Hudson Silva had already established his famous Sri Lanka Eye Donation Society. Thailand thereafter played a major role in stimulating the programme in Asia and the Pacific Region through the contribution of Dr. Y. Vajaradul. The first Regional Workshop was held in Bangkok in November 1989 under the auspices of the IAEA Regional Coordination Agreement (RCA). To coincide with this meeting was the foundation of the Asia Pacific Surgical Tissue Banking Association, of which Dr. Vajaradul was the first Secretary General.

IAEA: Past and Present

5

It was the RCA programme, which delivered the necessary impetus and financial support, with Dr. John Easy being the RCA Coordinator who supplied the push over a ten-year period. The other pioneering Technical Officers at IAEA were Dr. Ramen Mukherjee and Dr. Vitomir Markovic. It was their support and vision that initiated the development of the first Training Curriculum in Radiation and Tissue Banking, using distance learning anywhere in the world. More detail about the historical development is given in Phillips and Strong (1997 and 1999), Phillips and Tatsuzaki (1998), Phillips (1999), Phillips (2000) and Phillips and Morales (2002). 2. Economic and Health Care Impact In all, some 30 countries have subsequently been involved (but not all funded) in this initiative: in Asia/Pacific Region: Australia, Bangladesh, China, India, Indonesia, Japan, Republic of Korea, Malaysia, Pakistan, Philippines, Singapore, Sri Lanka, Thailand and Viet Nam; in Latin America: Argentina, Brazil, Chile, Cuba, Mexico, Peru and Uruguay; in Africa and Middle East: Algeria, Jordan, Libya, Zambia and Iraq and in Europe: Greece, Poland, Turkey and Slovakia. All these countries are using radiation sterilisation as the method of choice. Experts from Australia, Europe, Japan and USA have assisted the IAEA Radiation and Tissue Banking Programme. In the countries involved with the IAEA Radiation and Tissue Banking Programme, as experience has been gained, the growth had been exponential. To the end of the year 2000, more than 220,000 tissue allografts have been produced and used clinically. Taking the mean value of the cost levied by Tissue Banks in the USA and Europe, as a re-charge for the processing, the value of these grafts can be placed at US$51,768,553. The total expenditure associated with the IAEA Radiation and Tissue Banking Programme over the same period was US$6,313,335, which includes a training cost of US$2,036,034. Not only does these provide a benefit in health care to these

6

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countries but also avoid the costly importation of grafts into the country. In Mexico, for example, when a US$400 graft is imported from USA, the cost to the patient is at least US$3,000, due to value added taxes, import, agent, hospital charges, etc. It costs US$10,000 to import a whole femur from USA into the Republic of Korea. The following tables summarise the main outcomes of the IAEA Radiation and Tissue Banking Programme in general, and in the Latin America region in particular. The graft production over the period 1988-end 2000 in 16 countries for which data has been reported is the following (Table 1): Table 1. Graft production and cost benefits. Cancellous bone

Massive bone allografts

Skin and Amnion

Others*

69,195

8,588

96,645

50,278

*Pig skin, dura mater, demineralised bone, xenografts, pericardium, tendon, ligaments, and fascia. The total graft production is 222,580, valued at US$51,768,553 (at mean European and USA Tissue Bank prices).

In Latin America, the IAEA supported the formation of 7 Tissue Banks and trained 66 Doctors, Tissue Bank Operators and Nurses. Table 2 shows the distribution of the trainees who have set out to establish Tissue Banks and used the country nuclear center to carry out the radiation sterilisation of their grafts. It summarises the position at the end of 2001. The main benefit, however, is undoubtedly the improvement in health care in the individual countries. There exist major problems in the use of autografts, which is grafts taken from the patient and then transferred to another site during surgery, as already noted. Also, the available amount of autograft bone, particularly in child or infants, is limited. The second lesion offers a site for potential infection, which is particularly relevant in a developing country. Skin and amnion offer immediate cover

IAEA: Past and Present

7

Table 2. Catalysing the start up of new Tissue Banks in Latin America. Country

Number on Attended Number of Number National 1 year Fellowship of New Training Training Training Tissue Course Course Banks

Argentina Brazil Chile Cuba Mexico Peru Uruguay

3

10

16 4 4 5 6 2 2

Total

13

39

7 2 6 3 5 23

11 6 4 10 4 1 1

Date when joined IAEA Programme 1993 1998 1998 1994 1996 1994 2001

37

Note: Initial total Tissue Banks in the region is 7.

for burns, wounds or leprosy lesions, which can promote healing, prevent fluid and energy loss and the introduction of infections. Another major advantage of the IAEA Programme in these countries is the exposure that surgeons get to the newer methods of using allografts and creating a cultural change in their approach to surgical treatment in their country. In Western countries, the use of such grafts is now routine, and more than one million are used world-wide. The perceived need in a developing country is not always apparent, but as the technology is introduced, the health benefits become clear. When this occurs, the graft supply is far from sufficient. The IAEA Programme has inspired this revolution in many countries and the growth henceforth will be exponential. Finally, it is not possible to measure the benefits for giving more than 220,000 people restored limbs, prevention of amputation, repair of fractures and spinal defects, or to treat burnt children and people who have been burnt or suffer from diseases

8

/. Morales & G.O. Phillips

like leprosy, or paraplegics who are vulnerable to pressure sores. This is the true measure of the benefits of the IAEA Programme. 3. Innovative Training Distance learning training for tissue banking operators, managers and doctors within the IAEA Radiation and Tissue Banking Programme started in 1995. Initiated in the Asia and Pacific Region, with the support of international experts, the Singapore Government and the National University of Singapore, the first comprehensive IAEA/NUS Curriculum in Radiation and Tissue Banking was produced. It is now a tangible asset. This Curriculum, now available in English, Spanish and Korean, is an unique vehicle for delivering training of tissue bank operators, managers and doctors worldwide. The University Diploma extends over one year, starting with a two week faceto-face contact in Singapore or Buenos Aires, is the first available anywhere in the world. In Asia/Pacific, Latin America, Africa and Europe, 296 tissue bank operators, managers and doctors have been trained under the IAEA Radiation and Tissue Banking Programme, out of which 65 have been successful graduated in the University of Singapore and 9 at the University of Buenos Aires. For the 81 trainees from 18 countries who attained the University Diploma level, the total cost was US$557,117 compared with US$972,000 in the UK for the same level course, using conventional training methods. 4. The On-going Programme A Global Programme has been identified, which will place all activities within an inter-regional and regional structure. There is now an unified management and evaluation system for all activities. The inter-regional programme provides the basic expert and technical services, which can then be transferred to Latin America, Asia/Pacific and Africa Regions for implementation through the regional projects. There has been recently

IAEA; Past and Present

9

requests from a group of European countries, including Ukraine and Russia, which indicate a more active participation of the European region in the IAEA Programme. The IAEA Programme will ensure that all tissue banks have access to the latest international standards, and supported with documentation to enable them to mount an effective public awareness and tissue production programmes to promote health and economic benefits within their region and country. The activities will all be designed to ensure better public acceptance of the benefits of radiation sterilised tissue grafts and ensure that the same uniform international standards are being used throughout Asia/Pacific, Africa, Latin America and eventually the European region. The key initiatives will be: (a) The IAEA International Standards for Tissue Bank: Presently there is a great deal of variation. Expert and regional groups will review the present adopted USA and European practice and the planned activities will ensure that these, when adopted, are compatible with regional circumstances. (b) The IAEA Code of Practice for the Radiation Sterilisation of Biological Tissues. No international body has undertaken this important function, which is within the IAEA's technical competence. This Code of Practice will then be introduced into the regions for practical evaluation. (c) The IAEA Handbook for Public and Professional Awareness: Public and professional awareness is the major obstacle to even further extension of the use of radiation sterilised grafts in individual countries. Education is needed at all levels. Documentation to assist workshops and national programmes will be prepare and adapted to the regions in several languages. (d) Internet training: Internet training will be introduced for all "Radiation and Tissue Banking" training courses all over the world. This is a logical extension and will build upon the IAEA Distance Learning Programmes now being used. The resources of the multi-media curriculum and its recent update, needs to be exploited in this way to give full benefits

10

/. Morales & G.O. Phillips

to IAEA. Already the educational methods and technical needs have been analysed, and as a result an implementation strategy agreed. In April 2002 the first International Training Course for Tissue Bank Operators was held in Singapore with the participation of 23 students form 13 countries of Africa, Latin America, Asia and Europe. The Tissue Bank in Singapore will become the International Center for Training Tissue Bank Operators when a Memorandum of Understanding is signed with the National University of Singapore. Another training course at the regional level will be held in 2002 in Buenos Aires for the Latin American countries. Eight participants will be accepted from 7 countries. It is expected that a Memorandum of Understanding will be signed between the IAEA and the National Atomic Energy Commission of Argentina in the following months to allow the establishment of a Regional Training Center for tissue bank operators in the country for the Latin America Region. A Memorandum of Understanding was signed in May this year with the Musculoskeletal Transplant Foundation (MTF) in the USA to promote the co-operation with the IAEA in the field of tissue banking, in particular in the area of training medical doctors and transplant coordinators. The establishment of a Homepage on the Internet for the IAEA Programme was finalised in 2002 (www.tissuebanking.org). Areas for co-operation between the IAEA and WHO have been identified and a letter of intention have been prepared and submitted for approval to the competent authorities in both organisations. 5. Benefits to D e v e l o p e d Countries Before the IAEA Programme started using radiation to sterilise tissues, the situation elsewhere in the world was very mixed. There were misconceptions about the benefits of using this technology, which are considerable, such as:

IAEA: Past and Present

11

• No significant temperature, physical and chemical changes are induced which influence the required function of the tissues. • The high penetration enables the bulk of the hard or soft tissues to be sterilised in final packaged form. The systematic use of radiation sterilisation in final packaging reduces the cost of investment by approximately 80% compared with alternative methods. • The effect is instantaneous and simultaneous for the whole target. The process control is precise and can be applied accurately to achieve sterility. Irradiation time is the only variable to achieve a sterility assurance level of 10~6. A series of cases, particularly in the USA, where infections were transmitted through tissues, which had not been endsterilised, drew fresh attention to the experience of the IAEA Radiation and Tissue Banking Programme. Now at least half of the grafts in the USA are either being sterilised or decontaminated using radiation. The method is mandatory in Austria and widely practised in UK, Germany, Belgium, Slovakia, Poland and France. At a conservative estimate, it can be demonstrated that at least 600,000 tissues grafts in developed countries are now being annually radiation sterilised directly as a result of the IAEA Programme, through a type of reverse educational transfer to developed countries. Experts from developed countries are supporting the IAEA Programme. This is a confirmation that the benefits of collaboration between developed and developing countries are not all one-way. 6. References PHILLIPS, C O . , TALLENTIRE, A. and TRIANTAFYLOU, N. (eds.) (1978). Radiation Sterilisation of Irradiated Tissues and their Potential Uses, The North East Wales Institute, Wales, UK. PHILLIPS, G.O. and STRONG, D.M. (1997 and 1999). The Contribution of the International Atomic Energy Agency to Tissue

12

/. Morales & G.O. Phillips

Banking, American Association of Tissue Banks: Tissue and Cell Report, 1997, 4(1), 5-10, and Advances in Tissue Banking (ed. G.O. Phillips), 3(1999), 357-397. PHILLIPS, G.O. and TATSUZAKI, H. (1998). The Tissue Banking Programme supported by the International Atomic Energy Agency (IAEA), Transfusion Today, 37 (December), 24-25. PHILLIPS, G.O. (1999). Tissue Banking in the Asia Pacific Region, Advances in Tissue Banking (ed. G.O. Phillips), 3(1999), 399-402. PHILLIPS, G.O. (2000). The future role of the International Atomic Energy Authority (IAEA) in Tissue Banking, Cell and Tissue Banking, 1, 27-40. PHILLIPS, G.O. and MORALES, J. (2002). Catalysts of Better Health Care: Medical Tissue Banks Bring Multiple Benefits to Countries, International Atomic Energy Agency Bulletin, 44, 17-20.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

2 IAEA PUBLIC AWARENESS STRATEGIES FOR TISSUE BANKS

A N IAEA CONSULTATION D O C U M E N T

1. Public Awareness as a Component w i t h i n the Total Tissue Banking System The aim of this paper is to provide guidance on organising and running awareness campaigns and detail some promotional activities that have proved to be successful. It is not exhaustive and it cannot provide universal answers. Local conditions will dictate the appropriate approach and might also provoke innovative solutions. But it is a collection of ideas, contributed by people working against wide ranging ethnic and cultural backgrounds, which have been proved to work. Tissue Banking requires interaction between the public, the professional health care staff and the Tissue Bank. This interrelationship is shown in the following flow chart (Fig. 1). Recognising this integrated relationship, IAEA has addressed the following component needs: • Public awareness strategies for tissue banks. • International standards on tissue banks. • Code of practice for the radiation sterilisation of biological tissues. Additionally, training programs have been identified which support the activities and quality management within the tissue 13

IAEA Public Awareness Stategies for Tissue Banks

14

bank, and the professional education of surgeons and health care personnel who use the tissues and have a role to play in promoting donor availability. In this paper, strategies for public awareness are identified. But it is important to understand that public awareness alone \*i&m&®MW&e&

Public Awareness Strategies for Tissue Banks

Donor Source

•

National Transplant Systems > Specific Strategics

• •

Hospitals Coroner S> stems

•

Otlrci

Honor Referral .mil Transplant Cu-urdiimlion

Professional Education Doctors Health (.'we staff

• iJJ.

Surgical L'scrs

Tissue Banks Recovery I'rocessmj: Sterilisation Storage Labelling Distribution

Standaid* OIL I'lssue Ranks

~^

1AKA 1 raining Programs

Code of I'racuvc loi filiation SlciilisiitJOEi of Uiologkal Tissues

Fig. 1. A general tissue banking system.

•

IAEA Public Awareness Stategies for Tissue Banks

15

will not be successful unless it is a part of an integrated system as illustrated in Fig. 1. 1.1. Donor referral and transplant coordination systems A critical link between tissue banks and donor occurs within the donor referral and transplant coordination system. Public awareness and professional education activities aim to increase and facilitate tissue donation within the donor referral and transplant coordination system. This system is composed to two interdependent processes: donor referral and transplant coordination. These activities may not be distinct and often overlap, but in any event, the two functions need to be achieved. The tissue bank can either be involved in the control of the activities or contract out to another organisation or individuals. This paper deals with an activity that precedes both donor referral and transplant coordination with the objective of providing information to potential donors and their families. 1.1.1. Donor referral Donor referral is the process by which the tissue bank is informed or notified of potential donors when a death occurs at the donor sources (hospital, coroners system, organ procurement agencies, funeral homes, others). Donor referrals are made using some basic donor suitability criteria, which have been previously provided to the donor source staff as part of a professional education and awareness program. Every tissue bank will need to establish processes to ensure suitable potential donors are identified and referred to the tissue bank. This notification may be made because of a mandated requirement or it may be voluntary. Regardless of whether this system of referral is under the control of the tissue bank or not, it will require liaison with, and education of other healthcare personnel.

16

IAEA Public Awareness Stategies for Tissue Banks

In order to establish a donor referral system, the tissue bank will need to consider: • Identification and prioritization of referral sources (these may be external or internal). General criteria on which donor suitability is based may include: age, contra indications to donation, time limits between death and donation. • Establishment of relationships and agreements with those donor sources. • Identification and training of donor source staff in order to participate in the identification and referral of potential donors: explain the referral and donation process and the role of the staff members in the process; outline timelines for referral and recovery processes; provide basic donor criteria; explain tools necessary to make referrals (phone numbers, forms, etc.); and establish professional education that includes routine follow-up and feedback to the donor source staff. 1.1.2. Transplant coordination Once the donor referral is made to the tissue bank, the transplant coordination process can begin. Transplant coordination is the responsibility of suitably qualified personnel who have been trained in performing the various elements of the transplant coordination process. The transplant coordination process may include: • Confirming the suitability of the potential donor with the referral source. • Obtaining contact information for the next-of-kin. • Approaching next-of-kin to discuss donation options: tissue that can be donated; uses of the donated tissue; description of the recovery, testing, processing, storage, and distribution of tissue; consent process; medical social history; services available to the donor family. • Coordinating the recovery team. • Performing tissue recovery. • Completing documentation.

IAEA Public Awareness Stategies for Tissue Banks

17

• Following up with family (thank you letters, donor family services, etc.). • Following up with donor referral source (feedback on the process). 2. Planning a Public Awareness Campaign Tissue banking is the recovery, processing, sterilisation, storage, labelling and distribution of tissues for transplantation. Although public awareness about all these activities might not seem to be a core activity of tissue banking, without donors, users and recipients, banking itself will have no value. Therefore, it is vital to communicate what tissue banking is, why availability of tissues for transplantation is important, its role in the community and how individuals can benefit from its existence, and finally, how its services can be accessed. Some tissue banks have public awareness programs permanently in place. This strategy can be very successful. In Thailand, for example, the promotion of the social importance of tissue donation has been introduced into the ethos of the Scout movement which itself is a mandatory part of the education system. The Rotary movement has also been convinced of the importance of tissue banking and promotes the principle as part of its social development policy. Religious and cultural leaders can play an important role in promoting tissue banking in certain communities. In developed countries, for example the USA, it is the role of central government to promote awareness amongst the public. Although the major emphasis is on organ donation, it is now also being extended to tissue donation. Such initiatives take time to set up and require continuing effort, but the results more than justify the resources employed. However, persisting with a routine in communication with the public that might have been previously successful may have drawbacks. A familiar approach can lose impact. Over several years, there might be demographic or cultural changes that will make the approach less effective. It is also difficult to assess the

18

IAEA Public Awareness Stategies for Tissue Banks

result of the effort — are people responding because of the communication or would they respond anyway? And if it is not known exactly who is responding and why, it becomes impossible to decide who is not being reached and to find ways of getting the message to them. A coordinated public awareness program, run over a specific time scale, will enable those operating it to: • • • • •

Set targets. Assess results against the targets that were set. Work out how many hours and the budget to be spent. Decide priorities. Decide what is possible, given staff, time and cash restraints.

The key to a successful public awareness program is planning the communication — deciding what needs to be said, who needs to hear the message and how that message will be delivered. 3. Communication Strategy Although each tissue bank will have to establish its own communication strategy taking into account its geographical area, social reality, religious beliefs and specific needs, there will be some common elements in every communication strategy for every tissue bank. The following ten-point plan will provide an overview of how to develop a Communication Strategy and some of the elements that need to be considered: (1) goals; (2) situation analysis; (3) success factors; (4) objectives; (5) target audiences; (6) selection of media; (7) strategic messages; (8) delivering strategic messages; (9) feedback and evaluation; and (10) crisis management. 3.1. Goals Each tissue bank should identify the goals of its communication strategy and how it will achieve them. Goals may vary and might include:

IAEA Public Awareness Stategies for Tissue Banks

19

• To enhance the public acceptability of allografts. • To promote the clinical application of allografts among medical professionals. • To ensure a high level of accessibility of allografts. • To increase the number of tissue donors. • To ensure the production of high quality and safe allografts. • To maintain high ethical standards. Once established, these goals would set the foundations upon which the communication strategy will be built. The first step, however, is to evaluate the present position. The progress of any journey can best be gauged by looking back at the starting point. Just as important, it will ensure the strategy is based on current reality. 3.2. Situation analysis A thorough analysis of the situation upon which the communication strategy will be built is vital to understand the environment in which the work will take place. This will help to ensure its effectiveness. If the situation is not fully analysed during the planning stage of the communication strategy, a vital component may be overlooked leading to the failure of the entire plan. In order to understand the situation it might be advisable: (a) To analyse the Strengths, Weaknesses, Opportunities and Threats (SWOT analysis) of the operation. For example: Strength

— the tissue bank is run by the orthopedic department of which the assistants are supportive of its activities. Weakness — the tissue bank does not have access to cardiac surgeons. Opportunity — the scope of activities of the tissue bank can be widened by establishing relationships with cardiac surgeons. Threat — the cardiac surgeons might decide to use imported or artificial cardiac valves.

20

IAEA Public Awareness Stategies for Tissue Banks

(b) To conduct surveys (e.g. send out questionnaires to key stakeholders, etc). (c) To interview key people (e.g. surgeons, hospital administrators, government authorities, donor families, recipients, religious leaders, community leaders, etc). (d) To conduct workshops with key personnel. (e) To review the results of previous campaigns and experiences of other tissue banks. (f) To analyse already available data (e.g. number of potential users, tissue demand, numbers of imported tissues, etc). (g) To prepare a budget so that the financial implications of developing the communication strategy can be fully understood. 3.3. Success factors From the situation analysis it is necessary to identify factors that will be critical to the success of the communication strategy. These will then become targets in the communication strategy. Some examples are found in the Table 1.

Table 1. Success factor

Target

Low number of donations among a particular ethnic or religious group.

To gain the support of these communities or religious leaders for donation of tissues.

Access to surgeons.

To ensure that surgeons are aware of the safety, relative low costs and availability of allografts.

Low awareness about tissue transplantation in the general public.

To increase community acceptance and awareness of tissue donation by publicly recognising the contribution of donor families.

IAEA Public Awareness Stategies for Tissue Banks

21

3.4. Objectives While the goals of a tissue bank will reflect its general purposes, objectives are the means by which these goals will be achieved. Objectives that could be considered are: • To raise awareness on tissue banking in the community and among medical practitioners, funding bodies and sponsors. • To provide high quality information on the issues surrounding tissue transplantation to facilitate decision making by potential recipients, donors and their families. • To reassure medical professionals about the safety and clinical utility of allografts. • To publicise the availability of allografts. • To cultivate an ethical, professional and caring image of the tissue bank. • To provide donor families with a supportive communications network. • To make tissue donation a natural consideration at the time of death. • To form a strategic alliance with partners such as community groups, health authorities, corporations, etc., to promote tissue donation. • To keep government authorities informed of tissue banking activities. • To assist government authorities in promoting tissue banking. • To assist government authorities in developing regulatory systems for tissue banking. • To enlist government support for tissue banking. 3.5. Target audiences If the communication strategy is to be successful, the audiences that need to be addressed must be identified. This means recognising all relevant groups of people. This will help in establishing priorities within the limits of the budget or

22

IAEA Public Awareness Stategies for Tissue Banks

resources. It will also help in the selection of the appropriate media or techniques that will be effective with the audiences and the messages that each audience will be receptive to. But whatever the resources available, the principles set out here should be followed. If the audiences are not carefully selected, funds and resources will be wasted trying to reach everyone. The same message will be delivered universally. At best it will be ineffective for many groups; at worst it could be unsuitable or even objectionable. Depending upon the objectives, the audiences might include: (a) Donor families There would be no tissue donation without the consent of donor families. Consequently donor families should be given special prominence in a communication strategy when an objective focuses on donors. Research indicates that most families want the option of obtaining information, even if they do not read it until well after they have made their decision. Because of their personal experience donor families are in the best position to encourage community support for tissue donation. (b) Recipients Tissue banks need the assistance of recipients to promote the benefits they have received from tissue donation. The provision of high quality information to surgeons for distribution to recipients may be a useful tool in gaining their support for the activities of the tissue bank. (c) User surgeons Well-informed surgeons can greatly assist the tissue bank by talking to their colleagues about the high quality of allografts and service supplied. They are also responsible for informing recipients. Surgeons thus need to know about the services of the tissue bank, the kind of tissue available and their clinical usefulness. They also need to know the safety protocols of the tissue banks and their impact on costs.

IAEA Public Awareness Stategies for Tissue Banks

23

(d) Bereaved families Effort should be made to raise community awareness of tissue donation among bereaved families. (e) Hospital staff Finance managers, medical directors and chief executive officers may be involved in the administration of tissue transplantation. Intensive care and specialty directors may be responsible for developing and implementing policies on tissue transplantation. They may also refer potential donors and deal with their families. They therefore need to be kept informed of all the developments and requirements of the tissue bank. (f) The media While the media are a means by which strategic messages are communicated to target audiences, it is legitimate to regard them as an audience in its own right. Medical and science journalists and producers for radio and television are in a position to reflect and influence public opinion and must be appraised of the benefits of tissue transplantation, the status of tissue donation and advances in the field. (g) Tissue bank staff Anecdotal evidence suggests that personal contact with donor families with the back up of a strong corporate identity would help individuals to make a commitment to donation. Tissue bank staffs are potential ambassadors for tissue donation. (h) Community groups Community groups (e.g. Rotary, churches, scouting, scientific societies, etc.) can be important in raising public awareness about the activities related to tissue banking and transplantation. They can also be supportive of these activities through spreading the word a n d / o r fundraising. (i) Other transplant groups The cooperation of other transplant groups is essential to elevate the status of tissue donation to that of organ donation.

24

IAEA Public Awareness Stategies for Tissue Banks

(j) Government departments In some countries, government departments and other regulatory authorities may set the policies for donation and transplantation programs. They may also be responsible for budget allocation and regulation of activities. For these reasons, government departments and regulatory authorities should be kept informed about the activities of tissue banks and their achievements. 3.6. Selection of media Having agreed on the objectives and the target audiences to be addressed, the communication media must be selected. This is another area where choice will be dictated by local circumstances. To many people in the developed world, "the media" means the established channels of mass communication — radio, television, the press and, perhaps, cinema. However, in a far larger part of the world, television is regarded as an elitist and minority medium. The impact of newspapers might be restricted by low literacy, low purchasing power, distribution problems or a shortage of newsprint. Even where newspapers have few of these problems, they might have to serve several language groups and so the circulations of the different editions will be small. Radio might be widespread but it is patchy in areas without reliable electricity. Where such situations exist, public information messages have been successfully distributed by taking the message to the people by means of traveling cinemas and video shows, exhibitions and traditional or folk media. However, it would be well to regularly assess the scope and influence of the various media to ensure that the most effective means is being used to contact the audience selected. The media "balance" will change if there are increases in literacy, improvements in prosperity or technological advances. Political change can very quickly alter the way people receive information. Areas where controls on free expression have been

IAEA Public Awareness Stategies for Tissue Banks

25

relaxed have seen a huge and rapid increase in the number of newspapers published, all reflecting a wide variety of political and social attitudes. As purchasing a newspaper is a voluntary act, that is, people choose to pay for the product rather than take what is given as in radio or television, it follows that people buy a newspaper because it reflects their views. This is significant because the growth of the press offers an opportunity to target specific groups in a way that is difficult with any other media. In areas where controls on free expression have been introduced, individuals have used the World Wide Web to create newspapers and even run live radio stations in opposition. This creative use of the media is instructional. It is now possible to "webcast" live video and the technology that allows this is becoming cheaper and more reliable. The "new media" should at least be given consideration in any media communication program. 3.7. Strategic messages The key messages form the central core of the communication strategy. They should be reflected explicitly or implicitly in all communications with stakeholders so as to build within the target audience a broad appreciation of subjects which will ultimately lead to the fulfillment of the objectives. The following are suggestions that could be considered as primary and subordinate strategic messages: (a) Value of tissue transplants to community health One donor can benefit many lives; possibility of someone in the audience benefiting from a future transplant; types of tissue that can be donated; tissue donation can be possible even when organ donation is not; advantages of tissue transplants over alternatives; e.g. cost benefits, efficacy, etc.; leading edge medical technology.

26

IAEA Public Awareness Stategies for Tissue Banks

(b) Safety is the first priority There is a small but nonzero risk associated with all biological material; there is comprehensive quality control, including irradiation of tissue; need to fully inform recipients. (c) Focus on donor family Donor families could be important in raising community awareness; donor families may seek the support of the tissue bank; the body is respected and left intact. (d) Highly professional and ethical tissue bank Leading authority in tissue donation; fully accredited; fees are based on the cost of producing, storing and supplying safe tissue; user surgeons contribute to policy development; professional advice on all aspects of tissue donation and banking. (e) Technical aspects of tissue donation Suitability of potential donor; time frame; ethical and responsible disposal of tissue; restoration of the body after tissue removal; administrative procedures. (f) Technical aspects of tissue transplants Types and availability of tissues; procedure for obtaining and using tissues; procedure for sending tissues to the tissue bank. 3.8. Delivering strategic messages Strategic messages operate much like a mission statement. They reflect the value of what the tissue bank does, its technical and professional competence, compassion, sensitivity and ethical standards. Unlike a mission statement, however, strategic messages are not static but active. They can be used to project your values to audiences with differing needs. Finally, the strategic messages can be presented in different ways to make the message easily acceptable to a target audience. The delivery of a strategic message is the matching of an audience, the message and the resource, or delivery vehicle. For

IAEA Public Awareness Stategies for Tissue Banks

27

example, the strategic messages for user surgeons might be: safety is the first priority; highly professional and ethical tissue bank; the technical aspects of tissue donation. It might be decided that the most persuasive way of delivering these strategic messages to this group is by: recipient information booklet; articles in medical journals; professional development of medical specialists; or displays or posters at medical conferences. Strategic Messages might be part of a public awareness campaign but they will generally be used in a different way and for a different reason. They deliver a specific message to a selected group consistently and over a long period. They aim to change attitudes and maintain that change by constant reinforcement. Communication with user surgeons, for example, will always contain the strategic messages of safety, professionalism and technical information regardless of the main subject. As such they should be part of continuing effort of a tissue bank. The following (Table 2) shows how the three elements — target audience, strategic message and resources can be matched to ensure success in reaching the proposed objective. Public awareness campaigns are generally more focused, shorter term and aim to educate to change long-term behavior. This subject is discussed in more detail in the public awareness campaign section. 3.9. Feedback and evaluation There is a need for constant re-evaluation of the communication strategy employed in pursuing established objectives through a feedback system. Communication strategies are of a dynamic nature and must be modified and adapted in response to identified successes, failures or even subtle changes in the initial objectives. The outcomes from the communication strategy should be evaluated at regular time intervals established in the initial

28

IAEA Public Awareness Stategies for Tissue Banks

Table 2. Target audience

Strategic message(s)

Resource(s)

Donor families

Value of tissue transplants to community health. Safety is the first priority. Focus on the donor family. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

High quality donor information/help kit. Establish "Friends" to provide grief support and raise community awareness. Quarterly newsletter. Thanksgiving service.

Recipients

Value of tissue transplants to community health. Safety is the first priority. Highly professional and ethical tissue bank.

Booklet for distribution through surgeon. Quarterly newsletter. Thanksgiving service. Media coverage. Outward focused brief annual report.

User surgeons

Safety is the first priority. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

Recipient booklet. Articles in medical journals. Professional development of medical specialists. Displays/posters at medical conferences.

Bereaved families

Value of tissue transplants General information on tissue donation. to community health. Focus on the donor family. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

Media

Value of tissue transplants to community health. Safety is the first priority.

Establish contacts. Invite to tissue bank seminars.

29

IAEA Public Awareness Stategies for Tissue Banks

Table 2 (Continued) Target audience

Strategic message(s)

Resource(s)

Focus on the donor family. Highly professional and ethical tissue bank. Technical aspects of tissue donation. Technical aspects of tissue transplantation.

Provide newsletters and annual report. Invite to "Friends" events and thanksgiving service.

Tissue bank staff

Value of tissue transplants Regular presentations to tissue bank to community health. seminars. Safety if the first priority. Focus on the donor family. Encourage staff to join "Friends". Highly professional and Provide newsletter, ethical tissue Bank. donor registry forms Technical aspects of tissue and annual report. donation. Technical aspects of tissue transplantation. Role in promoting activities and professionalism of tissue bank.

Hospital staff

Safety is the first priority. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

Liaison with tissue bank. In service seminars. Newsletter. Invitation to tissue bank seminars. Provide information booklets.

Community groups

Value of tissue transplants to community health. Technical aspects of tissue donation.

Seminars, presentations, promotional activities, etc.

30

IAEA Public Awareness Stategies for Tissue Banks Table 2 (Continued)

Target audience

Strategic message(s)

Resource(s)

Other transplant groups

Safety is the first priority. Establish personal contact. Focus on the donor family. Invite to seminars. Highly professional and Market the advantages ethical tissue bank. of greater Technical aspects of tissue collaboration. donation. Negotiate inclusion of tissue donation in organ donation information. Send newsletter and annual report.

Government departments

Value of tissue transplants to community health. Safety is the first priority. Highly professional and ethical tissue bank. Technical aspects of tissue donation and transplantation.

Meetings with authorities. Provision of information. Participation in the medical advisory board.

planning process. This will require feedback from key stakeholders, using the same tools that were employed for the initial Situation Analysis. The evaluation may reveal whether there have been advances in certain areas of the communication strategy and also other areas where the communication strategy has had limited success. Adaptation of the communication strategy so as to incorporate the information obtained through feedback information and new Situation Analysis will ensure that the communication strategy remains effective.

IAEA Public Awareness Stategies for Tissue Banks

31

3.10. Crisis management The success of tissue banks relies upon the trust and goodwill of the public as well as the confidence of medical professionals. These are based upon a positive perception of such things as the contribution of a tissue bank to society, its procedures, the competence of its professionals, its ethical code and safety standards. If, because of something that is done, something that is not done, misinformation or malicious rumor, that positive perception is damaged, then years of education and promotion could be undone within days. The tissue bank would be facing a crisis. 3.10.1. Features of a crisis (a) Someone is to blame An incident that could not have been anticipated and was a result of natural forces will not usually cause a crisis for a tissue bank. However, if it is the result of someone's negligence, then the tissue bank will become the focus of public and, therefore, media attention and anger. (b) Something is at stake There is no crisis if there is nothing that can be damaged by the public anger and media exposure. In the case of a tissue bank, what is at stake might be its donor base or the cooperation of other medical professionals. (c) Someone finds out A crisis only begins when it becomes public. 3.10.2. Usual reaction The usual reaction to a crisis is characterised by panic and inaction. It is tempting to think that the crisis will go away if nothing is said or done — it won't.

32

IAEA Public Awareness Stategies for Tissue Banks

3.10.3. Result of inaction As a consequence of inaction, public anger grows; rumor replaces fact; regulatory bodies, politicians, etc., become involved; confidence collapses and the crisis spirals out of control. 3.10.4. Handling of the crisis (a) Stop whatever is causing the problem This might be costly and inconvenient, but it removes the cause of the emotion and shows the tissue bank to be sensitive and considerate of its public responsibilities. It can also demonstrate a commitment to safety, quality or whatever else the tissue bank's "brand" represents. (b) Put out holding statement Say something to show the problem has been identified, it is being dealt with, there is no cause for public alarm and, most importantly, that the tissue bank is a source of information on the subject. This will allow rumors to be stopped before they get into the media or into general circulation. (c) Assemble crisis team Unless care is taken, the crisis will take over the entire tissue bank. A small team must be assigned to handle the crisis while others get on with the day to day job of running things. (d) Decide on audiences These might be the general public, regulatory bodies, medical professionals, politicians, people living close to the building, professional bodies and associations, etc. And do not forget to inform the staff what is happening; they are an audience too! (e) Decide what will be said Separate messages will probably be needed for audiences.

different

IAEA Public Awareness Stategies for Tissue Banks

33

Your audiences will fall into one or more of three groups: Passive — they do not know. Informed — they know but are not active. Active — they know and they are taking action. The purpose of communication in a crisis is to: prevent the Passive from receiving misleading or emotive information and moving from Passive to Active. Persuade the Informed audience that there is no need to take action. Reassure the Active that the crisis is being dealt with and they no longer need to become directly involved. 3.10.5. Planning for crisis A crisis should not catch a tissue bank unprepared. Almost all eventualities can be anticipated and planned for. The first step is communication. If you have tissue banks or individuals that are not supportive of tissue banking talk to them and explain what is done and why. They might never be convinced but understanding might prevent them from moving from being an informed audience to an active audience in a crisis. The second step is anticipation. Decide NOW what defines a crisis for the tissue bank. There are only a handful of things that are likely to cause problems. Work out what they are. Ask: what might go wrong; what outside events might affect us; and which response should be made in each case. 3.10.6. Create a crisis team Decide NOW who will run the crisis, who will deal with the media, government, regulatory bodies, etc., and who will continue to run the tissue bank. 3.10.7. Identify your audiences Decide who will need to know.

34

IAEA Public Awareness Stategies for Tissue Banks

3.10.8. Work out what you will communicate What must be said to each audience. 3.10.9. Write it d o w n Create a document that includes contact numbers for all the audiences as well as home, mobile and holiday numbers for key staff and "friendly" journalists. Keep it up to date. 4. Public Awareness Campaigns Public awareness campaigns aim at creating a climate in which the immediate goals of the tissue bank — increasing donor registration numbers, obtaining corporate sponsorship, encouraging family discussion, etc. — become acceptable and result in increased donation. Unlike strategic messages, which are a statement of the worth and values of a tissue bank, public awareness campaigns are usually very focused. They set out to achieve a desired outcome, they often include the use of several different media to influence the target audience and they are finite, so the results can be measured against effort and expenditure. For example, in a program to increase enrollment of university students as donors the campaign organisers would select the target audience — in this case both students and their families. They would decide on the messages, which would include reassuring and educating parents, persuading the young people to apply for a donor card and impressing upon them the importance of informing their families. The organisers would set a target, perhaps how many more students would carry a donor card after a specific effort over a set time period. Finally, they would decide on the communication tools to be used. Given that the principal audience is the young people, the Internet might be selected together with videos. Within the university, students are a captive audience and so displays or

IAEA Public Awareness Stategies for Tissue Banks

35

exhibitions could be set up and lectures organised linked to the distribution of brochures. If resources are available, the communication tools might be extended to include information for would-be students and their families delivered with university entrance materials; advertisements on local and university radio stations or even a donor disco; the possibilities are limited only by money, effort and the imagination of the organising team. Examples that have already been found to work by other tissue banks worldwide are many and various. 4.1. Potential public awareness activities Potential public awareness activities include: a school program "teaching the teachers" workshop; a scout program; a kids club; a donor registry; a university/college student enrolment program; a community program; a corporate and community tissue banks program; donor and transplant recipient services; a drivers licensing bureau program; and an old folks' home. There should be constant monitoring of progress throughout the campaign so that, if necessary, effort can be redirected to ensure the objectives are met. Finally, there should be a careful appraisal of the results to identify what worked well, what could have worked better and what lessons have been learned. To recap, a successful public awareness campaign should include the following: an assessment of the problem or need; the setting of realistic targets or desired outcome; assessment of resources; the choice of target audience; choice of messages; selection of tools; a distinct start — possibly a public launch; constant monitoring; an agreed end; and appraisal of results. 4.2. Tables The following tables contain details of the potential public awareness campaigns including their desired outcome, messages and communications tools.

36

IAEA Public Awareness Stategies for Tissue Banks

Method of practice

School program "teaching the teachers" workshop

Target audience

Primary target: elementary and high school teachers, Secondary target: students and families.

Desired outcome

Train teachers to educate students about organ and tissue donation. To encourage a family discussion about a donation decision.

Messages

Overview of organ and tissue donation. Importance of sharing your decision with your family. Concept of giving, sharing and receiving.

Tools

Two-hour workshop.

Recomendations

Seek support from the education authority, promoting joint programs among the relevant authorities.

Teacher curriculum. Pre-test. Video (highlights recipients). Brochure.

Scout program

Method of practice Target audience

Scouts,

Desired outcome

Educate scouts about donation so they can promote and discuss the donation decision with their families and the community.

Messages

"Serving Life". Provide information and promotion of donation to their community through the scouts service commitment.

Tools

Brochures/leaflets. Video (transplant recipients). Lecture.

Recomendations

Seek scout leaders' support to implement a program with reward (badges).

IAEA Public Awareness Stategies for Tissue Banks

Method of practice

37

Kids club

Target audience

Children (ages 4-12 years old) and parents.

Desired outcome

To promote donation discussion among the child's family, classmates and their families.

Messages

Benefits of donation and transplantation.

Tools

Videos and posters. Donor cards ("donation promoter card"). Stickers. Always stress importance of child's involvement in donation discussion.

Recomendations Method of practice

Donor registry

Target audience

General Public (age 18 and older).

Desired outcome

Develop a database to record individual donation decision to facilitate the donation process at the time of death.

Messages

Make your decision now, so your family can follow through with your wishes later.

Tools

Brochures. Form with appropriate signatures. Data base system.

Recomendations

Seek government legislation to implement and maintain a donor register.

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IAEA Public Awareness Stategies for Tissue Banks

Method of practice

University/College Students/Enrolment Program

Target audience

Students and their families.

Desired outcome

Every student within the university/college will make a decision about donation and carry a donor card.

Messages

Educate the students about donation and the importance of making a donation decision and communicating it to their families.

Tools

Video. Lecture. Brochure/donor card. Internet. Display/exhibit.

Recomendations

Seek support from the university or college authorities.

Method of practice

Community program

Target audience

General public.

Desired outcome

Educate the general public about donating, the importance of making a donation decision and communicating that decision to their family.

Messages

Importance and benefits of donation and transplantation. Make a decision now, so your family can carry out your wishes later.

Tools

Media campaign. Special events. Celebrity endorsement. Displays/health fairs. Internet. Donor cards. Drivers License Program (and other official identification documents). Toll free number. Brochure.

Recomendations

Keep message simple and consistent.

IAEA Public Awareness Stategies for Tissue Banks

Method of practice

Corporate and community tissue banks programs

Target audience

Corporations (executives, staff). Service groups. Community groups.

Desired outcome

Educate members, staff or customers about donation. Seek sponsorship. Make a decision now, so your family can carry out your wishes later.

Messages

Benefits of donation and transplantation.

Tools

Brochures. Video. Internet. Program materials with recognition of sponsorship. Lectures. Scholarships.

Recomendations

Keep message simple and consistent.

Method of practice

39

Donor and transplant recipient services

Target audience

Donors, recipients and their families, General public.

Desired outcome

Increase donation through recognition of the donor and support of the family who generously donated. Highlighting the improved quality of life of the recipients.

Messages

"Celebrate life".

Tools

Transplant recipient Olympic games. Donor memorial services. Donor recognition (day). Donor medal/certificate. Donor quilt.

Recomendations

Enlist volunteer donor families and recipients to plan coordinate and participate.

40

IAEA Public Awareness Stategies for Tissue Banks

Method of practice

Drivers licensing bureau program (or official identification document)

Target audience

Driver License staff. Drivers/general public.

Desired outcome

Drivers License staff consistently offers the option of a donor designation to each individual who renews or applies for Drivers License or official identification document.

Messages

Educate the Drivers License Bureau staff about donation and importance of the donation decision and the family discussion. Ensure Drivers License staff gives a donation brochure to individuals who have questions.

Tools

Posters (posted in the Drivers License Bureau office). Lectures. Videos (donor families and transplanted recipients). Brochures (staff and public).

Recomendations Provide information/materials related to donation to Drivers License Bureau staff. Support of the Bureau staff is key in offering drivers the option of donor designation. Method of practice

Old folks' home

Target audience Senior public. Desired outcome

Educate senior people to participate in promotion of program. Promote tissue (e.g. Cornea) donation.

Messages

Nobody is too old to participate in promotion of donation program.

Tools

Media campaign. Special events. Posters. Brochure.

Recomendations Keep message simple and consistent.

IAEA Public Awareness Stategies for Tissue Banks

41

5. U s i n g the Media Most public awareness programs include some attempt to involve the media. Sometimes the attempt results in an enthusiastic response from the journalists and sensitive, valuable coverage. Sometimes it results in disinterest and little or no coverage. Occasionally it results in inaccurate or sensational coverage of one unimportant aspect of the program, which misinforms the public and damages what is being attempted. Many people who have occasionally had to deal with the media — medical professionals, academics, businessmen, scientists, public service workers and charities — have experienced one or more of these responses. One factor that unites them all is they don't know why. Those who have had a good experience are pleased, but those who have had a bad experience are angry. Frequently both groups are mystified by the response. It is true that taking the media as a whole, newspapers, radio and television, it does seems to have contradictory and conflicting needs. This section of the document aims to explain why the media acts as it does and how to anticipate that response. From this it will become easier to identify the media most suitable for the Target Audience. First, the media is not there to right wrongs, to shine light into dark places or to highlight those things that are important in life although it might do all of those things. It exists principally to make money and occasionally, also, to extend influence. To do this it must generate an audience. Moreover the media creates readers, viewers or listeners by identifying a target group or groups and feeding them what they want. 5.1. The audience A large national broadcaster will produce a range of programs to appeal to a selection of audiences. But the more media

42

IAEA Public Awareness Stategies for Tissue Banks

competition there is in any sector, the more specific the media becomes in the audience it targets. Where there is a lot of competition, typically in newspapers, both national and local, cable television and local radio, the media outlets will target a very narrow group. They will know the lifestyle of that group very well indeed and know their hopes, fears, beliefs prejudices, drives and aspirations. They also know that people will read the newspapers and listen to programs that reflect their values and beliefs. Thus, they will provide their Target Audience with information that reflects the world as that audience sees it. There are two significant points arising from this. Firstly, it is usually a waste of time sending the same information to all sections of the media. Secondly, even when a Target Audience has been identified, the media that appeals to that Target Audience might not be interested in what you want to say. For example, let us suppose that an objective is to persuade more 18-25 year-olds to opt to be tissue donors. Possibly the group is being targeted because it has been identified as having a general lack of awareness and interest in the subject. However, if that is the case, why should a magazine or radio station run a story or feature on something they know their readers or listeners are unaware of and probably not interested in! 5.2. Give them what they want! The answer is that the information must be linked with something that the Target Audience will find interesting. This might sound like a lot of effort for no guaranteed return but trying to sell the media something they don't want is effort for no return. 5.3. Case study one — Quit and W i n A public health organisation decided to run a campaign to try and persuade young people — teenagers and early twenties — to

IAEA Public Awareness Stategies for Tissue Banks

43

give up smoking. The group was chosen because it was considered that although many would have been smoking for several years, this habit might be easier to break and the health benefits would be easier to "sell" and more readily recognised. For economy, the campaign had to be run largely through the news columns of local newspapers and the news programs of local TV and radio rather than by advertising. The campaign organisers looked at what the young people in that group valued. It was recognised that among the few things that this group felt important enough to save money were for cars and holidays. A car distributor was persuaded to give a car as a first prize on the promise of publicity and several holiday companies gave holidays. The campaign became "Quit and Win". People pledged to give up smoking by filling in a form, signed by a friend. After three months they became eligible to take part in a free draw for a holiday. The campaign ended after six months with the final draw for the car. Pictures and interviews with the happy winners were regular features in the media in months four, five and six while the car was handed over by the Minister for Health with great publicity. But what of those who didn't win? A subsidiary message of the campaign was how much could be saved by not spending money on cigarettes — about the equivalent of $7 US a packet in the United Kingdom. There were other interviews at regular intervals about people who had saved for their own holidays or other consumer goods by giving up smoking. There were some up-front costs. There were posters to be printed for the workplace, entry forms and administrations costs. But the value of the publicity was many times the fixed costs. More importantly, the campaign succeeded in its three aims: a number of people stopped smoking; they became role models — their success was likely to influence others;

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IAEA Public Awareness Stategies for Tissue Banks

giving up became a "smart" thing to do — not something to be mocked. 5.4. Case study t w o — Medic Alert Medic Alert is a charity that was founded by a parent whose child almost died of an allergic reaction to a food while at a playgroup. The child's allergy had been explained but the information was not passed on. Consequently, when the child became unconscious, no one recognised the cause and it was only by chance that the child was saved. Medic Alert provides a database of the medical conditions and current treatment for hundreds of thousands of people. People at risk wear a disc bearing a personal identification number and a 24-hour emergency telephone number. It ensures that if that person is taken ill or is injured vital information can be given which might save their lives. Since its inception, hundreds of lives have been saved. Obviously, the more the people who know about it, the more the people that can benefit. The charity decided that television was the ideal media for spreading the message. They approached the producers of several popular drama series with true stories of people who would have been unwittingly killed by routine treatment when they were unconscious but at the last minute someone noticed the badge and called the emergency number. They stressed the dramatic nature of this — moments away from death. The result was that Medic Alert was featured in three television series — a hospital drama and two police dramas all with an audience of several million. Very different from the first case but again, the charity recognised their product had something television producers wanted — dramatic impact. The advantage of using fictional television or radio series is not only publicity but, in cases where there are ethical or religious objections, the argument itself can become part of the program story line.

IAEA Public Awareness Stategies for Tissue Banks

45

5.5. D e a l i n g w i t h journalists Start by building a relationship with journalists. Getting material into a newspaper or on local television and radio is often as much about personal contact as the subject matter. Each day in a newsroom, hundreds of pieces of information jostle for attention. If the journalist making a decision about what to use knows and trusts the tissue bank and believes that what it is trying to do is a good thing, they are more likely to use the information when they come across it. Remember, journalists are not necessarily friends — they have a job to do. But a relationship can be built up from which both sides benefit. The Quit and Win campaign was a good one but it got more publicity than it might have done because the people running it were on first name terms with a large number of news journalists and health correspondents. 5.6. Getting in touch The most common way of giving journalists information is the news release, mailed or e-mailed to interested journalists and news desks. Bearing in mind the previous section it should be directed to a journalist contact that has been cultivated. However, there are a few things you can do to help it further. (a) Write an eye-catching headline The headline you write is unlikely to be the headline that appears in the newspaper — the object is to make the journalist receiving it read on. (b) Start with the most important points What is actually happening? Where is it happening? Why? When? How? Answering as many of these questions in the first paragraph will usually have the effect of ensuring the most important information comes first.

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IAEA Public Awareness Stategies for Tissue Banks

(c) Explain how it will benefit people Events are only news because they affect people. The event might be agreement to receive hundreds of corneas from the tissue bank of another country. But the effect will be the gift of sight for hundreds of people who would otherwise be blind. That makes a much more emotive and stronger story for the media. (d) Add a strong "quote" from a senior figure Quotations can make a story more personal and, if from a prominent person, more authoritative. (e) Use one side of a standard sheet of paper If a news release looks long and complicated a journalist might not begin reading it. If more information is wanted, they will call. (f) End with a contact number Make sure someone who has authority to speak to the media is available at that number. 5.7. The interview It is flattering to be approached to give a media interview, but anyone approached should ask themselves honestly if they are the right person to be interviewed. If not, who should be? Then the journalist should be asked: (a) Which program/publication? This will indicate the sort of audience — readers, viewers or listeners — that will receive the message. (b) What do you want to talk about? This is necessary anticipated questions.

to allow preparation

of answers

to

IAEA Public Awareness Stategies for Tissue Banks

47

(c) Are you talking to anyone else? This might be the only opportunity to learn if the journalist is also talking to someone critical of tissue banking. Then decide, is it in the interests of the tissue bank to do this interview? If so, prepare. Work out what the journalist is likely to ask; and what messages must be put out in the interview? Remember, an interview is an opportunity to put over positive points about the tissue bank and what it is trying to do. Don't put an opposing point of view to be "reasonable". If there are two sides to the issue, there will be plenty of others willing to make the opposing point of view and the journalist has probably found them. Another key to preparation is being clear about what the interview should achieve. Is it to inform? To encourage people to take a course of action? To calm people's fears? Think of two or three main points; things that are at the heart of what must be achieved and make sure that they are used. They must be delivered with enthusiasm and energy — broadcast journalists like interviews that sound good but it will also encourage a press journalist to use that as a "quote". Do not be nervous. They need your expert knowledge. In some cases, a journalist will arrive having had very little opportunity to learn about your tissue bank or its work. This is an opportunity to brief them and even suggest some questions they might want to ask. Finally, will journalists always be open and honest about what they want from the interview? The answer is no. Interviewees must try and work that out for themselves, which returns to the beginning and the audience. What audience this journalist is writing or broadcasting for; and what does that audience think of the tissue bank and tissue banking in general? Remember, journalists are not seekers after truth — they're seekers after stories! And that's not necessarily the same thing.

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IAEA Public Awareness Stategies for Tissue Banks

6. Promotional Tools A reference section has been compiled to illustrate how a database can be developed and provide a resource for tissue bank and organ transplant professionals. Through it, they can access examples of good practice, publicity, promotional and advertising materials that have proven to be successful in programs throughout the world. It is by no means exhaustive. Users may access information via the IAEA website or by contacting the relevant institutions that have contributed to that database. The information has been categorised as follows: Public Education; Professional Education; Donor Appeal and Donor Management. The material is further grouped into the following sub-categories: fliers; posters; stickers; audio-visual aids; newsletter; calendar themes, etc. The reference section can be obtained from IAEA on request.

SECTION II: SAFETY OF TISSUE ALLOGRAFTS

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

3 INFECTIOUS DISEASE TRANSMISSION THROUGH TISSUE TRANSPLANTATION

TED EASTLUND Division of Transfusion Medicine Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455, USA D. MICHAEL STRONG Puget Sound Blood Center/Northwest Tissue Centre Seattle, Washington 98104, USA

Abstract The incidence of tissue allograft-transmitted infection is unknown and can best be inferred from prospective studies — that have not yet been performed and reported. Viral, bacterial and fungal infections have been transmitted via tissue allografts such as bone, skin, cornea and heart valves. Bone allografts have transmitted hepatitis C, human immunodeficiency virus (HIV-1), human T-cell leukaemia virus (HTLV), tuberculosis and other bacteria. Corneas have transmitted rabies, Creutzfeldt-Jakob disease (CJD), hepatitis B virus, cytomegalovirus (CMV), herpes simplex virus, bacteria and fungi. Heart valves have been

51

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T. Eastlund & D.M. Strong

implicated in the transmission of tuberculosis, hepatitis B, bacteria and fungi. HIV-1, CMV and bacteria have been transmitted by skin allografts. CJD has also been transmitted by dura mater and pericardium transplants. Over the past several years, improvements in donor screening criteria, such as excluding potential donors with "risky lifestyles" for HIV-1 and hepatitis infection, and donor blood testing, have greatly reduced the risk. The addition of viral nucleic acid testing of the donor will further enhance the ability to detect pathogens and reduce the risk of disease transmission. During tissue processing, many allografts are exposed to antibiotics, disinfectants and terminal sterilisation such as irradiation, which further reduce or remove the risk of transmitting disease. Because the effectiveness of some tissue grafts depends on cellular viability, not all can be subjected to sterilisation steps, and, therefore, the risk of infectious disease transmission remains. For these situations, preventing the transmission of infection with the graft depends on careful donor selection and testing; aseptic technique during surgical removal of the tissue from the donor; aseptic processing; microbiological testing of the allograft to ensure that virulent organisms are not acquired during processing, or if present, are identified and eliminated as much as possible. To further ensure safety in the use of allografts, the physician and hospital should select tissue banks that follow national professional standards, as their source. 1. Introduction Tissue transplantation therapy, which has been utilised for over 50 years (Strong, 2000), is a rapidly developing field carrying with it great promise for ameliorating or curing many diseases. One of its drawbacks, however, is the potential for donor-torecipient disease transmission. This risk is greatly reduced by excluding donors at risk of carrying infection, and by testing the

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Infectious Disease Transmission Through Tissue Transplantation

donor for transmissible infectious disease. Aseptic surgical technique in a quality environment, when removing the tissue from the donor, when processing and storing the tissue, and during implantation, is critically important to prevent bacterial and fungal contamination. Non-viable tissue grafts such as bone can undergo disinfection and sterilisation steps. During the past two decades, the disease transmission risk associated with tissue transplantation has been greatly reduced by implementation of standards set by professional organisations, such as the American Association of Tissue Banks (AATB, 2001), the European Association of Tissue Banks (EATB), the Eye Bank Association of America (EBAA, 2001), and governmental regulations. However, the incidence of transplant-transmitted infection is uncharted, and the studies needed to determine this have hitherto not been performed. Cadaveric donations (Table 1) and clinical transplants (Table 2) of cornea, bone, skin, heart valve and other tissue allografts in the United States greatly exceed that of organs (United Network for Organ Sharing, 2001). Organ transplantation flourished in the early 1980s following the discovery and introduction of cyclosporin as an effective immune suppressant. This brought a large supply of cadaveric donors that could also be used for tissue donation. Unlike the limitations of organ transplants, tissue transplantation generally is not limited by HLA histocompatibility barriers (Choo and Eastlund, 1996) or by ABO Table 1. Cadaveric organ and tissue donation in the United States. Type of donor tissue

Donations per year

Cornea donors* Bone, skin, or other tissue donor* Organ donor*

46,729 18,021 6,082

*Eye Bank Association of America, 2001. +American Association of Tissue Banks, 2001. ^United Network for Organ Sharing, 2001.

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T. Eastlund & D.M. Strong

Table 2. Estimated number of allografts transplanted annually in the United States. Tissue CADAVER TISSUE* Bone Corneat Skin (sq. ft.) Heart Valve Vessels Pericardium CADAVER ORGAN ALLOGRAFTS* Kidney§ HeartH Liver Pancreas Lung Intestine LIVING DONOR TISSUE AND CELLS Red Blood Cells" Unrelated Bone Marrow Stem Cells*** Peripheral Blood Stem Cells Cord Blood Stem Cells

Transplants 675,370 50,868 11,222 5,500 433 5,327 17,601 9,094 2,210 4,667 465 1,034 113

13,361,000 1,160 524 59

'American Association of Tissue Banks — 2000; Office of the Inspector General — 2001. tEye Bank Association of America — 2001. ^United Network for Organ Sharing — 2001. ^Includes 884 combined kidney and pancreas transplants. ^Includes 27 combined heart and lung transplants. ''National Blood Data Resource Centre — 2002. '"National Marrow Donor Program — 2001. blood group incompatibility (Eastlund, 1998a). No longer being a scarce resource, the widened availability of tissue allografts encouraged new clinical use and brought attention not only to their effectiveness and advantages over autografts, but also to

Infectious Disease Transmission Through Tissue Transplantation

55

their drawbacks, side effects and complications. One complication of tissue transplantation has been transmission of disease of donor origin to the recipient (Gottesdiener, 1989; Eastlund, 1995). Although transmission of malignant tumour of donor origin is a recognised complication of organ transplantation in immunosuppressed recipients (Perm, 1993), there has been only one report of a tissue graft transmitting a malignancy: over 50 years ago, a single case of a retinoblastoma transplanted with a cornea was reported (O'Day, 1989). On the other hand, bacterial, fungal and viral infectious diseases have been transmitted more often and by several types of tissue. Most reported tissue transplant-transmitted infections have been of donor origin. Viral infections can be transmitted if the donor has a viral infection with viral levels too low for detection. In asymptomatic donors who were recently infected, a transient viremic phase can exist prior to development of a positive donor screening test for antibodies. Preventing donorto-recipient infectious disease transmission relies heavily on selecting safe donors not only through testing, but also by medical and social behaviour screening, to select donors more likely to be free of transmissible infections. Bacteria and fungi can be introduced into the tissue allograft as a contaminant during surgical removal of the tissue from the donor, or from processing, storage or implantation. During allograft processing, the use of sterile supplies and aseptic technique in an environment as free as possible of microbial contamination is important. Environment monitoring, adequate sampling techniques and final sterility testing are also essential. The characteristics of each tissue allograft, and whether it can be exposed to disinfection and sterilisation, have a large bearing on whether disease transmission is likely. Some tissue allografts, e.g. corneas, heart valves and skin, need to remain viable and cannot be exposed to disinfectants or sterilisation without an unacceptable loss of viability. Other grafts are non-viable, largely comprised of acellular connective tissue, and can be disinfected or sterilised more freely resulting in a greater assurance of

T. Eastlund & DM. Strong

56

Table 3. Allograft characteristics affecting ability to transmit disease. Nonviable allograft

Viable allograft Type Heart valve and vessels Cornea Skin Marrow Blood stem cells Vascularized organs Semen and oocyte Foetal tissue

Bone Dura mater Pericardium Tendon Costal cartilage Fascia Ear ossicles Characteristics Non-viable Acellular Connective tissue Can be processed, sterilised

Contains viable cells May be antibiotic treated Cannot be sterilised

sterility (Table 3). This review focuses on infectious disease transmission through transplanted tissues, and the steps taken for its prevention. Disease transmitted through organ and cellular transplantation and through tissue and cells donated by living individuals (i.e. blood, marrow, semen, and oocytes) will not be addressed. 2. Infections Transmitted by Cornea Allograft 2.1. H u m a n immunodeficiency virus As thin avascular tissue, cornea comprises a well-hydrated transparent layer of connective tissue, and a single-cell layer of viable endothelial cells. Consequently, it is not very immunogenic; nor is it often rejected by the recipient unless it becomes vascularised. Similarly, it is not very efficient in transmitting

Infectious Disease Transmission Through Tissue Transplantation

Table 4. Infectious disease transmitted by tissue allografts. Allograft

Infectious disease

Bone

Hepatitis C Hepatitis, unspecified type HIV Bacteria Tuberculosis HTLV

Tendon

Bacteria Hepatitis C HIV

Cartilage

Bacteria

Cornea

Hepatitis B Rabies Herpes simplex virus Creutzfeldt-Jakob disease Cytomegalovirus (?) Bacteria Fungus

Dura

Creutzfeldt-Jakob disease Bacteria

Heart valve

Hepatitis B Tuberculosis Fungus Bacteria

Skin

HIV (?) Bacteria Cytomegalovirus (?) HCV (?)

Pericardium

Creutzfeldt-Jakob disease Bacteria

57

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T. Eastlund & DM. Strong

viral infectious disease from the donor. Diseases transmitted through corneal transplants are listed in Table 4. The cornea is not efficient in carrying or transmitting HIV. Based on assumptions about HIV antibody test sensitivity, Goode et al. estimated that 3 per 10,000 cornea allografts will be from HIV-infected donors despite HIV antibody testing (Goode et al, 1988). There have been several documented cases in which cadaveric organ and tissue donors were infected with HIV, but the cornea recipients did not become infected. Although HIV has been isolated from tears, retina, cornea, aqueous humour, iris, and conjunctiva (Cantrill et al, 1988; Fujikawa et al., 1985a; Fujikawa et al, 1985b; Heck et al, 1989; Salahuddin et al, 1986), HIV from infected cadaveric donors has not been transmitted to cornea recipients (Pepose et al, 1987; Schwarz et al, 1987). This should not be surprising because the inoculum of HIV is small in the relatively avascular, hypocellular cornea as compared to that in an organ transplant or a blood transfusion from an HIV infected donor.

2.2. Hepatitis B virus Failure to transmit viral infection from hepatitis B surface antigen (HBsAg)-positive donors has been reported in two recipients of corneas. This suggests that the cornea is inefficient as a mode of HBV transmission, although in these cases the administration of hepatitis B immune globulin and vaccine to the recipient may have prevented infection (Raber and Friedman, 1987). Khalil et al. assessed the presence of HBsAg and HBV DNA in corneal buttons taken from HBsAg-positive donors (Khalil et al, 1995). They found HBV in a small percentage of corneas. Others studied 31 donors infected with HBV or HCV but were unable to detect HBV DNA or HCV RNA in the corneas (Sengler et al, 2001). Despite this inefficiency, HBV transmission by corneal transplantation has been reported. In earlier reviews by O'Day (O'Day, 1989) and Raber and Friedman (Raber and Friedman, 1987), there were brief reports of hepatitis

Infectious Disease Transmission Through Tissue Transplantation

59

B transmission to cornea recipients from HbsAg-positive donors. Two cases of recipient HBV infection after transplants from two different HBsAg-positive donors were eventually reported (Hoft et al, 1997). Corneal donations took place from two donors; one in 1984 from an alcoholic man, and one in 1985 from an injecting drug user. Tests for HBV were performed on the donors retrospectively after recipients developed HBV infections. Both donors were positive for HBsAg. Only one of the two recipients of corneas from each of the two donors developed symptomatic HBV infection. The use of current professional standards and federal regulations would have prevented these cases since exclusion of donors with hepatitis risk behaviours, and testing for HBsAg, are now required. 2.3. Fungal disease Cornea transplants have transmitted fungal and bacterial infections (O'Day, 1989). Fungal growth can develop during storage of corneas in refrigerated liquid media containing bacterial antibiotics. Fungal contamination does not arise from a clinicallyapparent eye infection of the donor, but may originate as a contaminant on the eye surface, or acquired during collection, processing or storage. Clinically important fungal infections in recipients are rare but can be serious (Pels and Vrensen, 1999). Donor-to-recipient transmission of Torulopsis glabrata (Larsen et al, 1978; Cameron et al, 1991; 1998), Exophilia (Benaoudia et al, 1999), Cryptococcus neoformans (Beyl and Waltman, 1978), Aspergillus flavus (Cameron et al, 1991), Candida tropicalis (Behrens-Baumann et al, 1991), and Candida albicans (Insler and Urso, 1987; Stuart and Linn, 1984; Merchant et al, 2001; Sutphin et al, 2002; Cameron et al, 1991) have been reported. Merchant et al. reported that, in 40 of 44 cases, Candida albicans was cultured from both the recipient and the donor corneal rim (Merchant, 2001). In many of these cases, current donor selection practices would have excluded these donors, including a donor who died of polymyositis and was being treated with

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immunosuppressive drugs and who transmitted Cryptococcus neoformans; and an alcoholic donor with disseminated Candida tropicalis, bronchopneumonia, and pancreatitis who had the same yeast cultured from his throat and from his organs postmortem (Behrens-Baumann et al, 1991). 2.4. Bacterial disease Post-transplant bacterial infection of the cornea can be serious and can lead to wound dehiscence, corneal perforation and emergency regrafting. In cases where the infecting organism is found in corneal tissue, or in the corneal storage medium, or where both corneas of one donor had transmitted the same bacterial infection to two different recipients, the likelihood that the bacterial infection was from the transplanted cornea is high. In two reported cases, both implicated donors would not have been accepted as donors using today's donor eligibility criteria, because of the presence of bilateral bronchopneumonia in one, and Hodgkin's disease, small bowel perforation, peritonitis, and high fever in the other (Gandhi et al., 1981). Leveille et al. reported four cases of bacterial endophthalmitis in 1,876 recipients of corneal transplants (Leveille et al., 1983). Three of these infections were from an organism that was also isolated from the donor cornea. It was found that the presence of sepsis in the cornea donor appeared to be a risk factor for bacterial transmission. Others found that mechanical ventilatory support of the corneal donor was not a risk factor by itself (Seedor et al., 1987). Some studies have not found a correlation between the use of a septic donor and recipient corneal infection, and propose that corneal grafts from septic donors be available for transplantation (Spelsburg et al., 2002). This proposal was supported by others who found that external bacteria on donor cornea are mainly skin bacteria; and internal bacteria are mainly gut bacteria. It is speculated that these gut bacteria arose from peri-mortem bacteraemia and not from an underlying infection of the donor (Robert et al, 2002). They observed that bacterial

Infectious Disease Transmission Through Tissue Transplantation

61

infection in the donor at the time of death had no effect on the incidence of endophthalmitis in cornea transplant recipients. In some cases of bacterial endophthalmitis, it was difficult to determine whether the bacteria were from the donor, or acquired during surgical removal of the cornea (Moore et ah, 1989). Bacterial contamination is more likely with the use of suboptimal techniques during the surgical removal of the tissue from the cadaveric donor. In one programme, the occurrence of several post-transplant infections by Streptococcus pneumoniae prompted an investigation. After collection techniques were modified to reduce contamination, there were no further infections by the same microbe during the following year (Moore et ah, 1989). A corneal disinfection step with povidone-iodine can lessen the risk (Pels and Vrensen, 1999). Antonios et ah, reported that 0.23% of corneal transplants became infected due to bacterial contamination of the allograft during storage, and that the risk was highest if the cornea had been stored for five or more days prior to use (Antonios et ah, 1991). Bacteria on contaminated corneas can survive refrigerated storage in media containing antibiotics (Baer et ah, 1989; Glaros et ah, 1991). Recently, gentamicin has become a widely-used antibiotic for storage (Gopinathan et ah, 1994). Gentamicinresistant bacteria have been found in 14% of stored corneas, but generally have not resulted in recipient infections (Farrell et ah, 1991). Streptococcus, Propionibacterium, Staphylococcus and diphtheroids were commonly found. Recently, Khokhar et ah reported a bacterial infection from a corneal transplant from an organism which was resistant to gentamicin, which was the only antibiotic in the storage medium (Khokhar et ah, 2002). Failure of corneas to transmit the syphilis spirochete has been demonstrated in animal experiments in which corneas were transplanted from rabbits with latent or active syphilis (Randolph, 1952). The spirochete causing Lyme disease has been isolated from human cornea, but it is not expected to be a risk to cornea recipients because prospective corneal donors with active Lyme disease would be excluded. In addition, infected

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donor corneas show infiltrative birdshot keratitis, and this would be discovered and be a cause for discard prior to use (SuttorpSchulten et al, 1993). 2.5. Other viruses Cytomegalovirus does not appear to be readily transmitted by cornea transplantation from seropositive cadaveric donors to seronegative recipients. Of the 25 seronegative patients receiving a corneal graft from a seropositive donor, only two seroconverted (Holland et al, 1988). Herpes simplex virus, type 1, has been found to be widespread in corneal stromal cells but only one case of transmission by a cornea allograft has been reported. The infection caused corneal deterioration in the recipient by the fifth day after transplant (Cleator et al, 1994). HSV DNA was found in two of five cornea allografts from other donors (Tullo et al., 1990). 2.6. Rabies Rabies virus infection in humans is often found in the cornea. Because of this, a cornea impression test has been useful for early diagnosis (Koch et al., 1975). Corneal allografts are also capable of transmitting rabies. Seven cases of fatal rabies transmission from cornea transplantation had been reported in the US, France, Thailand and India during 1979-1988, and in Iran in 1994 (Gode and Bhide, 1988; Centres for Disease Control, 1979-1981; Houff et al, 1979; Baer et al, 1982; Javadi et al, 1996). The first case involved a 39 year old man in the US with ascending paralysis (Centers for Disease Control, 1979), and the second involved a donor in France who died from paraplegia, encephalitis and myocarditis (Centers for Disease Control, 1980). In 1997 Javadi et al, and Gode and Bhide each reported rabies developing in two patients who received corneal transplants from the same donor. (Javadi et al, 1996; Gode and Bhide, 1988). Each of these cadaveric donors had an obvious acute neurological illness

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clinically consistent with rabies. National professional standards used by tissue banks today prohibit the use of these donors and would have prevented these cases of rabies transmission. 2.7. Creutzfeldt-Jakob disease The ability of the cornea to transmit Creutzfeldt-Jakob disease (CJD) has been reported in humans (Duffy et al, 1974), and confirmed experimentally using hamsters and guinea pigs (Manuelidis E. et al, 1977). CJD was transmitted by a cornea donated by a 55 year old man who died with bronchopneumonia following a two-month history of incoordination, myoclonic jerks, and neurological decline (Duffy et al, 1974). Cornea donors implicated in CJD or in rabies transmission had acute antemortem neurological illnesses with signs and symptoms such as fever, asymmetric weakness or paralysis, swallowing difficulties, ophthalmoplegia, diplopia, absent reflexes, abnormal sensation or coma. In all cases of rabies and CJD transmission, each donor was clearly not eligible to donate under criteria in place today, which exclude persons with acute and chronic neurological illnesses (AATB, 2001; Campagnari and O'Malley, 1994; Eye Bank Association of America, EBAA, 2001). Recent reviews again emphasise the importance of excluding prospective donors with neurological illness as a measure of preventing CJD (Hogan and Cavanaugh, 1995; Hogan et al, 1999). Overall, the risk of transmitting CJD by corneas is very low. 3. Infections Transmitted by Bone Allograft 3.1. Bacterial disease Bacterial infection due to use of a contaminated bone allograft has rarely been reported. Lord and associates (Lord et al., 1988) observed one of 283 massive frozen bone allografts to be a cause of a postoperative infection. Tomford and colleagues (Tomford et al, 1981) found one of 303 small freeze-dried bone

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grafts from the Navy Tissue Bank to be implicated in a recipient Staphylococcus epidermis infection. These bacteria may have been donor-derived or may have been acquired at the time of procurement. Tomford and co-workers (Tomford et al., 1990) reported an infection rate of about 4 to 5% in use of 324 culturenegative, non-sterilised unprocessed frozen bone allografts at Massachusetts General Hospital. These rates of infection were not different from those found following surgery using prosthetic devices. Bone allograft appeared responsible for infections in only three patients, all of whom developed infection with Serratia marcescens and received bone from the same donor. Others have also reported bacterial infection as a complication of spinal surgery using bone allograft but, as a whole, the incidence was similar to that when using autograft (Aurori et al., 1985; Knapp and Jones, 1988; McCarthy et at., 1986; Transfeldt et at., 1985). Aho et al., reported two deep bacterial infections during use of 63 large allografts, apparently caused by transplantation of the unprocessed frozen large bone allografts (Aho et al., 1998). The same bacteria (Pseudomonas aeruginosa and Staphylococcus epidermidis) were isolated from the allograft prior to surgery and from the recipient site of infection. These two allografts had no growth on bacterial testing at the time of procurement but the bacteria were identified when the frozen allograft was thawed and retested at the time of surgery. These cases demonstrate the potential risk: even though the cadaveric bone donor may appear free of infection, bacterial contamination missed due to inadequate bacterial sampling or testing technique, can be a cause of infection. The bacterial infection risk appears to be similar to that of other major orthopaedic surgeries not using allografts. About 40 years ago, several cases of tuberculosis of the bone in patients undergoing spinal fusion were reported from the use of frozen rib allografts (James, 1953). The source of bone allograft was the rib resected during chest surgery from patients with active pulmonary tuberculosis. As one of the current donor screening criteria, individuals with active or previously treated tuberculosis are excluded from donating (AATB,

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2001; Campagnari and O'Malley, 1994). This exclusionary criterion is important because there is no blood screening test for tuberculosis that offers a good predictive value.

3.2. Hepatitis Hepatitis has been reported from use of unprocessed refrigerated and frozen bone allografts, but not from bone grafts that were cleaned of cells and fat with water jetting and ethanol soaks prior to being freeze-dried or treated with sterilants such as gamma irradiation or ethylene oxide. In 1954, prior to the availability of viral hepatitis testing of donors, a Yale medical student received a refrigerated bone graft to treat a depressed fracture of the proximal tibia, and developed hepatitis with jaundice 10 weeks later (Shurkin, 1954). The bone graft was obtained from the amputated leg of a patient with occlusive vascular disease and gangrene. Otherwise, the donor was in good health, with normal liver function and without a history of jaundice or liver disease. The donor had received blood transfusions three years previously. Three reports from nearly a decade ago documented that hepatitis C virus (HCV) can be transmitted from donor-torecipient through the use of frozen, unprocessed bone allografts (Eggen and Nordbo, 1992; Pereira et ah, 1993; Conrad et al.r 1995). In the first case, donor testing for HCV antibodies was not available. HCV was transmitted by the use of a femoral head allograft after it was donated by a donor undergoing hip arthroplasty and stored frozen for two months (Eggen and Nordbo, 1992). In a second report, HCV was transmitted from an infected cadaveric tissue donor through frozen, unprocessed bone and tendon grafts, but not through freeze-dried bone allografts that were treated with gamma irradiation (Conrad et ah, 1995). In this study, the cadaver bone donor tested negative for HCV antibodies using the first generation test available at the time of donation in 1990, but stored serum tested positive when

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a new, more sensitive test was introduced in 1992. Testing for HCV RNA by polymerase chain reaction (PCR) was also positive. In a third brief report involving five HCV-infected organ and tissue cadaveric donors, a minority of the recipients of frozen bone allografts became infected with HCV (Pereira et al, 1993). In a more recent case an HCV-infected organ and tissue donor was tested and found negative for HCV antibodies (Tugwell et al, 2002). Despite this negative screening test for HCV, several organ and tissue recipients became infected. When blood samples from the donor were tested later for HCV,RNA, the results were positive, and this confirmed the link between the donor and multiple infected recipients. The donor had been recently infected and was viremic but had not yet produced detectable antibodies. Bone allografts from the same donor that had been treated with gamma irradiation did not transmit HCV. With the implementation of HCV RNA as a donor screening test in the future, cases such as this will be prevented. There have been no reports of HBV transmission through bone transplantation, although it has been recognized as a complication of organ, cornea, and heart valve transplantation. It is quite probable that there have been transmissions, but none have been recognised and published. 3.3. H u m a n immunodeficiency virus HIV-1 has been transmitted through blood, tissues, and organs (Petersen et al., 1993; Simonds et al, 1992). Viable HIV-1 can be recovered from bone, marrow, and tendons of patients with acquired immunodeficiency syndrome (Buck et al, 1990; Merz et al, 1991; Nyberg et al, 1990; Marthy and Richter, 1998). In 1984, a fatal HIV-1 infection was transmitted to a woman undergoing spinal fusion for scoliosis, through the use of a frozen femoral head allograft several weeks after it had been donated during hip arthroplasty from a donor who had a history of

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intravenous drug abuse and who had an enlarged lymph node that had been biopsied the previous year (CDC, 1988b). Both the donor and the bone allograft recipient subsequently died of AIDS. A test for HIV-1 antibody was not available at the time of donation. This donor would not have been eligible to be a donor today due to his history of intravenous drug abuse and lymphadenopathy. There have been other cases of HIV infection in recipients of bone allograft, derived from HIV-infected donors who were not tested for HIV at the time of donation. Prior to HIV antibody test availability in Germany, 12 recipients had had frozen bone allografts from an infected cadaveric donor during November 1984 through January 1985 (Schratt et al, 1996). Only four of these recipients became HIV-positive. Seven remained HIVnegative. In Taiwan a man donated a femoral head during hip replacement surgery but was not tested for HIV. The bone allograft was used in a 34 year old woman in 1996 during knee reconstructive surgery. She seroconverted with HIV antibodies when tested five months later (Li et al, 2001). Another reported case of HIV transmission through the use of frozen bone allograft involved a seronegative but infected cadaveric donor but the test was new and not very sensitive. Multiple organs, corneas, bones, and connective tissues were transplanted (Simonds et al, 1992). Three organ recipients and three recipients of frozen bone and tendon allografts became HIV-infected. These allografts had not been sterilised with gamma irradiation or ethylene oxide gas prior to use. The donor tested negative for HIV antibodies at the time of donation in October 1985, which was a few months after the first, relatively insensitive HIV-antibody testing kits became available. Between 1985 and 1991, several modifications greatly improved test sensitivity. Prior to 1989, HIV antibodies were detectable after a median of 63 days from initial infection (Bowen et al, 1988; Horsburgh et al, 1989). A study of HIV-infected blood donors between March 1987 and 1991, when whole viral lysate enzyme

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immunoassays were used to detect HIV antibodies, showed an average seronegative window period of 45 days (Petersen et al, 1994). A report in 1992 showed that HIV-antibody test kits in use at that time detected twice as many infected individuals as did the test kits available in 1985 (Nowicki, 1992). Since 1992, HIV-antibody tests have become even more sensitive, detecting IgM, the earliest form of antibodies, earlier by an average of 8 20 days (Stramer et al, 1989; Zaaiger et al., 1992) and resulting in a seronegative window period of approximately 22 days (Busch, 1994; Busch et al., 1995). Since then, blood donor testing for HIV RNA by nucleic acid testing (NAT) has been implemented, and has further reduced the risk of a transfusion; and when validated and implemented for cadaver tissue donors, will reduce the risk in tissue transplantation (Stramer et al., 2000). The prevalence of HIV antibodies in bone donors is low, and when medical history screening and selection processes are applied vigorously, it should not be greatly different from that of voluntary blood donors. This may be true for living bone donors (Hamilton et al, 1990; Scofield et al, 1993a; 1993b) but not necessarily for cadaveric donors. Of 9,000 living bone donors who donated femoral heads at the time of hip arthroplasty surgery, none were found to have confirmed positive tests for HIV-1 antibodies at the time of donation (Hamilton et al, 1990). Prevalence of infectious disease markers in surgical bone donors was not different from that of blood donors, except for a higher prevalence of false positive syphilis tests and antibodies to HBV core protein (Scofield et al, 1993a). Retesting of 1,608 living bone donors 180 days later yielded none with confirmed positive HIV or HCV tests (Scofield et al, 1993b). Of 5,513 cadaver bone donors tested throughout the United States in 1992, three confirmed positive for HIV antibodies (Eastlund et al, 1994), but these three were from a single tissue bank that later disclosed accepting donors with risk factors for HIV. A more recent survey by AATB (AATB, 2000) revealed a higher prevalence rate of infectious disease markers than have been reported for blood donors, with rates ranging from two to 40

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Table 5. Positive infectious disease markers in tissue donors and blood donors. Test

Tissue donors*

Blood donors'1'

Anti HIV-1/2 HBsAg Anti-HCV Anti-HTLV-I/II Syphilis

0.4% 1.2% 1.5% 0.8% 1.0%

0.05% 0.03% 0.12% 0.12% 0.5%

'American Association of Tissue Banks. 18,021 cadaveric donors in 2000. (AATB, 2000) tPuget Sound Blood Centre. 182,138 blood donors in 2001. (Strong, 2002)

times higher (Table 5). The addition of viral nucleic acid testing since 1999, in screening blood donors for HIV and HCV, has further reduced the risk from blood donors. Among first time blood donors, there is a well known higher infectious disease marker rate, and such donors may be more similar to organ and tissue donors (Schreiber et ah, 2003). Because prospective tissue donors with HIV risk behaviours and positive tests for HIV are excluded, and most bone graft processing removes blood and marrow cells and applies disinfectants and sterilants, the risk of HIV transmission by bone transplantation is now very remote, or nearly absent (Asselmeier et at, 1993). The risk of transmitting HIV through bone grafting has been calculated to be less than one in a million grafts (Buck et ah, 1989; Carlson et ah, 1995), and is even less if the graft has been subjected to processing and sterilisation steps using gamma irradiation or ethylene oxide. However, the HIV transmission risk is higher in the less frequently used frozen unprocessed bone allograft. An accurate estimate of the risk cannot be made until a more accurate determination of the prevalence of HIV infection in the donor and recipient population is available, and prospective studies have been done on recipients.

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3.4. H u m a n T-Lymphotrophic virus Asymptomatic HTLV-I infection has been transmitted by transplantation of a fresh-frozen unprocessed femoral head bone allograft (Sanzen and Carlsson, 1997). A 62 year old man became infected by HTLV from a blood transfusion in 1987 during hip surgery. One month later he developed fever, a rash and a transient right-sided radial nerve palsy. Frozen sera obtained during this illness (but tested later) demonstrated HTLV seroconversion. In 1991 he donated a femoral head without antiHTLV testing during a second surgery for a hip prosthesis. The unprocessed frozen femoral head was used as a graft in a different patient one month later. This bone graft recipient developed HTLV-I antibodies but had no HTLV-I associated disease. 4. Infections from Cartilage and Osteochondral Allografts Costal cartilage allografts are routinely disinfected or sterilised prior to their use as allografts, and, provided in a freeze-dried or frozen form. There have been no reports of processed costal cartilage transmitting infection from the donor to the recipient. Donald and Cole surveyed 312 surgeons who used cartilage allografts preserved by eight different methods for facial reconstructive surgery (Donald and Cole, 1982). They found a postoperative bacterial infection rate of 19% that was similar to the 16% reported following use of autologous cartilage. 4.1. Bacterial infection Until recently, the use of articular cartilage, e.g. in the form of a fresh or frozen unprocessed femoral condyle, has not been a significant risk of transmitting bacterial infections. An articular cartilage allograft, used in knee surgery, is minimally processed to maintain cartilage mechanical properties and chondrocyte

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viability, although it is not known whether chondrocyte viability is important. Thus, the allograft is often only briefly exposed to antibiotic solutions, and frozen with or without a cryoprotectant such as dimethyl sulfoxide. These cartilage allografts that have not been exposed to disinfectants or sterilisation as a final step, are capable of transmitting bacterial infection. In the US during 2001, a fatal bacterial sepsis case involved an osteochondral allograft (femoral condyle) and led to an investigation linking the fatality to an allograft contaminated by Clostridium (CDC, 2001a; 2001b; 2002a). In this case a 23year old male patient had surgical-site pain three days after a knee allograft implant procedure, leading rapidly to hypotension and death 24 hours later. The cartilage allograft had been aseptically processed, packaged and distributed by a for-profit tissue processing facility that is not accredited by AATB. A thorough investigation by the CDC and US FDA revealed several factors that contributed to the distribution and use of the contaminated allograft (CDC, 2002a) (Table 6). Bacterial testing at Table 6. Contributing factors leading to contaminated osteochondral allograft and fatal Clostridium sepsis.* Donor body not refrigerated until 19 hours after death — procurement 23.5 hours after death 1) The tissue processing facility did not obtain pre-processing bacterial testing of tissues. 2) The tissue was processed aseptically without use of disinfectants or terminal sterilisation. 3) Antibiotic soaks were used, and antibiotic residue may have interfered with detection of Clostridium. 4) No bacteriostasis testing was performed at the time of final sterility testing. 5) Processing steps were not validated to achieve an acceptable level of sterility assurance. 'Centres for Disease Control, MMWR 2002, 51 (March 15), 207-210.

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the time of removal from the cadaveric tissue donor, and prior to processing in the tissue facility, was not performed. AATB standards require this step to identify virulent microbes that might not be eliminated despite antibiotic soaking during processing (AATB, 2001). The cadaveric donor body was stored at room temperature for 19 hours, and at refrigerated temperatures for an additional 4.5 hours prior to tissue removal. National professional standards limit the storage of the cadaveric body to 12 hours or less if stored at room temperature (AATB, 2001). If refrigerated, the tissue can be surgically removed from the donor u p to 24 hours after death. C. sordelli was also found in fluid bathing the tissues during processing. The tissue was processed aseptically with antibiotic soaks but included no other disinfectants or sterilants. Antibiotic residues may have interfered with detection of C. sordelli during final sterility testing. No bacteriostasis testing was performed by the tissue processing facility to determine whether antibiotic residues caused falsely-negative final sterility test results. Tissue processing had not been validated to achieve an acceptable level of sterility assurance. C. sordelli was found in two of 19 other unused tissues from the same donor. Two of nine patients who received other tissue allografts from the same donor became infected (CDC, 2002a). The tissue processing facility was prohibited by the US FDA from further processing and distribution until it agreed to make required changes, including following AATB standards. The investigation of this case led to the discovery of Clostridium infections in several other recipients of frozen tendons provided by the same tissue processing facility (CDC, 2002a). 4.2. Ear ossicles Tympanic membrane and middle ear ossicles are transplanted to restore hearing, but there have been no reports of disease transmission (Lesinski, 1977). The bacteriologic sterility of ear ossicles after extraction from a cadaveric donor, is unlikely. These tissues

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are frequently obtained after an intracranial autopsy, and the tympanic membrane is normally exposed to the external environment and frequently colonised with bacteria. Consequently, treatment with disinfectants or sterilants during processing is standard practice (Glassock et al., 1988). 5. Infections Transmitted by T e n d o n Allograft 5.1. Viral diseases The use of the patellar tendon allograft to replace the knee's injured anterior cruciate ligament has become commonplace (Noyes et al., 1990). HIV has been isolated from tendons in HIV-infected persons (Buck et al, 1990; Buck and Malinin, 1994) and has been transmitted from a seronegative cadaveric tissue donor through a donated patellar tendon used in knee surgery (Simonds et al, 1992). HCV was transmitted to recipients of frozen tendon allografts from an anti-HCV positive cadaveric donor (Conrad et al, 1995). It is possible that HIV and HCV were harboured in the unprocessed bone blocks at either end of the tendon allograft. These allografts had not been processed to remove blood and marrow cells. Despite these cases, the risk to recipients is presumably low as long as donor screening steps are applied as required by national standards (AATB, 2001; Campagnari and O'Malley, 1994) and federal regulations (FDA, 1993; 1997; 1999). In addition, tendons can be treated with gamma irradiation to further reduce the risk of disease transmission. Selecting donors without risk factors and without HCV antibodies, makes the risk of spreading HCV by transplant an exceedingly rare event. However, an early HCV infection in a cadaveric organ and tissue donor not yet producing antibodies, was reported recently (Tugwell et al., 2002). A patellar tendon allograft recipient developed acute, symptomatic hepatitis C in May 2002, six weeks after transplantation. No other potential sources of infection were identified. The tissue donor was

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anti-HCV negative but stored serum showed HCV RNA when tested later. Thirty-nine other persons received tissues or organs from this same donor. Early results of a partially completed investigation showed that of 18 recipients tested, six showed HCV infection, including a lung recipient who became HCV RNA positive on day 4 and died of liver failure 14 months later. Presumably, the cadaveric donor was in a viremic stage early in infection prior to antibody development. To date, cadaveric testing for HCV RNA is not available for routine use. HCV RNA should be considered for cadaveric tissue donors as soon as test reliability has been evaluated, particularly using cadaveric samples obtained up to 24 hours after death. 5.2. Bacterial infection Recently, there have been several reported cases of bacterial infection arising from the use of contaminated patellar and Achilles tendon allografts. These cases were primarily a cluster of Clostridium infections in connective tissue provided by a single tissue processing facility. After a fatality due to contaminated cartilage allograft (CDC, 2002a) the resulting investigation of the tissue bank and tissue recipients found Clostridium infections in several other tendon recipients, thus prompting changes within the tissue processing facility to prevent further cases. Several other non-clostridial infections were subsequently discovered in patients who had received tendons from other tissue banks, but only a few were demonstrated to be caused by the tendon allograft (CDC, 2002a). Two cases involved non-clostridial bacterial infections in patients who received frozen tendon allografts for anterior cruciate ligament repair, and in these cases a tissue bank distributed the contaminated allografts due to human error (CDC, 2001c). The intended terminal sterilisation by gamma irradiation did not take place. Another case involved pseudomonas infection in two recipients using frozen tendon allografts derived from the same donor, suggesting that the tendon caused the infection

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(CDC, 2001c). However, the source of the bacteria was not determined. An independent investigation later revealed that the tendons had received sterilisation treatment with 1.8 Mrad gamma irradiation (Eastlund et ah, 2003). Because the pseudomonas bacterium is exquisitely sensitive to gamma irradiation, the tendon seemed unlikely to be the source, and acquisition of the contaminant during the surgical procedure needed to be more thoroughly considered as the potential cause. A recent investigation of suspected tendon allograft-related infections was made at six US tissue banks that process bone and connective tissue allografts (Eastlund et ah, 2003). Of the 27 suspected cases, only five bacterial infections were determined to be probably caused by the allograft: a contaminated patellar or Achilles tendon in three cases; an osteochondral allograft in one case; and aseptically processed cancellous bone chips in another case. In each of these five cases, investigators found evidence of the same infecting organism either in samples of tissue taken at the time of surgical removal from the cadaveric donor, or in sampling during final sterility testing at the end of tissue processing by the tissue bank. Subsequently, these tissue banks implemented procedures to prevent use of tissue with evidence of virulent organisms found at the time of tissue procurement or at final sterility testing. Of 26 other cases of musculoskeletal allograft-associated bacterial infections investigated by the CDC, data was not provided to confirm that the allograft was the cause. Thirteen cases involved Clostridium infections, and eleven of the implicated allografts — mostly frozen tendons — were distributed by a single tissue processing facility (CDC, 2002a). As a consequence of this, the US FDA implemented an emergency federal regulation requiring that allograft processing facilities have written procedures that are validated for the prevention of infectious disease contamination (or cross-contamination) by tissue during processing (FDA, 2002). It also required maintenance of records concurrently with the performance of each significant step.

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Validation data must demonstrate that procedures reliably prevent infectious disease contamination during processing. 6. Infections Transmitted by Cardiovascular Allograft 6.1. Viral disease The capacity of human heart-valve allografts to transmit HBV was demonstrated in a study of 31 patients who received heart valve allografts from HBsAg-positive donors. Twentytwo recipients were HBsAg-positive prior to transplant, or were immune to HBV and not susceptible to HBV infection. Of the nine recipients susceptible to HBV infection, only one developed HBV viral markers. None developed clinically apparent hepatitis. However, four susceptible recipients received hepatitis B immune globulin, and one received HBV vaccine following transplant, which may have prevented infection (Morris et al., 1990). Currently, all donors are tested for HBsAg, and if positive, are excluded. Despite testing of donors for HBsAg and anti-HBc, HBV transmission can still occur because some donors can have circulating HBV at levels not detectable in routine tests. Thijssen et al., found one of 676 heart valve allograft donors to have HBsAg detectable with routine screening tests (Thjissen et al, 1993). In addition, they found 10% to have anti-HBc. Fifty-two of 63 donors with anti-HBc also had antibodies to HBV surface protein (anti-HBs), indicating a resolved HBV infection and a recovered, immune non-infectious status. Three of those with anti-HBc but without anti-HBs were positive for HBV DNA via a more sensitive liquid-phase DNA hybridisation assay. This would suggest a possible use of anti-HBc donor testing to prevent transmission of HBV; however, one study of blood donors has shown a lack of predictive value in preventing post-transfusion hepatitis (Blajchman et al, 1993). More recently, however, several reports have confirmed that some anti-HBc positive donors will be positive for HBV DNA and will transmit HBV (Roth et al, 2002).

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6.2. Mycobacterium and other bacterial disease Postoperative bacterial endocarditis is a complication of cardiac valve replacement surgery, occurring at similar rates for valves of human, porcine or mechanical origin. There have been several reports that implanted heart valves may be capable of transmitting mycobacterium of donor origin. Several cases were reported of the transmission of non-tuberculous mycobacterium by use of contaminated non-human prosthetic heart valves. In one report, a patient manifested a Mycobacterium chelonei infection five months after implantation of a porcine xenograft heart valve (Tyras et ah, 1978). The valves used in this patient, and in seven others, were sampled prior to implantation, and the same bacteria grew from all. Only one patient had a clinical infection. These porcine xenograft valves apparently became contaminated with M. chelonei during production at the manufacturer. Another patient who received a prosthetic valve from the same manufacturer during the same time period also developed M. chelonei endocarditis, but the infection was not evident until three years later (Rumisek et ah, 1985). A total of four patients were reported as getting infected by M. chelonei from the same manufacturer's contaminated prosthetic valves. Khanna et ah, reported a case of miliary tuberculosis developing 8 months after receiving a human valve allograft, and concluded that the infection arose from the transplanted valve (Khanna and Monro, 1981). Anyanwu et ah, reported seven cases of miliary tuberculosis among 934 patients receiving human heart valve allografts (Anyanwu et ah, 1976). There was unconfirmed suspicion that the source of the infection was from the implanted valves. Of the seven cadaveric donors implicated, all were over 60 years of age; two had a history of treated tuberculosis, and one had active pneumonia at the time of death. Each of these donors would have been excluded from cadaveric donation of heart valves due to age or medical history, under today's donor criteria (AATB, 2001; Campagnari and O'Malley, 1994).

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The addition of antibiotics effective against tuberculosis in the processing steps, along with routine culturing of donor heart tissue for Mycobacterium, and excluding prospective donors with a history of treated tuberculosis, all contribute to today's low risk of transmitting tuberculosis by heart valve transplantation (Hopkins, 1989; Yankah et al, 1988). 6.3. Fungal disease There has been a recent well-documented case of fungal infection transmitted by heart valve allograft (Kuehnert et al, 1998). A 61 year old male with long standing aortic insufficiency was given a human cryopreserved aortic valve and was discharged doing well after five days. Sixteen days later, he had high fevers, nausea and diarrhoea. An infected valve allograft was removed. Candida albicans was found in his blood, on valvular vegetations and from an intramyocardial abscess (Kuehnert et al, 1998). An investigation of the tissue processing facility's records revealed that the same yeast with similar DNA fingerprinting had been identified from a sample obtained prior to processing the valve. The heart valve had been temporarily soaked in antibacterial solutions (imipenem, netilmicin, vancomycin) and antifungal solutions (amphotericin B, fluconazole) during processing. After processing of the valve, a final sterility test showed no growth but fungistasis procedures were not performed to assure that residues of antifungal solutions did not interfere with identifying microbial contamination (Kuehnert et al, 1998). Although the fungus isolated at the tissue bank was identical to that from the patient, the latter was more resistant to amphotericin and fluconazole. According to Joly and Carbon (Joly and Carbon, 1998) the resistance was induced during in vitro disinfection at the tissue bank that was using these two antifungals. These authors concluded that since fungal endocarditis is severe, and that the disinfection process of soaking the valve in an antimicrobial solution containing amphotericin B and fluconazole can be ineffective, it is necessary to discard the

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allograft if fungal contamination is detected from samples taken prior to processing. This underscores the importance of performing tests for fungal contamination prior to processing, a practice routinely carried out by AATB-accredited heart valve banks, but not by the tissue processing facility that was involved with this patient's Candida endocarditis. In addition, the authors recommended avoiding disinfection of fungal contamination because of the lack of effectiveness; the severity of fungal infection in the presence of a foreign body; and the risk of inducing resistance to the anti-fungals used (Joly and Carbon, 1998). 7. Infections Transmitted b y Pericardium Allograft There was one case of CJD reported in a recipient of human pericardium allograft used to reconstruct a tympanic membrane (Tange et al, 1990). Transmission from the donor was not confirmed. Three cases of post-transplant bacterial meningitis due to Ochrobactrum anthropi were caused by the use of human pericardium allograft to patch dural defect (CDC, 1995; Chang et al, 1996; Christenson et al, 1997). The bacteria was not of donor origin but was acquired during processing. The organism was also found in an unused pericardium allograft from the same donor, and in an unused bottle of Hanks Balanced Salt Solution from the same lot that was used for processing. 8. Infections Transmitted by Dura Mater Allograft Freeze-dried dura mater allografts have been used for closure of dural defects caused by resection of brain tumour, or repair of traumatic injury. Long-term follow-up of 1,364 dura allografts donated, processed and used in the United States and sterilised either with ethylene oxide (Malinin et al, 1978; Prolo, 1981; Rosomoff and Malinin, 1976) and 804 dura grafts sterilised with gamma irradiation (Cantore et al, 1987) revealed no infections attributable to the graft. Surgeons have reduced their use of dura

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allograft in favour of autologous fascia because of concern for transmitting CJD. 8.1. Creutzfeldt-Jakob disease Creutzfeldt-Jakob Disease (CJD) occurs at a rate of one per million in the general population, and is a fatal, transmissible, dementing spongiform encephalopathy usually accompanied by myoclonus and characteristic electroencephalographic abnormalities. It is caused by a prion, a small proteinaceous infectious particle. CJD has been transmitted from cadaveric donors through the use of pituitary-derived growth hormone and gonadotrophin (Brown, 1990; Brown et al, 1985; Cochius et al, 1990), cornea (Duffy et al, 1974), pericardium (Tange et al., 1990), and dura mater allografts (CDC, 1987b; 1987c; 1989; 1993; Lane et al, 1994; Masullo et al, 1989; Thadani et al, 1988; Yamada et al, 1994). Many cases of CJD were transmitted by use of dura allograft that was obtained from the same German medical device manufacturer, and transplanted worldwide mainly in 1984 and 1985 (Yamada et al, 1994; Lang et al, 1995). Several factors may have contributed to this outbreak, such as pooling of dura from many different cadavers during processing, and possibly obtaining dura from individuals who may have been demented. Other than dura, cornea and possibly pericardium, there have been no reports of CJD from use of other cell, tissue, or organ allografts. Because CJD is resistant to the usual sterilants, and there is no blood test for CJD, tissue banks have adopted donor selection procedures to exclude those who have received pituitary-derived growth hormone or dura allograft, or have had dementia or other symptoms of neurological disease, or have a family history of CJD, and those who have been diagnosed with or are suspected to have CJD (Table 7). The importance of excluding prospective donors with dementia alone, is demonstrated by studies showing that 18% of 230 patients with CJD initially presented with dementia as an isolated symptom (Brown et al., 1986), and

Infectious Disease Transmission Through Tissue Transplantation Table 7. Donor exclusions for CJD and nvCJD risk behaviour. 1) Persons with a diagnosis of CJD or known family history (blood relative) of a person with non-iatrogenic CJD. 2) Persons with a history of dementia or degenerative neurological disorder of viral or unknown cause. 3) Persons who have received injections of human pituitary derived growth or gonadotrophin hormone. 4) Persons who received transplants of human dura mater.

5% of those diagnosed with Alzheimer's disease were shown to have CJD at autopsy (Boiler et ah, 1989). National professional standards in the United States prohibit pooling of tissue from multiple donors and during processing, and prohibit accepting donors who have received human pituitary derived growth hormone (AATB, 2001). This is important because the hormone has transmitted CJD when used to treat short stature, and because the latency period between acquisition of the infection and development of the disease can be over a decade (Preece, 1983). With the current donor screening practices used by tissue banks, it seems unlikely that CJD will pose a serious threat to the safety of organ, tissue, or cell allograft use. The CJD agent is surprisingly resistant to the usual sterilants and disinfectants, but is fairly susceptible to sodium hydroxide (Brown et ah, 1984; Kearney JN and Johnson C, 1991). Some tissue banks have added sodium hydroxide exposure during dura processing, but have not adopted it for bone or other tissue allografts. The use of sodium hydroxide has not been widely adopted because the prevalence of CJD is very low in the general population, and even lower in the carefully screened tissue donor selected to be free of dementia, other neurological symptoms, or growth hormone treatment. Sodium hydroxide solubilises proteins, and it is not known whether it penetrates dense bone, impairs mechanical or osteoinductive properties of bone allograft, or persists in bone after processing (Kearney and

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Johnson, 1991). Due to its toxicity to cell viability, sodium hydroxide treatment is not suitable for viable tissue, cell or organ transplants. 9. Infections Transmitted by Skin Allograft 9.1. Bacterial infection Skin allograft — cryopreserved in glycerol — has been reported to carry bacterial infection (Monafo et ah, 1976). A frozen skin allograft contaminated with Pseudomonas aeruginosa was applied to a 5-year old burn patient, causing a severe infection with high fevers and Pseudomonas sepsis. In two other patients, contaminated skin allograft caused Herellea burn wound infection. National professional standards currently in place require bacteriologic sterility testing of frozen skin allografts, mandating disposal if virulent bacteria are found (AATB, 2001). In contrast to cryopreservation procedures, where skin is usually processed within 24 hours of recovery and stored at -80 degrees C or colder temperatures, skin allograft can also be stored for several days in liquid antibiotic solutions at refrigerated temperatures. At the end of the storage period, and prior to application on burned patients, the skin is often bacterially tested. Kealey reported that during 10 years of a skin bank's operation, positive results arose in only two samples of skin stored for many days at a cold temperature, in a solution of gentamicin and penicillin (Kealey, 1997). These test results, available after clinical use of the skin allograft, have sometimes shown the presence of virulent bacteria. Despite occasionally transplanting skin with virulent bacteria, clinically significant infections caused by the allograft are rare. In one small study, refrigerator-stored skin was cultured immediately prior to use, and virulent bacteria was found in a few. No adverse clinical outcome was found in recipients (Clayton et al.r 1995). Others store skin allografts in 85% glycerol solution. Of these skin allografts, 10% initially had bacterial contamination, mostly due to Staphylococcus epidermidis; but after

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prolonged storage in glycerol, bacteria were no longer detectable (Van Baare et al, 1998). 9.2. Viral infection Viral disease transmission by skin allografts has been reported. Epidermal cells can be infected with HIV-1, and the epidermis of HIV-infected individuals can transmit HIV to white cells in vitro (Berger et al, 1992; Gala et al, 1997). In one study, HIV RNA was found in only one of 12 infected patients (Kanitakis et al, 1982). Clarke reported, in a brief letter, a weakly positive test for antibody to HIV-1 in a burn patient after receipt of skin from an HIV-positive donor (Clarke, 1987). The results of donor testing were not known before the skin was used. The authors did not report whether other recipient risk factors were present, or supply the results of confirmation testing. HIV transmission from skin allograft has been recently reviewed (Van Baare et al, 1997; Pirnay et al, 1997). Transmission of hepatitis from skin allograft has not been reported although HCV nucleic acid has been demonstrated in skin from infected donors (Conrad et al., 1995). There are recent reports implying transmissibility of human cytomegalovirus (CMV). Animal models clearly demonstrate that skin grafts are capable of transmitting CMV (Abecassis, 1994; Abecassis et al., 1993; Shelby et al, 1988; Shelby and Stanley, 1987). Earlier studies by Kealey et al, showed that burn patients acquire CMV during hospitalisation, and that blood transfusions may be a contributing factor (Kealey et al, 1987). A subsequent study eliminated blood transfusion as a contributor, by studying patients who received skin allografts but no CMV-positive blood. The study showed that CMV-negative burn patients who receive skin allografts from CMV-positive donors can seroconvert to become CMV-positive (Cederna et al, 1994). CMV resides in peripheral blood leukocytes in asymptomatic CMV antibody-positive donors, long after their initial infection. Asymptomatic CMV-positive donors can transmit CMV infection

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through transfusion and transplantation if the recipient is CMV negative. Most healthy adult prospective donors have CMV antibodies: therefore, excluding CMV positive donors to prevent CMV transmission would exacerbate the already existing shortage of skin allografts, and would not be practical. Testing donors for CMV antibody is not required by national professional standards. Immunosuppressed individuals such as organ recipients have a high mortality and morbidity rate from transplant-transmittedCMV of donor origin. The burned patient also acquires CMV infection but does not generally experience serious complications as regularly as organ recipients, perhaps because burn-related immunosuppression may be less profound than that produced by drugs used to prevent organ rejection. As burn patients begin to receive potent immunosuppressants (such as cyclosporine) to block rejection of skin allografts, CMV may become a more serious complication of burn care and related blood transfusion and skin allografting. Further studies of skin allograft recipients are needed to determine whether transmission of CMV by skin allograft is associated with symptomatic disease as seen in organ transplantation recipients, or whether the infection is asymptomatic, as generally seen in transfusion-transmitted CMV infections in immunocompetent blood transfusion recipients. Prior to knowing the outcome of these studies, it is premature to assume that it is beneficial to base selection of skin donors on CMV antibody testing. 10. Risk of Transmitting Other Diseases 10.1. Syphilis There have been no reports of syphilis transmission via organ, tissue or cell transplants — probably due to the low prevalence of infection in the screened donor population; frequent use of antibiotics during tissue processing and storage; the absence of the spirochete in many tissues and cells; and low temperature

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storage of allografts, which kills or suppresses growth of the spirochete. Because Treponema pallidum, the agent causing syphilis, does not survive when stored at 4° beyond 48-72 hours (Barnes, 1992), transmission of syphilis by tissue allografts is not expected. Most transplanted tissues are initially stored refrigerated or frozen for several days prior to processing and use. Syphilis could possibly be transmitted if the tissue allografts were used shortly after donation, and if the donor were spirochetemic. Donor testing for syphilis has major limitations, and will not reliably detect spirochetemia. During the short early period of spirochetemia at initial infection with syphilis, the syphilis screening test is negative. Most, but not all, tissue banks perform the syphilis screening tests on donors despite its uncertain value (AATB, 2001; Campagnari and O'Malley, 1994; Strong et al., 1991). Most positive syphilis screening tests in donors are falsely positive. Organs and tissues have been transplanted in the face of positive donor syphilis tests without transmitting the disease (Gibel et al., 1987). Professional tissue banking standards permit use of tissues from donors with falsely positive syphilis screening tests: reactive nontreponemal tests with nonreactive treponemal confirmation tests (AATB, 2001; Campangnari and O'Malley, 1994). 11. Emerging Infection Risks Infection risks of tissue transplantation have usually been identified after first being recognised as blood transfusion-transmitted infections. Many real or theoretical risks of tissue transplantation can be considered by looking at the emerging infections that threaten to affect transfusions (Chamberland, 2002; Strong and Katz, 2002). Most recently, West Nile Virus (WNV) infection has swept through the United States with nearly 4,000 human cases identified, and 254 deaths in 2002 (CDC, 2002b). In addition to being mosquito-borne, WNV has been transmitted through organ transplantation, blood transfusions, transplacental intrauterine spread, and percutaneous route from laceration and needlestick

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(CDC, 2002b). There are, on average, four malaria transmissions annually in the US (Mungai et al, 2001). Approximately 40 cases of transfusion transmitted babesiosis have been reported from parasitemic blood donors (Dobroszycki et al., 1999). Trypanosoma cruzi transmission from transfusion has been newly reported (Leiby et al., 1999). Parvovirus B19 has been transmitted to a few recipients of plasma derivatives, red cells, platelets and solvent-detergent-treated plasma (Koenigbauer et al., 2000). 12. Reducing the Risk Through Donor Selection To minimize the risk of transmitting infectious disease, several important approaches are taken by transplanting surgeons and tissue banks. An initial approach by the surgeon is to judiciously use tissue allografts, and from accredited organisations; use sterilised allografts whenever possible; and consider use of autografts and alternative non-human graft material. However, the most important steps are exercised by the tissue bank in excluding those prospective donors suspected to be at risk for HIV, hepatitis (Table 8), CJD (Table 7) and bacteriologic and Table 8. Donor exclusions for Hepatitis and HIV risk behaviour. HIV and Hepatitis Risk Behaviour Exclusions 1) 2) 3) 4) 5) 6) 7)

8) 9)

Persons with clinical or laboratory evidence of HIV infection. Men who have had sex with other men even once in past five years. Nonmedical injections of drugs in past five years. Persons with haemophilia or related clotting disorders, who have received human-derived clotting factor concentrates. Persons who engaged in sex for money or drugs in past five years. Persons who have had sex with any of the above in past 12 months. Exposure to blood that is suspected to be HIV- or hepatitis-infected through percutaneous inoculation of open-wound or mucous membrane contact in past 12 months. Inmates of prisons for at least seven days in past 12 months. Tattoo received in past 12 months.

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fungal infections. Tissue transplantation is generally considered a non-urgent surgical procedure, permitting a careful tissue donor selection process. Tissue donor selection by tissue banks has evolved to include a direct interview with the donor's next-ofkin concerning the donor's medical history and risk behaviours for HIV and hepatitis, along with blood infectious disease testing, a physical examination, and the results of an autopsy examination, if performed (Table 9). These donor selection steps are essential activities that result in a low risk of transmitting disease. In addition, many tissue allografts can undergo further processing and exposure to antibiotics, disinfectants or sterilants (Table 10), all of which further reduce the hazard of disease transmission (Asselmeier et ah, 1993). Although there have been no carefully-controlled prospective studies of allograft recipients to determine the incidence of disease transmission, there is good reason to believe that established donor screening procedures, infectious disease testing and the effectiveness of processing and sterilisation to reduce or eliminate bacteria and virus, results in a very low risk of transmitting disease. Donor blood testing, physical examination, autopsy reports, and surgical removal through aseptic technique and use of sterile disposable supplies, are further aids in providing tissue with a low risk of transmitting disease. Lastly, sterilisation of certain tissues can be performed, and is very effective. The bone graft disinfection and sterilisation step most often used by bone banks in the United States are gamma irradiation at doses of 15 to 30 kGy (Strong et ah, 1995). 12.1. Donor selection One important contribution to recipient safety is to seek voluntary, non-remunerated donors. Monetary inducement to the nextof-kin of cadaveric tissue donors is prohibited by professional standards (AATB, 2001), but it is being considered as a means of reducing the severe organ supply shortage in the United States (Peters, 1991). Monetary reimbursement of semen and oocyte

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donors is not prohibited by national professional standards (AATB, 2001) or governmental guidelines (CDC, 1988a; 1994) or regulations (FDA 1993; 1997; 1999), but is considered necessary by most US reproductive tissue banks in the United States. Monetary payment to donors for blood donation increases the risk of disease transmission (Eastlund, 1998b). Monetary incentives to donate may cause donors or their next of kin to be untruthful about the donor's health history information, and to donate when they should not. Published data clearly show a 5-10 times increase in incidence of donor-to-recipient posttransfusion hepatitis B virus infection with the use of paid blood donors (Eastlund, 1998b). In addition, there is an 11-15 times increase in prevalence of HCV antibodies, and 3-14 times increase in prevalence of HIV antibodies in paid blood donors compared to voluntary blood donors (Eastlund, 1998b). HCV RNA was detected more often in clotting factor concentrates derived from paid donor plasma than volunteer donor plasma (Markris et al., 1993). Transfusion transmitted malaria has been documented from a paid donor who lied about a history of malaria to sell his blood (Guerrero et al., 1983). To minimize the risk of transmitting infectious disease, tissue donor eligibility requirements have been set by national professional standards (AATB, 2001; Campagnari and O'Malley, 1994; EBAA, 1994; EATB, 2000), United States Public Health Service guidelines (CDC, 1988a; 1994), and United States federal regulations (FDA, 1993; 1997; 1999). Donor selection is an important first step taken to ensure that the resulting allograft is safe and effective. The cadaver tissue donor selection process includes a donor's medical and social history obtained from the next-of-kin and medical care providers; blood tests; a physical examination performed by tissue bank personnel; and an autopsy, if performed (Table 9). Preliminary donor selection is based on the donor's medical history and circumstances surrounding death. Donors are excluded if elements of the past medical and social history or recent hospitalisation indicate a risk of infection, malignant disease, or inadequate quality of donated organ or

Infectious Disease Transmission Through Tissue Transplantation Table 9. Selection steps to prevent disease transmission from cadaver tissue donor. Voluntary donation without monetary inducement Health History Review • Review of medical records. • Interview of next of kin. • Exclusion of those with infection, malignancy. • No human pituitary-derived growth hormone. • No HIV, hepatitis risk behaviour. • No nvCJD risk behaviour. Blood Tests • Hepatitis B surface antigen. • Antibody to HIV-1, HIV-2, HCV. • Antibody to HTLV-I, HTLV-II, syphilis.* • Blood culture (optional). • Antibody to HBV Core Antigen (for living donors only).* Physical Examination • Unexplained jaundice. • Evidence of injectable drug use. • External signs of infection, including HIV. Autopsy Examination (if performed) • Exclude those with infection, malignancy. Maternal HIV testing and risk factor exclusion if donor < 18 months old (56). *AATB, EBAA, United Network for Organ Sharing and American Red Cross requirements. *AATB requirement.

tissue. The living donor, the legal c o n s e n t i n g next-of-kin or life p a r t n e r of a cadaveric donor, or both, m u s t be directly interviewed to d e t e r m i n e w h e t h e r HIV, hepatitis or CJD risk b e h a v i o u r s are p r e s e n t (Tables 7 a n d 8). Persons w i t h HIV a n d hepatitis risk b e h a v i o u r s are e x c l u d e d from d o n a t i o n . A g e criteria are set to e n s u r e functional quality of the d o n a t e d graft.

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For example, donated bone from a very young donor may not be satisfactory for use in load bearing graft applications due to the presence of nonmineralized epiphyseal growth plates. The presence of many other tissue and organ specific exclusionary criteria, such as chronic corticosteroid use and advanced age (which can weaken donated bone), or diabetes indicating a non-functional pancreas, will exclude donation of that part, but not necessarily other grafts from the same donor. The tissue bank physician makes the final determination of suitability of a cadaveric tissue donor as required by national professional standards (AATB, 2001; Campagnari and O'Malley, 1994). 12.2. Physical examination of donor The next screening step is a limited physical examination of the tissue donor by procurement staff at the time of cadaveric donation (AATB, 2001; Campagnari and O'Malley, 1994). The body is examined for signs of injecting drug use, and signs of HIV, hepatitis or other infection or trauma over bodily sites that can affect the quality of donated tissue. 12.3. Blood testing of donor Donor blood testing for disease markers plays an important role in reducing the risk of disease transmission. By eliminating prospective donors with infectious disease risk factors prior to blood testing, the risk of a seronegative but infected donor is minimized. Testing for HBsAg, anti-HIV and anti-HCV is required by federal regulations (FDA, 1993; 1994; 1999) and national professional standard setting organisations (AATB, 2001; Campagnari and O'Malley, 2002; EBAA, 2002). Other tests required by standard setting organisations are syphilis and anti-HTLV-I/ II (AATB, 2001; Campagnari and O'Malley, 1994; EBAA, 2002). Blood cultures are not required, but are commonly performed for cadaveric tissue donors to aid in determining whether the donor is infected, particularly if the donor was hospitalised, and

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ventilation was mechanically assisted. Their predictive value is very uncertain, but can be used to augment bacterial testing of tissues. HIV antigen (p24 antigen) testing of the donor is not performed by most tissue or organ banks. Large scale studies of low risk (Alter et al, 1990) and high risk (Busch et al, 1990) blood donor populations demonstrated a lack of utility for HIV antigen screening. These blood donor studies, and similar studies on smaller numbers of cadaver bone (Harrell et al, 1993) and cornea (Pepose et al, 1992) donors did not detect HIV infected donors beyond those already detected by testing for HIV antibodies. Studies are underway to determine whether testing donors for HIV RNA and HCV RNA by nucleic acid testing is practical. One study of 1,424 cadaver bone donors showed that the use of HIV DNA (not HIV RNA) and p24 antigen blood testing did not detect additional HIV infected cadaveric bone donors (Harrell et al, 1993). All 1,424 donors negative for HIV-1 antibodies were also negative for HIV DNA. Although viral nucleic acid testing is more sensitive than antibody assays, it may be premature to apply it routinely to cadaver donor testing due to the low HIV prevalence in the donor population, its uncertain predictive value, its false positive rate, and its false negative rate due to haemoglobin contamination and other interfering substances in cadaveric post-mortem blood samples. Testing of living blood donors for HIV and HCV RNA has markedly improved the safety of the blood supply even though screening has been done using pools of 16 to 24 samples (Stramer et al., 2000). Initially, viral nucleic acid testing was not feasible in blood donor screening applications due to lack of automation, time and space restrictions and cost. Recently, however, two test systems are being used to test over 13 million blood donations annually in the United States: the Roche Molecular Systems COBAS AMPLISCREEN tests for HCV and HIV, and the Gen-Probe/ Chiron Pooled Plasma HIV-1/HCV Amplified Assay. Testing is being done on pooled samples using pools of 24 or 16. Testing of pooled samples reduces the number of tests required on a

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daily basis, and also the time to perform testing and the cost. It also takes into account the rapid rise of viral nucleic acid in recently infected individuals, so that pooling has a minimal impact on the sensitivity of these assays. The increased sensitivity of these two systems over previously available PCR tests has also made this possible. Both systems have now been licenced for blood donor screening, and efforts are underway to qualify them for organ and tissue donor screening. The same approach is now being tested in trials for HBV, and soon, for the West Nile Virus. Many bone banks test donors for anti-HBc, a test originally introduced for blood donors as a surrogate for detecting non-A, non-B hepatitis carriers. The utility of this test as a surrogate has been diminished since the addition of specific tests for HCV (HCV antibodies and HCV RNA), the major cause of non-A, non-B hepatitis (Blajchman et ah, 1993). Although not required by AATB Standards, and in the absence of HBV DNA testing, the use of anti-HBc in donor testing likely had a safety benefit in reducing HBV infections. Several reports have documented the presence of HBV in the sera of HBsAg-negative, anti-HBcpositive blood donors (Yotsuyanagi et ah, 2001; Roth et ah, 2002; Wang et ah, 2002). These reports also suggest that the addition of HBV DNA testing will increase the sensitivity of HBV detection, but may not entirely replace the need to test for anti-HBc or HBsAg. For example, recipient directed lookback procedures have revealed recipients of HBsAg negative, NAT negative, anti HBc-positive blood components to have been infected with HBV (Roth et ah, 2002). 12.4. Hemodilution of donor b l o o d sample Massive blood loss and intravascular volume replacement by transfusion of blood, colloid, and crystalloid solutions can cause hemodilution, and result in unreliable donor test results for infectious diseases (Eastlund, 2000). In 1987, a case of HIV transmission to multiple recipients of organs derived from an infected

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donor was reported. Testing of the donor was negative when blood was sampled immediately after receiving blood transfusions amounting to two to three total blood volumes, and an additional large volume of crystalloid solution over an elevenhour period (CDC, 1987). When a blood sample was obtained 48 hours later and tested for anti-HIV, it was positive due to intravascular replenishment of immunoglobulin from extravascular sites. In 1993, US federal regulations (FDA, 1993) were first published, with subsequent modification (FDA, 1997; 1999), requiring quarantine of tissue from adult donors who had blood loss and received greater than two litres of blood and colloids within 48 hours of blood sampling; or greater than one litre of crystalloid within a one hour of sampling. The donated tissue was not to be used unless a pre-transfusion sample was available for testing, or an algorithm was used to evaluate blood and colloid volumes administered in the 48 hours prior to blood sampling, to ensure that any plasma dilution sufficient to alter test results, had not occurred. AATB Standards also require tissue banks to follow written procedures the set hemodilution limits to prevent use of falselynegative results when testing post-transfusion blood samples for infectious disease (AATB, 2001). Acceptability limits must be part of written procedures. Standards of the American Red Cross Tissue Services require that in the case of blood loss and transfusion within 48 hours of death, a pre-infusion sample must be used (Campagnari and O'Malley, 1994). A post-infusion sample may be used in patients with major clinical blood loss, provided that the tissue bank physician has evaluated whether blood and crystalloid infusions have compensated for blood loss; estimated hemodilution is 50 percent or less of the total blood volume; and the tissue bank physician has given written approval. The estimated amount of hemodilution depends upon the type of fluid infused, and the amount of time elapsed since infusion.

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12.5. Quality of cadaver donor b l o o d samples The testing of cadaveric tissue donor serum for viral markers may be complicated by false positive tests when sampling is delayed after death (Pepose et al, 1992) or when there is haemolysis (Novick et al, 1993; Pepose et al, 1992). False positive results for HBsAg and p24 antigen due to haemolysis may be found depending upon which manufacturer's test kit is used (Novick et al, 1993). Sample quality and presence of haemoglobin can also cause false negative results during testing for HIV and HCV by NAT (Adams et al, 1993; Comeau et al, 1992). Frozen storage and multiple freeze-thaws do not have a major effect on detectability of antibodies to infectious agents in serum, but they may reduce the reliability of testing for microbial nucleic acids by NAT. Busch et al, showed that multiple freeze thaws can reduce the detectability of HCV RNA by NAT (Busch et al, 1992). Published studies are too few for any firm conclusions to be drawn, other than a possible deleterious effect of frozen storage and freeze thawing on tests of serum for HCV RNA, and tests of peripheral blood white cells for HIV DNA, by NAT. Despite this, NAT was used to detect and retrospectively diagnose HIV infection using marrow specimens from an organ and tissue donor, and frozen sera from organ recipients five years after donation and transplantation (Simonds et al, 1992). HCV testing by NAT was also useful in confirming HCV infection in one cadaveric donor (Conrad et al, 1995) and was essential in another (Tugwell et al, 2002). Although NAT for hepatitis and HIV will be very useful, the tests are under development for organ and tissue donor testing. 12.6. Autopsy Autopsies of donors are not generally required, tissues that can be stored, a final donor suitability is not made by the tissue bank physician until the autopsy, if performed, have been reviewed

but for those determination the results of (AATB, 2001;

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Campagnari and O'Malley, 1994). Autopsy findings that have disqualified donors, include previously undiagnosed malignancy, widespread granulomas, abscess, and pneumonia. 12.7. Living bone donor selection Femoral heads can be donated by persons undergoing hip arthroplasty. Donor medical history screening and testing requirements are similar to those for cadaver bone donors, except that the donor medical history interview can be made directly, and a retest for HIV and HCV antibodies is required 180 days following donation (AATB, 2001; Campagnari and O'Malley, 1994; CDC, 1988a; 1994). The retest aims to identify recently infected donors who were seronegative for HIV and HCV antibodies at the time of donation. This 180-day retest is required for semen and bone donors but not for living donors of blood, marrow, amnion, umbilical veins, or foetal tissue. Retesting the low risk voluntary living bone donors has not detected any additional infections despite thousands of donations and retests (Scofield et al., 1993b), whereas it may have utility in the higher risk population of paid semen donors. Most tissue banks have ceased collecting surgical femoral heads, partly due to lower quality, but also due to the difficulties of acquiring the 180-day sample for retesting. Testing of samples for HCV and HIV RNA from living donors at the time of donation would enhance safety and could eliminate the need for a 180-day retest. 13. Reducing the Risk During Cadaveric Tissue Collection Removal of tissue allografts takes place up to 24 hours after the death of a cadaveric donor. Most transplantable tissue such as corneas, bone, tendon and skin are recovered from donors in whom there has been complete cardiac and respiratory cessation for some time period. The collection of tissue allografts from

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the cadaveric donor begins as soon as possible after death, to reduce the risk of endogenous or exogenous bacterial contamination, and to maintain functional characteristics or viability of the tissue. National professional standards require bone, tendon and heart valve recovery to be completed within 12 hours of death, or within 24 hours of death if the body is refrigerated (AATB, 2001; Campagnari and O'Malley, 1994). Recovery of corneas is preferred within four to six hours of death, to ensure endothelial cell viability. Viable skin allografts are usually collected within six hours of death if the body is not refrigerated but within 24 hours otherwise (AATB, 2001; Campagnari and O'Malley, 1994). Delayed removal of tissue from the cadaver donor can possibly result in an increase of anaerobic and spore forming bacterial pathogens which are more resistant to disinfecting and sterilisation procedures. The majority of tissue banks which procure hearts for heart valves, culture the transport media which is filtered prior to processing; the filter is then cultured, thus increasing the sensitivity of detection. The results are used to determine whether to release the allograft for clinical use. Physical manipulation, environmental exposure and storage conditions of the body prior to tissue removal can contribute to bacterial or fungal contamination of the tissue allograft. It is possible that in some of these donors there is post-mortem translocation or transmigration of bacteria from the intestines or respiratory system to other parts of the body, and to the tissue removed for transplant purposes. The epithelial mucous membrane of the intestine is a very fragile barrier protecting a person from intestinal bacteria. This intestinal barrier is particularly sensitive to ischemia and reperfusion, and a second insult — even mild hypotension — can affect intestinal barrier function, leading to loss of mucosal defence, and consequently, escape and spread of luminal bacteria to other organs. This sequence is called bacterial translocation (Fukasima et ah, 1992; Steffen and Berg, 1983; Mejima et ah, 1984). It is unknown whether bacterial

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Table 10. Additional steps taken to control microbial contamination of tissue allografts. Aseptic Procurement • Monitoring and control of microbial contamination of air, surfaces. • Sterile barrier dress for personnel. • Aseptic technique. • Sterile instruments and supplies. • Cleaning, disinfecting donor skin. • Procurement cultures. Aseptic Processing • Monitoring and control of microbial contamination in air, surfaces. • Sterile water, reagents, supplies, instruments, equipment. • Aseptic Technique. • Sterile barrier dress for personnel. • Removal of extraneous tissue and blood. • Exposure to disinfectants, antibiotics. • Sterilisation. • Sterility Testing. Final Packaging • Sterile supplies, sealed impervious packaging.

translocation occurs consequent to cardiopulmonary resuscitative attempts, autopsies or other post-mortem manipulation of the cadaveric tissue donor. A surgical autopsy is performed in approximately half of cadaveric tissue donors prior to tissue allograft removal, and this can contribute to bacterial contamination. The environment in which the tissue allograft is removed may also have an impact of whether the tissue contains microbial contamination. Tissues are removed at various environmental sites: hospital operating rooms, autopsy suites in hospital morgues and regional forensic medical examiner facilities; funeral homes, and in dedicated tissue procurement facilities at medical examiner facilities or tissue banks. To reduce the risk of bacterial contamination, the room and table top is cleansed;

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Table 11. Surgical removal of tissue — Cadaver tissue donor. Operating room or morgue. Cleaning and preparing of room. Cleaning and preparing of body. Sterile drapes and equipment. Aseptic surgical technique. Dissecting and removing of tissue. Preserving blood vessels for embalmer, Funeral Director. Bacteriologic testing. Restoring anatomic shape of body.

sterile drapes are used; donor excision sites are disinfected; sterile equipment and supplies are used; and aseptic technique is practised (Table 11). One study reported that prior organ donation and the location of the procurement site have been correlated with the discover of virulent bacteria on tissue allografts removed from cadaveric donors (Johnson et al, 2002). They found virulent bacteria on at least one tissue in 58 (21%) of 275 donors, and on 125 (3.5%) of 3586 individual tissues. In contrast, non-pathogenic bacteria were found on 622 (17.3%) of tissues. Bacterial contamination is a common finding of tissues freshly removed for purposes of transplantation (Bettin et al, 1998; Deijkers et al., 1997; Martinez et al, 1985; Vehmeyer et al, 1999; Vehmeyer and Bloem, 1999; Farrington et al, 1998; Chapman and Villar, 1992; Sutherland et al, 1997; Novick et al, 1991; Bennett et al, 1991; Scofield et al, 1994; Journeaux et al, 1999). Swabbing of the bone allograft surface is routinely performed by most tissue banks at the time of procurement, but the rate of bacterial contamination — when swab culturing the entire allograft — was found by Veen et al, to be one tenth that of entire bone that had been immersed in culture media (Veen et al, 1994). The limited sensitivity of swab culturing techniques has been confirmed (Vehmeyer et al, 2001).

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14. Reducing the Risk During Cadaveric Tissue Processing Tissue allografts can also be contaminated during processing from air and environment surfaces, from personnel, and from contaminated reagents, surgical instruments, supplies and processing equipment (Table 12). In rare cases, contamination has been acquired during liquid nitrogen storage (Hawkins et al., 1996). An additional site of acquired contamination can be the hospital or surgical centre operating room where microbial contamination can develop during opening of the container, handling of the allografts, and exposure to the air in the operating room. Table 12. Possible sources of bacterial contamination. Donor infection prior to death. Postmortem translocation of bacteria in body to the tissue donated. Effect of prior autopsy. Length of time and temperature during body storage prior to procurement. External contamination during procurement • Morgue versus OR versus special facility. • Surgical technique. Contamination during processing • Air (particulate and bacteria monitoring). • Water (bacteria monitoring). • Surfaces: (tables, walls: bacterial testing). • Equipment (sterilised). • Supplies (sterilised). • Reagents, solutions (sterilised). • Staff (aseptic technique, sterile barrier clothing, bacterial testing). Contaminated during storage • Liquid nitrogen storage.

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In cases of HIV and HCV transmission by tissue allografts, the origin of the virus has been from an infected donor. In contrast, bacterial contamination of tissue allografts can arise from infected donors; or contamination may occur during surgical removal of tissue from the donor, or from contaminated environments, equipment or supplies during processing at a tissue processing facility; or from implantation at the time of surgery. Electrolyte solutions purchased commercially (CDC, 1995), or deionised water prepared by the tissue bank (Farrington et ah, 1996), can be contaminated by bacteria which consequently contaminate the tissue allograft when used for processing. Some tissue banks have been able to demonstrate removal of low virulence bacteria during processing of heart valves, by soaking the allograft in antibiotics (Lau and Lazaro, 2002). Others processing and storing corneas (Antonios et ah, 1991) and skin (Clayton et ah, 1995) have demonstrated that bacteria can survive in refrigerated antibiotic solutions. The recent outbreak of Clostridium infections from minimally-processed cartilage and tendon allografts, and fungal infection from a heart valve from a single facility, demonstrate that antibiotic soaking is not effective against all virulent microbes, and that residual antibiotics can interfere with, and existing sampling techniques can miss, detection of the microbe during final sterility testing (CDC, 2002; Kuehnert et al., 1998). This is particularly true for spore-forming organisms where the vegetative forms can be eliminated by antibiotics, but the spores are not. Complete absence of bacterial and fungal contamination is far greater in importance for bone, cartilage, tendon, and heart valve allografts that are surgically implanted within the body, than for cornea and skin allografts that are implanted at non-sterile sites on the surface of the body. 15. Recipient Safety and Federal Regulations In 1993 the US Food and Drug Administration (FDA) investigated the importation of human tissue allograft from foreign

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countries (FDA, 1993). The FDA documented that full medical history screening and interviews with the next-of-kin (to exclude those with risk behaviours for HIV and hepatitis), had not been performed. Donor medical records were not available, and consent to donate had not been obtained. Although donor blood testing for HIV and hepatitis were alleged to be negative, the FDA retested samples and found at least one to be markedly positive for HBsAg. FDA also found that imported tissue from some foreign tissue banks were recovered without aseptic precautions (FDA, 1993). It was not ascertained that imported tissue allografts caused hepatitis or HIV infection in recipients. However, a decade earlier many human dura mater allografts that had been processed in pools, were imported into the US by a German medical device manufacturing firm, and these were implicated in the spread of CJD to recipients. This raised the issue of pooling, and the increased risk of contaminating all tissue in a batch. In addition, the ability to trace recipients of a particular donor's tissue becomes nearly impossible if multiple donors are involved. Although the US Public Health Service Centres for Disease Control had published recommendations for screening organ, tissue, and semen donors (CDC, 1988a; 1988b; 1994), the FDA added legal requirements to provide further protection for the public from HIV and hepatitis infection through transplantation of imported tissue. The FDA issued an "Important Alert" to health professionals in December 1993, so that tissue allografts would not be imported without prior notification to the FDA. In addition, the FDA published an emergency regulation on December 14, 1993, which took effect immediately (FDA, 1993). The regulation, entitled "Human Tissue Intended for Transplantation" applies to bone, tendon, skin and cornea allografts, but not semen or organs. The regulation required tissue donor testing for HBsAg and antibodies to HIV and HCV, by a CLIA-registered laboratory using FDA licensed test kits, and using a blood sample that has not been diluted enough from recent transfusion

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to alter test results. Other requirements include donor medical history screening to assure absence of risk factors or clinical evidence of HBV, HCV, or HIV infection; written operating procedures; and record keeping to ensure traceability (FDA, 1997; 1999). The FDA has proposed requirements for Good Tissue Banking Practices modelled after GMP requirements for biologies, drugs and blood transfusion (FDA, 1997; 2001a; 2001b). In November 2001, the sudden death of a man who had received a cartilage allograft contaminated with Clostridium led to an investigation by CDC and FDA. In 2002, the US FDA enacted emergency regulations to require allograft processing facilities to observe written procedures that are validated and certified to reliably prevent infectious disease contamination and cross-contamination during processing (FDA, 2002). Equivalent regulation exists in Europe. In addition to federal regulations stipulating donor screening requirements to ensure safety, there are other federal regulations for cellular transplants and tissue composites, to ensure clinical effectiveness and to require compliance with good manufacturing practices for medical devices (Kessler et ah, 1993; FDA, 2001b). These regulations cover extensively processed cells, and gene therapy. 16. Conclusion Transplantation of tissues has resulted in the transmission of bacterial, viral, fungal and prion diseases from donor to recipient. When the first truly effective immunosuppressant, cyclosporin, became available in 1981, organ transplantation flourished. The numbers of organs transplanted each year grew rapidly, leading to implementation of effective programmes to develop public support for organ donation. As organ donation grew, so did tissue donation. With the rapid growth of transplantation, early cases of transplant transmitted infection arose from infections of the donor, but these could have been

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prevented if tests were available. Donor testing only reduced the risk. Early cases of transmitted viral infection involved donors who had HIV and hepatitis risk behaviours. Exclusion of these as prospective donors has played an important role in reducing the risk of transmission. More recently, several cases have been reported, wherein the infecting organism is bacterial and fungal instead of viral, and the microbes did not arise from the donor but was a contaminant acquired during the procurement, processing or storage of the tissue. Newer national professional standards and US FDA regulations are addressing this area. To prevent, or at least minimise, the risk of transmission of infectious disease, several approaches are important. Surgeons should use allografts only when needed, and obtain them from accredited tissue banks. They should consider use of autografts, alternative nonhuman graft material, or processed and sterilised tissue allografts. Whenever possible tissue banks reduce the risk by rejecting those donors suspected to be susceptible to HIV, hepatitis, or other viral or bacterial infections; performing physical examinations of donors, reviewing autopsy reports, and using aseptic technique during surgical removal of donated tissue. Bacterial testing of tissues donated and other laboratory testing procedures, help identify potentially infectious donors. Preventing the acquisition of microbial contamination and of cross-contamination during processing of tissues is required. Final sterility testing needs to be sufficiently sensitive to provide assurance of sterility. Lastly, sterilisation of certain tissues can be very effective, but it is not universally applicable, because some infectious agents such as CJD are impervious to sterilants, and because the clinical effectiveness of many tissues (such as skin, cornea, valves, veins) can be altered by sterilisation procedures (Pruss et ah, 2001). Application of all these steps will ensure a very low risk of transmitting disease from the donor to the recipient.

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17. References ABECASSIS, M. (1994). Transmission of CMV by skin allografts — A review, Tissue Cell Rep. 2, 15-20. ABECASSIS, M., KAUFMAN, D. and STUART, F. (1993). Direct evidence that murine cytomegalovirus (CMV) can be transferred via skin grafting using novel oligonucleotides in the polymerase chain reaction (PCR). Proceedings 19th Annual Meeting, American Society of Transplant Surgeons. Houston, TX. ADAMS, M., LEE, T.H., BUSCH, M.P., HEITMAN, J., MARSH, G.J., GJERSET, G.F. and MOSELY, J.W. (1993). Rapid freezing of whole blood or buffy coat sample for polymerase chain reaction and cell culture analysis: Application to detection of human immunodeficiency virus in blood donor and recipient repositories, Transfusion 33, 504-508. AHO, A.J., HIRN, M., ARO, H.T., HEIKKILA, J.T. and MEURMAN, O. (1998). Bone bank service in Finland. Experience of bacteriologic, serologic and clinical results of the Turku bone bank 1972-1995, Acta Orthop. Scand. 69, 559-565. ALTER, H.J., EPSTEIN, J.S., SWENSON, S.G., VAN RADEN, M.J., WARD, J.W., KASLOW, R.A., MENITOVE, J.E., KLEIN, H.G., SANDLER, S.G. and SAYERS, M.H. (1990). Prevalence of human immunodeficiency virus type 1 p24 antigen in US blood donors — An assessment of the efficacy of testing in donor screening, N. Engl. J. Med. 323, 1312-1317. AMERICAN ASSOCIATION OF TISSUE BANKS (2000). Annual Registration Survey of Accredited Tissue Banks. McLean VA: American Association of Tissue Banks. AMERICAN ASSOCIATION OF TISSUE BANKS (2001). Standards for tissue banking. McLean VA: American Association of Tissue Banks.

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ANTONIOS, S.R., CAMERON, J.A., BADR, I.A., HABASH, N.R. and COTTER, J.B. (1991). Contamination of donor cornea: Post penetrating keratoplasty endophthalmitis, Cornea 10, 217-210. ANYANWU, C.H., NASSAU, E. and YACOUB, M. (1976). Military tuberculosis following homograft valve replacement, Thorax 31, 101-106. ASPENBERG, P. (1998). Backbone, infections and HIV, Acta Orthop. Scand. 69, 557-558. ASSELMEIER, M.A., CASPARI, R.B. and BOTTENFIELD, S.A. (1993). Review of allograft processing and sterilisation techniques and their role in transmission of human immunodeficiency virus, Am. J. Sports Med. 21, 170-175. AURORI, B.J., WEIERMAN, R.J., LOWELL, H.A., NADEL, C.I. and PARSONS, J.R. (1985). Pseudoarthrosis after spinal fusion for scoliosis. A comparison of autogeneic and allogeneic bone grafts, Clin. Orthop. 199, 153-158. BAER, G.M., SHADDOCK, J.H., HOUFF, S.A., HARRISON, A.K. and GARDNER, J.J. (1982). Human rabies transmitted by cornea transplant, Arch. Neurol. 39, 103-107. BAER, J.C., NIRANKARI, V.S. and GLAROS, D.S. (1988). Streptococcal endophthalmitis from contaminated donor corneas after keratoplasty. Clinical and laboratory investigations, Arch. Ophthalmol. 106, 517-520. BAER, J.C., NIRANKARI, V.S. and GLAROS, D.S. (1989). Survival of Streptococcus viridans in gentamicin-supplemented McCareyKaufman medium, Cornea. 8, 131-134. BARNES, A. (1992). Transfusion transmitted treponemal infections. In: Transfusion Transmitted Infections, D. Smith and R.Y. Dodd, eds., ASAP press, Chicago, pp. 161-166.

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(1993). Birdshot chorioretinitis and Lyme Borreliosis, Am. J. Opththalmol. 115, 149-153. TANGE, R.A., TROOST, D. and LIMBURG, M. (1990). Progressive fatal dementia (Creutzfeldt-Jakob disease) in a patient who received homograft tissue for tympanic membrane closure, Eur. Arch. Otorhinolaryngol. 247, 199-201. THADANI, V., PENAR, P.L., PARTINGTON, J., KALB, R., JANSSEN, R., SCHONBERGER, L.B., RABKIN, C.S. and PRICHARD, J.W. (1988). Creutzfeldt-Jakob disease probably acquired from a cadaveric dura mater graft, /. Neurosurg. 69, 766-769. THIJSSEN, E.J., KROES, A.C., BOX, E., PERSIJN, G.G. and ROTHBARTH, P.H. (1993). The significance of complete serological testing for hepatitis B in heart valve banking, Transplantation 56, 82-84. TOMFORD, W.W., STARKWEATHER, R.J. and GOLDMAN, M.H. (1981). A study of the clinical incidence of infection in the use of banked allograft bone, /. Bone Joint Surg. 63A, 244-248. TOMFORD, W.W., THONGPHASUK, J., MANKIN, H.J. and FERARO, M.J. (1990). Frozen musculoskeletal allografts. A study of the clinical incidence and causes of infection associated with their use, /. Bone Joint Surg. 72A, 1137-1143. TRANSFELDT, E.E., LONSTEIN, R., WINTER, D. and BRADFORD, D. (1985). Wound infections in reconstructive spinal surgery, Orthop. Trans. 9, 128-129. TUGWELL, B.D., PATEL, P.R., WILLIAMS, I.T., THOMAS, A., HOMAN, H., HEDBERG, K. and CIESLAK, P.R. (2002). Hepatitis C Virus (HCV) Transmission to Tissue and Organ Recipients from an antibody negative donor — United States, 2002. In: 42nd Ann. Interscience Conf. Antimicrob. Agents

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Chemother. (ICAAC) (September 28), San Diego, CA, Poster presentation LB-17. TULLO, A.B., MARCYNIUK, B., BONSHEK, R., DENNETT, C , CLEATOR, G.M., LEWIS, A.G. and KLAPPER, P.E. (1990). Herpes virus in a corneal donor, Eye 4, 766-767. TYRAS, D.H., KAISER, G.C., BARNER, H.B., LASKOWSKI, L.F. and MARR, J.J. (1978). Atypical mycobacteria and the xenograft valve, /. Thorac. Cardiovasc. Surg. 75, 331-337. UNITED NETWORK FOR ORGAN SHARING (2001). Cadaveric donors and cadaveric organ transplantation in the US, UNOS Update 10, 29. VANBAARE, J., MACKIE, D.P. and MIDDELKOOP, E. (1997). HIV transmission by transplant of allograft skin: A review of the literature (letter), Burns 23, 460. VEEN, M.R., BLOEM, R.M. and PETIT, P.L.C. (1994). Sensitivity and negative predictive value of swab cultures in musculoskeletal allograft procurement, Clin. Orthop. 300, 259263. VANBAARE, J., LIGTVOET, E.E. and MIDDELKOOP, E. (1998). Microbiological evaluation of glycerolised cadaveric donor skin, Transplantation 65, 966-970. VEHMEYER, S.B.W. and BLOEM, R.M. (1999). Bacterial contamination of post-mortal bone allografts. In: Advances in Tissue Banking, G.O. Phillips, J.M. Kearney, D.M. Strong, R. VonVersen and A. Nather, eds., World Scientific, Singapore, Vol. 3, pp. 33-41. VEHMEYER, S.B.W., BLOEM, R.M., DEIJKERS, R.L.M., VEEN, M.R. and PETIT, P.L.C. (1999). A comparative study of blood and bone marrow cultures in cadaveric bone donation, /. Hosp. Infect. 43, 305-308.

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VEHMEYER, S.B., BLOEM, R.M. and PETIT, RL. (2001). Microbiological screening of post-mortem donors — two case reports, /. Hosp. Infect. 47, 193-197. WANG, J., LEE, C , CHEN, P., WANG, T. and CHEN, D. (2002). Transfusion-transmitted HBV infection in an endemic area: The necessity of more sensitive screening for HBV carriers, Transfusion 42, 1592-1597. YAMADA, S., AIBA, T., ENDO, Y., HARA, M., KITAMOTO, T. and TATEISHI, J. (1994). Creutzfeldt-Jakob disease transmitted by a cadaveric dura mater graft, Neurosurgery 34, 740744. YANKAH, A.C., HETZER, R., MILLER, D.C., ROSS, D.N., SOMERVILLE, J. and YACOUB, M.H. (eds.) (1988). In: Cardiac valve allografts 1962-1987, Current concepts on the use of aortic and pulmonary allografts for heart valve substitutes. SpringerVerlag, New York. YOTSUYANAGI, H., YASUDA, K., MERIYA, K., SHINTANI, Y., FUJIE, H., TSUTSUMI, T., NORJIRI, N., JUJI, T., HOSHINO, H., SHIMODA, K., HIRO, K., ILINO, S. and KOIKE, K. (2001). Frequent presence of HBV in the sera of HBsAg-negative antiHBc-positive blood donors, Transfusion 41, 1093-1099. ZAAIJER, H.L., EXEL OEHLERS, P., KRAAIJEVELD, T., ALTENA, E. and LELIE, P.N. (1992). Early detection of HIV-1 by third generation assay, Lancet 340, 770-772.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

4 BACTERIAL CONTAMINATION OF BONE ALLOGRAFTS IN THE NETHERLANDS

JEROEN VAN BAARE Netherlands Bone bank Foundation Leiden, The Netherlands STEPHAN VEHMEIJER Department of Orthopaedics Leiden University Medical Centre Leiden, The Netherlands ROLF BLOEM Department of Orthopaedics, Reinier de Graafgasthuis Delft, The Netherlands

1. Introduction The increased need for allograft bone tissue in The Netherlands resulted in the establishment of the Leiden Bone bank Foundation in 1988. This was initiated by the Department of Orthopaedics, Leiden University Medical Centre and Bio This paper is a short summary of the recently published PhD. thesis of Dr. Stephan Vehmeijer (Vehmeijer, 2002). A hard copy of this thesis is available on request. 133

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Implant Services, a daughter organisation of Euro Transplant, Leiden. In 1998, the name of the bone bank was changed to Netherlands Bone bank Foundation (NBF), as a result of the implementation of two new laws on tissue banking. The Act on Organ Donation, July 1, 1998, concerns the central registration of consent for the donation of organs and tissues. In addition, the Royal Degree on Quality Requirements for Organ Banks was implemented on November 1, 1998. This Degree regulates tissue banking in The Netherlands and should further improve the quality and safety of human allograft tissue by specific regulations, accreditation of tissue banks by the government and the introduction of a certified quality system for tissue banks, such as ISO 9001. 2. Investigations by NBF NBF has been retrieving musculoskeletal tissues from living donors as well as from post-mortem donors (see Figs. 1 and 2). Currently, approximately 1,500 femoral heads are donated each year by patients undergoing primary total hip arthroplasty. The allograft femoral heads are stored as unprocessed deep-frozen tissue. The use of femoral head allografts in orthopaedic surgery

Femoral Heads from Living D o n o r s

•g 1500 £ 1200 « 900 | 600 £• 300

L UL11 1997

1998

1999

2000

2001

Year

Fig. 1. Number of Femoral heads received by NBF.

Bacterial Contamination of Bone Allografts in the Netherlands

Post-mortem Donors 120 -|

ilixiil 1997

1998

1999

2000

2001

Year

Fig. 2. Number of post-mortem donors received at NBF.

is widely accepted, particularly in revision hip and knee arthroplasty. Favourable results of the impaction grafting technique have increased the demand for this type of allograft (Sloof, 1996; Gie, 1993; van Donk, 2002). Also, NBF has been receiving approximately 100 post-mortem bone donors each year. Large bone segments can be retrieved from these donors, and used for reconstructive surgery in orthopaedic oncology or in revision arthroplasty. Grafts can also be processed into smaller units and applied in spinal fusions, or used for the filling of bone defects, for example, defects associated with bone cysts. 3. The Role of Tissue Banks Tissue banks play an important role in the safety and quality of allograft tissue. To ensure safety of bone allografts, many procedures are carried out to minimise the risk of transmission of infectious diseases. The medical and social history of potential donors is extensively screened. The procurement of donors is performed in the operating theatre with strict aseptic procedures; and serology and bacteriology tests are performed. Despite all these precautions, complications after bone transplantation may occur. Non-union, fracture and infection

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are relatively common, particularly in reconstructive surgery following resection of bony tumours. Infection is the most devastating complication, often leading to failure and resection of the graft. Infection can be caused either during surgery or through graft contamination. Tissue banks can only control the possible contamination of allografts, which can be divided into two main sources. Contamination can be superficial, e.g., caused by the procurement team during retrieval of the grafts, or through the skin of the donor. On the other hand, the contamination can be of deep origin, e.g. caused by endogenous donor sources. To detect possible contamination of each individual transplant, several culture techniques can be used. NBF has been using the swab technique, in which the entire surface area of each individual graft is swabbed carefully. The swab is taken directly after retrieval, and before any other processing procedures. The swab stick is inoculated onto culture plates and the swab stick itself is incubated in broth, which is then subcultured. This method allows for a semi-quantitative assessment of the bacterial load of the graft. A major disadvantage of the swab culture technique is that only the external surface is sampled. Although currently not implemented in the procedures at NBF, tissue banks may perform tissue cultures, as they consider this a more direct method to determine the bacterial load. Samples from representative areas of the graft are incubated in broth, which is then subcultured. This method is highly sensitive in cases where samples are contaminated with micro-organisms. It is, however, unlikely that micro-organisms present on an allograft are evenly distributed on the surface. As samples are taken from a limited number of sites on the graft, sampling error is evident. Micro-organisms inside the graft can remain unnoticed. To detect these micro-organisms, NBF has been performing post-mortem blood cultures. Although the value of these cultures remains unclear, and the interpretation of the results is difficult, it is the opinion of NBF that contamination by endogenous

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137

Table 1. Analysis of blood cultures of 550 bone donors at Netherlands Bone bank Foundation. 550 donors

1

i

1 140 donors blood culture +

410 donors blood culture -

1 70 donors skin contaminants

1

70 donors non-skin contaminants

1 16 donors swab & blood culture with identical micro-organism

donor sources can be detected through this technique. Therefore the blood culture of 550 bone donors were reviewed. The results are given in Table 1. Prior to bone retrieval, blood was taken from the subclavian vein or artery under aseptic conditions. For multi-organ donors, samples were taken through venal puncture before organ perfusion; and if post-mortem heart valve procurement preceded bone procurement, blood samples were taken after mid-sternal thoractomy from the interior cava vein under aseptic conditions. Blood samples were then cultured for aerobic and anaerobic micro-organisms for seven days. The swabs were inoculated within 24 hours onto blood agar and chocolate plates, and then cultured under aerobic and anaerobic conditions. The swab sticks themselves were incubated in broth which was inoculated after five days, onto blood agar and chocolate plates, and cultured for 48 hours. A positive blood culture was detected in 140 donors. From these donors, 70 cultures were tainted with skin contaminants. This can be explained by the fact that samples were contaminated during the blood retrieval procedure. The other

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Table 2. Details of a donor with positive swab & blood culture of same micro-organism. Donor A > Age: 28 years > Cause of death: drowning 15 minutes in water attempt to resuscitate failed > No open wounds nor fractures, no autopsy > No prior illness > No other procurement procedures before bone retrieval > Procurement 13 hours after circulatory arrest, completed within two hours > Total number of grafts: 12

70 donors showed contamination of the blood culture with non-skin contaminants, which is difficult to explain by the retrieval procedure. From these donors 16 donors were identified with the same micro-organism for both the swab culture and blood culture. These 16 donors could be divided into the following groups: a traumatic cause of death was observed in 12 donors; three donors received pre-mortem streptokinase; and one donor died by drowning. To stress the importance of the blood culture as an additional safety measurement, one donor will be described below in detail (Table 2). 4. Blood Cultures as an Additional Safety Measurement The blood culture of the donor was positive for Aeromonas species. Aeromonas is an organism of high pathogenic potential, and known for its presence in water. As the donor was discarded for this reason, there was a possibility to culture the entire grafts individually in toto and compare these results with the swab culture results. The results are shown in Table 3. The swab

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139

Table 3. Culture results of grafts obtained from donor mentioned in Table 2. Graft

Swab Culture

Culture of Entire Graft

Femur proximal left

-

CNS

Femur distal left

-

CNS

Tibia proximal left

-

Fibula proximal left

CNS

CNS

Achilles tendon left

-

Not cultured

Fascia lata left

-

CNS

Aeromonas spp CNS

CNS

CNS

Aeromonas spp

Tibia proximal right

-

CNS

Fibula proximal right

CNS

Staphylococcus aureus

Achilles tendon right

CNS

Aeromonas spp

-

CNS

Femur proximal right Femur distal right

Hemipelvis right

Staphylococci schleiferi

CNS = Coagulase Negative Staphylococci - = no growth culture showed only one graft positive for Aeromonas species and some grafts had no bacterial contamination. Cultures of the grafts in toto resulted in four positive grafts contaminated with Aeromonas species. However, also with this technique, many grafts showed only positive contamination results for Coagulase Negative Staphylococci, a frequently observed skin-contaminant in bone grafts. The pulmonary and heart valves were procured after the bone retrieval. The culture results of the heart valves were also positive for Aeromonas species. Therefore, considering the blood culture results; the cultures of the entire graft; and also

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the cultures of the heart valves, it is very likely that a haematogenous spread of Aeromonas species occurred pre-mortem, contaminating most organs, including the bones. One of the latest reports of infection after bone transplantation was published in the Morbidity and Morbidity weekly Report (4). One patient underwent reconstructive knee surgery with a femoral condyle allograft. The patient died the next morning. Pre-mortem blood cultures grew Clostridium sordelli. Another patient also underwent reconstructive knee surgery with an allograft femoral condyle and meniscus. He developed high fever the next day (39.7°C) but recovered with antibiotics. Cultures were not obtained. The allograft tissue was, however from the same tissue donor. Other bone allograft tissue at the tissue bank, not yet released for clinical use, also showed positive cultures for Clostridium sordelli. Reviewing the 550 bone donors at NBF for any Clostridium cases, three donors were found to be positive for Clostridium sordelli. However, none of the swab cultures of any graft showed contamination for Clostridium sordelli. All grafts of these donors were discarded due to the positive blood culture. 5. Conclusions Blood cultures can help to identify unnoticed haematogenous spread of (high pathogenic) micro-organisms. Grafts with positive swab cultures of non-skin contaminants, or, grafts obtained from donors with positive blood cultures from which non-skin contaminants are isolated, should not be used as large minimally-processed grafts. 6. References GIE, G.A., LINDER, L. and LING, R.S.M. et al. (1993). Impacted cancellous allografts and cement for revision total hip arthroplasty, /. Bone Joint Surg. (Br.) 75-B, 14-21.

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MMWR, November 23 (2001), 50(46); and March 15 (2002), 51(10). SLOOFF, T.J., BUMA P. and SCHREURS, B.W. et al. (1996). OAcetabular and femoral reconstruction with impacted graft and cement, Clin. Orthop. 324, 108-15. VAN DONK, S. (2002). Experimental and clinical data on the incorporation of impacted morsellised bone grafts. Nijmegen. ISBN 90-9015620-8. VEHMEIJER, S.B.W. (2002). Bacterial contamination of bone allografts. Leiden. ISBN 90-9015577-5.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

5 MICROBIOLOGICAL SCREENING OF CADAVER DONORS AND TISSUES FOR TRANSPLANTATION

OCTAVIO V. MARTINEZ University of Miami Tissue Bank

1. Introduction The risk of transmission of infectious agents by means of cadaver-derived osteoarticular and other tissues, has been the subject of concern since the earliest reports of the use of human tissues for transplantation. Over the years, tissue banks have developed multiple interdisciplinary approaches for screening prospective cadaver donors for the presence of infectious agents, as well as for the examination of the tissues and grafts derived from the same. While some form of a microbiological culture protocol is applied routinely to tissues obtained from cadavers or from living donors at the time of tissue retrieval, it is to be noted that such cultures alone are insufficient to reliably identify all non-septic donors, or to detect all contaminated tissues derived from these sources. Microbiologic screening is part of a collective of tests and procedures designed to minimise the likelihood of processing tissues harbouring infectious agents. Complementary procedures may include obtaining a medical and social history on the donor; performing serologic or molecular amplification tests for bacterial and viral pathogens; 143

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performing histopathological studies of lymph nodes or other organs; or a diagnostic autopsy; and implementing an effective strategy for tracking graft recipients. There is a two-fold purpose to the performance microbiological cultures of the blood and tissues of cadaver donors. First, to search for evidence of clinically unapparent sepsis in donors judged to be non-septic by medical history and physical examination or autopsy. The guidelines of the American Association of Tissue Banks state that tissues from donors exhibiting evidence of significant active infection, including septicemia at the time of donation, are not to be released for transplantation (AATB, 2001). Second, cultures are intended to identify specific tissues contaminated with bacteria or fungi at the time of excision. Microbiological cultures at the time of tissue retrieval influence the subsequent processing of tissues in other ways. Some tissue banks utilise the results of these cultures to determine preprocessing treatment of bones to reduce bioburden, or to determine the processing method, ascertain the need for endotoxin testing, and monitor quality assurance for procedures and processing personnel (Anderson and Vessey, 1996). 2. Sampling Methods The most common method for culture of the bones, cartilage or tendons employs the use of cotton swabs to sample both the surface, and whenever possible, the medullar canal of long bones. Other methods include the immersion of whole tissues or small segments of bone in a liquid culture medium. Occasionally, tissues are immersed in a diluent solution such as physiological saline, agitated to release any microbial contaminants, then a small portion of the diluent is cultured in liquid or agar medium or both under aerobic and anaerobic conditions (Farrington et al, 1998; LaPraire and Gross, 1991). There are legitimate concerns regarding the sensitivity of swab cultures for the detection of low numbers of microorganisms in the tissues. Veen et al. (1994) compared the results

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145

of three types of cultures from 75 fibular segments. These included surface swabs inoculated directly on agar medium; surface swabs inoculated into agar medium followed by immersion in a culture broth; and full immersion of bone segments in broth medium. The results showed a significantly lower rate of recovery of microorganisms by the direct agar plating method and the combined agar-broth method (culture sensitivity of 10% and 39% respectively) compared to the full broth immersion procedure. Mills and Roberts (2001) reported the recovery of microorganisms from 5.9% of surface swabs cultures from processed bone grafts (sensitivity, 22.1%) compared to 12.7% of tissue segments from grafts cultured by broth immersion. These investigators concluded that the destructive culture method based on the immersion technique, was approximately 3.5 times more sensitive than methods that relied on surface swab cultures. It is not known whether the sensitivity of a method that relies on single swab cultures can be increased by performing multiple cultures from each of the tissues. 3. Culture Media and Length of Incubation There is little agreement on the optimal choice of culture medium and the length of incubation for cultures obtained from cadaver musculoskeletal tissues at the time of procurement or from prepared allografts. Several investigators have reported the use of liquid thioglycollate medium (Barrios et al, 1994), brain heart infusion broth (Deijkers et al, 1994) or a combination of agar and broth culture media (Vehmeyer et al, 2001). The reported length of incubation ranges from 48 hours (Barrios et al, 1994) to as long as 10 to 14 days (Bettin et al, 1998). Bennett et al (1991) analysed the results of cultures of cadaver connective tissues and bone from living and cadaver donors, and reported that an incubation time of 14 days allowed for the detection of an additional 6% of contaminated tissues compared to a seven-day incubation protocol. In contrast, other investigators have reported that seven days of incubation were sufficient for

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detecting most organisms considered by the authors to be significant pathogens; therefore, extending the incubation period beyond seven days was judged to be unwarranted (Reik et ah, 1998). The microbiological screening protocol at the University of Miami Tissue Bank (UMTB) specifies the use of swab cultures of the surface and, when appropriate, marrow canal of bones and soft tissues obtained at procurement. The swabs are inoculated into tubes of thioglycollate medium previously degassed by boiling, and incubated at 35°C until microbial growth is evident or, if negative, for up to 14 days. The organisms most commonly recovered from these sources include species of coagulase-negative staphylococci, alpha-haemolytic streptococci and species of aerobic and anaerobic diphteroids (Table 1). Most isolates of organisms considered to be of significant pathogenic potential such as beta-haemolytic streptococci, Clostridia, Staphylococcus aureus and gram-negative enteric organisms, were

Table 1. Microorganisms isolated from the bones of 649 cadaver tissue donors at the time of tissue retrieval. Organism

Donors

% Positive

Cultures

% Positive

279

42.9

734

3.33

151

23.2

342

1.56

Streptococcus species

91

14.0

249

1.13

Corynebacterium species

69

10.6

97

0.44

Aerobic, Gram Negative Bacilli Clostridium species

54 30

8.3 4.6

192 173

0.87 0.79

Staphylococcus species Propionibacterium species

Total 21,971 cultures. Mean 33.8 cultures/donor. Total positive cultures = 1,946 (8.9%).

Microbiological Screening of Cadaver Donors

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Table 2. Time of detection by culture of microorganisms recovered at procurement from cadaver musculoskeletal tissues. No.

Organism

Positive

Percent detected on culture days <2

3-4

5-6

>7

Clostridium species

173

97

3

Staphylococcus species

734

44

28

14

14

Streptococcus species

249

69

23

4

4

Corynebacterium species

96

5

22

29

44

Propionibacterium species

342

1

11

28

59

1,917

47

20

14

20

All isolates

detected within 48 hours of incubation. However, up to 40% to 60% of corynebacteria and propionibacteria required seven or more days of incubation for detection (Table 2). It is to be noted that the time of detection of microorganisms in culture is, to an extent, dependent on the technology employed. Kostiak and Michlowski (2002) reported that the application of an automated microbial detection system (for blood cultures) to cultures of musculoskeletal tissues, allowed for the implementation of a 7-day incubation protocol with results comparable to those obtained with a reference 14-day incubation conventional culture system. In summary, the establishment of an optimal incubation period for cultures from cadaver tissue donors requires consideration of the need for a rapid turn around time, the type of microorganisms targeted for detection, and the type of detection system employed.

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4. Blood Cultures The role of postmortem blood cultures as indicators of sepsis and tissue contamination in cadaver donors of organs and tissues, has been the subject of extensive debate. A number of investigators have stated that postmortem blood cultures can yield useful information (Silver and Sonnerwirth, 1969) with a degree of reliability equivalent to that of antemortem cultures (Hove and Pencil, 1998) provided specific techniques were utilised for the collection of the blood. Others have suggested that postmortem blood cultures rarely, if ever, provide information that is not already known, or can be interpreted from other sources (Wilson et ah, 1993). One study (Gocke et al., 1998), addressing the use of blood cultures as predictors of tissue contamination in cadaver bone donors, revealed little concordance between blood culture results and the outcome of cultures of the tissues from the same donors. In that study, less than onehalf of donors with positive blood cultures also had positive tissue cultures, and only 13% of positive tissue cultures yielded matching isolates from the blood. The interpretation of blood culture results from cadaver tissue donors can be difficult not only because of the unavailability of clinical findings suggestive of sepsis, but also because the results can be influenced by a variety of perimortem and postmortem events. Lengthening postmortem interval, and a traumatic manner of death, have been reported to increase the likelihood of obtaining positive findings from cultures in cadavers judged to be non-septic a the time of death (Martinez and Malinin, 1996). Harvesting of viable organs also influences the outcome of blood cultures. A review of blood culture results from 1,923 donors examined at the UMTB in a five-year period revealed that only 20 of 501 (4%) donors of viable organs had microorganisms isolated from the blood, compared to 442 of 1,422 (31.1%) donors of devitalised tissues only. These differences in the rate of positive blood cultures between the two donor populations may be the result of significant differences in the timing of blood

149

Microbiological Screening of Cadaver Donors Bone and Organ Donors

Bone donors

Brain Death

Organ Retrieval

Physical Death

Physical Death

Blood cultures <-

Tissue Retrieval

-> Tissue Cultures •*-

Tissue Retrieval

Fig. 1. Timing of the collection of blood and tissue specimens for culture from cadaver donors of bones and organs.

collection. Among donors of musculoskeletal tissues only, blood is obtained for culture following a postmortem period of variable length. In contrast, blood cultures are performed on beatingheart cadaver organ donors following the determination of brain death but prior to the excision of physiologically viable organs (Fig. 1) when the natural clearing mechanisms of the blood may still be functional. It is of interest that, in contrast to the effect that organ donation exerts on the outcome of blood cultures, the excision of viable organs does not seem to have a significant effect on the results of cultures of the bones. In the experience of the UMTB, the rate of positive tissue cultures among donors of musculoskeletal tissues only ranged from 9% for donors with negative blood cultures, to 20% for those with positive findings in the blood. The corresponding rates for combined donors of organs and tissues were 6% and 17% for donors with negative

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and positive blood cultures respectively. Although the organisms recovered from the blood did not always match the identity of those isolated from the tissues, donors with positive blood cultures were more likely to yield positive cultures from the tissues than those with negative blood culture results. 5. Correlation of Blood and Tissue Culture Results Efforts to relate the results of postmortem blood cultures to those of tissues as indicators of disseminated sepsis in prospective donors, have not yielded encouraging results. Martinez et al. (1985) evaluated the culture results from 297 cadaver donors and concluded that blood cultures alone were unreliable predictors of tissue sterility. A more recent study, however, suggests a role for bone marrow aspirate cultures as an aid in the interpretation of blood cultures from clinically non-septic cadavers (Martinez et al., 2003). A retrospective review of the records of 185 selected cadaver donors of musculoskeletal tissues only with positive blood cultures or positive cultures of a bone marrow aspirate from the iliac crest, showed that a majority of the subjects (100 donors; 54.1%) had cultures positive with one or more organisms from both sites. The positive predictive value (PV+) of blood cultures alone for the recovery of the same type of microorganisms from the bones was 38%. For bone marrow aspirates alone, PV+ was 42%. Yet, the recovery of the same type of organisms from both the blood and the bone marrow aspirate had a PV+ of 72%. It is reasonable to assume that not all microbial contaminants of tissues from clinically non-septic donors are the result of haematogenous dissemination. The high prevalence of organisms from the skin or mucous surfaces normally recovered from the tissues, suggests that to a significant extent, contamination can occur at the time of tissue excision in spite of the use of sterile techniques. This data limits the use of blood or bone marrow aspirate cultures as absolute predictors of tissue contamination. As many as 21% of the donors studied had potential pathogens in the tissues that were not recovered

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from the blood or marrow aspirates. Similarly, pathogens were cultured from the tissues only in 14% of donors from a control group of 185 subjects with negative blood and marrow aspirate cultures (Martinez et al, 2003). These results suggest that neither blood nor marrow aspirates can be considered reliable substitutes for cultures of the tissues, but their combined results may serve as a complementary diagnostic modality for the evaluation of the donor. 6. Interpretation of Culture Results The interpretation of culture results from blood and tissues of cadaver donors has a direct impact on the disposition of the donor, as well as the subsequent processing of the grafts. A number of authors have reported on the various criteria used for donor deferral and tissue acceptance or rejection. The basis of the selection criteria depends on diverse factors such as the presence or absence of micro-organisms of high pathogenic potential in the blood or tissues; the presumed source and distribution of the isolates; identity among isolates from the tissues and the blood; or the magnitude of the microbial load in the tissues. Malinin et al. (1985) recommended rejection of donors who harboured identical species of micro-organisms in the bloodstream and the bone marrow of two or more anatomically unrelated bones. Recovery of multiple organisms from the internal or external flora, or the isolation of highly pathogenic organisms from one or more sites, also justified rejection of the donor. Other investigators accepted tissues for subsequent processing and allograft preparation only if cultures of the blood and surface swab cultures of the tissues were negative for growth after seven days of incubation (Tomford et al, 1990). La Praire and Gross (1991) described the culture of segments of femoral heads obtained from patients undergoing total hip arthroplasty, as a screening procedure for banked bone. No blood cultures were performed. The segments were incubated in broth medium for up to six days, and any evidence of microbial growth was interpreted as

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an indication for discarding the bone. Jackson (1987) recommended the combined use of blood and tissue culture results as a guide for acceptance or rejection of tissues. The excised bones were cultured in liquid medium at 30°C to 32°C and at 20°C to 25°C for 14 days. If the blood cultures were positive, the donor was assumed to be septic, and deferred for donation. Infection also was assumed to have occurred if 20% or more of the tissues yielded the same organisms. Tissues contaminated with organisms common to the skin flora were judged suitable for processing by secondary sterilisation methods. A quantitative approach to the interpretation of tissue culture results has been described (Farrington et ah, 1998). This method involved the use of an immersion technique for the determination of the microbial burden in femoral heads and other skeletal tissues obtained from living and cadaver donors. Each tissue was immersed in sterile saline solution and small samples of the diluent were cultured for the determination of microbial counts. Tissues were rejected if the microbial load of various selected pathogens (Staphylococcus aureus, Clostridium species, Pseudomonas species, beta-haemolytic streptococci and the Enterobacteriaceae) exceeded 50 colony-forming units (cfu) per mL. Tissues also were rejected if cultures yielded > 100 cfu/mL of any other organisms. All tissues with lower bioburden were considered acceptable for further processing and secondary sterilisation. 7. Conclusions The establishment of criteria for the interpretation of cultures from cadaver tissue donors is dependent on a number of factors. Tissue banks select suitable donors and obtain and process bone tissues in a variety of ways. Acceptance or rejection of such tissues is ultimately a risk-benefit judgment. The foremost objective of a microbiological screening protocol for prospective donors should be the exclusion of high-risk subjects from the donor pool. To this end, it may be prudent to consider the distribution

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of certain microorganisms in the blood and tissues as evidence suggestive of haematogenous dissemination, hence, a cryptic septic state. The selection of specific tissues for continued sterile processing, or the application of sterilisation methods, could be based, in part, on an assessment of the pathogenic potential of the microorganisms recovered by culture. Finally, the reliability of available sterilisation methods also needs to be considered in assessing risk. 8. References AMERICAN ASSOCIATION OF TISSUE BANKS (2001). Standards for Tissue Banking. McLean, Virginia, pp. 35-36. ANDERSON, M. and VESSEY, A. (1996). Cadaveric procurement cultures — What do they mean and how they are used. Abstr. 20th Meet. Amer. Assoc. Tisssue Banks. Washington, D.C. Abstract S-4. BARRIOS, R., LEYES, M., AMILLO, S. and OTEIZA, C. (1994). Bacterial contamination of allografts, Acta Orthop. Belg. 60, 293-295. BENNET, M., JOHNSON, J., NOVICK, S., HILGREN, J., RABE, F. and EASTLUND, T. (1991). Prevalence and growth rate of microbes found at procurement of cadaveric and living donor bone and connective tissue. Abstr. 15th Ann. Meet. Amer. Assoc. Tissue Banks. Clearwater, Florida, p. 35. BETTIN, D., HARMS, C , POLSTER, J. and NIEMEYER, T. (1998). High incidence of pathogenic microorganisms in bone allografts explanted in the morgue, Acta Orthop. Scand. 69, 311314. DEIJKERS, R., BLOEM, R., PETIT, P., BRAND, R., VEHMEYER, S. and VEEN, M. (1997) Contamination of bone allografts: Analysis of incidence and predisposing factors, J. Bone Joint Surg. 79B, 161-165.

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FARRINGTON, M., MATTHEWS, L, FOREMAN, J., RICHARDSON, K. and CAFFEY, E. (1998). Microbiological monitoring of bone grafts: Two years' experience at a tissue bank, /. Hosp. Infec. 38, 261-271. GOCKE, D., YEAGER, J., DOUGHRTY, C. and OSBORNE, J. (1998). Lack of correlation of blood culture and tissue culture in cadaver donors: The MTF experience. Abstr. 22nd Meet. Amer Assoc. Tissue Banks. New Orleans, Louisiana. Abstract S-16, p. 64. HOVE, M. and PENCIL, S. (1998). Effect of postmortem sampling technique on the clinical significance of autopsy blood cultures, Hum. Pathol. 29, 137-139. JACKSON, B. (1987). Bone banking: An overview, Lab. Med. 18, 830-833. KOSTIAK, P. and MICHLOWSKI, M. (2002). Validation of an automated seven-day culturing methodology for the detection of aerobic and anaerobic microorganisms from retrieved musculoskeletal tissues. Abstr. 26th Ann. Meet Amer. Assoc. Tissue Banks. Boston, Massachusetts. Abstract PR-11, p. 63 LaPRAIRIE, A. and GROSS, M. (1991). A simplified protocol for banking bone from surgical donors requiring a 90-day quarantine and an HIV-1 antibody test, Can. J. Surg. 34, 41-48. MALININ, T., MARTINEZ, O. and BROWN, M. (1985). Banking of massive osteoarticular and intercalary bone allografts — 12 years, experience, Clin. Orthop. Rel. Res. 197, 44-54. MARTINEZ, O., MALININ, M., VALLA, P. and FLORES, A. (1985). Postmortem bacteriology of cadaver tissue donors: An evaluation of blood cultures as an index of tissue sterility, Diag. Microbiol. Infec. Dis. 3, 193-200. MARTINEZ, O. and MALININ, T. (1996). The effect of postmortem interval and manner of death on blood and bone

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marrow cultures from non-septic cadaver donors of tissues for transplantation. Abstr. 96th Gen. Meet Amer. Soc. Microbiol. New Orleans, Lousiana. Abstract C-84, p. 96. MARTINEZ, O., BUCK, B., HERNANDEZ, M. and MALININ, T. (2003). Blood and marrow cultures as indicators of bone contamination in cadaver donors, Clin. Orthop. Rel. Res. 409, 317-324 MILLS, A. and ROBERTS, M. (2001). Evaluation of culturing methods at predicting allograft sterility for aseptically processed tissue. Abstr. 25th Ann. Meet. Amer. Assoc. Tissue Banks. Washington, D.C. Abstract S-9, p. 49. REIK, R., WOWK, S., MEADE, D. and BLAIR, K. (1998). Procurement culture study. Abstr. 22nd Meet. Amer. Assoc. Tissue Banks. New Orleans, Louisiana. Abstract S-22, p. 70. SILVER, H. and SONNERWIRTH, A. (1969). A practical and efficacious method for obtaining significant postmortem blood cultures, Amer. J. Clin. Pathol. 52, 433-437. TOMFORD, W., THONGPHASUK, J., MANKTN, H. and FERRARO, M. (1990). Frozen musculoskeletal allografts. A study of the clinical incidence and causes of infection associated with their use, /. Bone Joint Surg. 72A, 1137-1143. VEEN, M., BLOEM, R. and PETIT, P. (1994). Sensitivity and negative predictive value of swab cultures in musculoskeletal allograft procurement, Clin. Orthop. Rel. Res. 300, 259-263. VEHMEYER, S., BLOEM, R. and PETIT, P. (2001). Microbiological screening of post-mortem bone donors — Two case reports, J. Hosp. Infec. 47, 193-197. WILSON, S., WILSON, M. and RELLER, B. Diagnostic utility of postmortem blood cultures, Arch. Pathol. Lab. Med. 117, 986988.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

6 SAFETY OF VIRUS INACTIVATION METHODS FOR ALLOGENEIC AVITAL BONE TISSUE TRANSPLANTS

AXEL PRUSS Institute for Transfusion Medicine (Tissue Bank) University Hospital Charite, Berlin M O U J A H E D K A O a n d GEORG PAULI Robert Koch-Institut, Berlin, G e r m a n y

1. Introduction At present, several procedures are used for the inactivation of viruses in the production of avital allogeneic bone transplants. Among these are gamma irradiation (Bright, 1987; Ostrowski, 1968; Sautin, 1963), thermal treatment with moist heat (Hofmann et al, 1996; Knaepler et al, 1994; von Garrel et al, 1997) and peracetic acid-ethanol treatment combined with negative pressure (Starke and von Versen, 1984; von Versen et al., 1992). Apart from the varying irradiation doses reported in the literature, ranging between 15 and 40 kGy, the problems with gamma irradiation appear to be, above all, the development of toxic radicals, in particular with non-processed transplants (Moreau et al., 2000), and the negative effect on biomechanical parameters (Rock, 1991). Additionally, logistics management is complex. Definite assessments regarding the efficiency of the 157

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sterilisation procedure of contaminated bone tissue, so far exist only in sporadic reports, and are limited to the human immunodeficiency virus (Campbell and Li, 1999; Hernigou et al, 2000). Experiences with the irradiation of medical products have led to the statement that an irradiation dose of 25 kGy is obviously sufficient for inactivating relevant pathogens, including viruses (Botzenhardt and Thofern, 1988). A reduction of infectivity titres by 6 logio for bacteria, fungi, and spores is recommended for the "industrial sterilisation of medical products" (IAEA, 1990; European Committee for Standardization, 2000) in order to reach the "sterility assurance level" (SAL) (Gaughran, 1985). For example, an SAL of 10" 6 means that the probability of a single viable micro-organism being present on a product unit is one in one million after the unit has undergone a terminal sterilisation process validated to this SAL. However, the concept of SAL is not applicable for assessing the viral safety of human tissue because of the difficulty in standardising such materials. Thermal treatment by means of the Lobator sd-2 system (telos, Marburg/Germany), which is widespread in Germany, is used to disinfect femoral heads which are collected in the context of total endoprosthesis operations of the hip joint. Validation methods described for this procedure (Knaepler et al, 1994; von Garrel et al., 1997) meet the requirements of national and international standards (Paul-Ehrlich-Institut and Bundesinstitut fur Arzneimittel und Medizinprodukte, 1994; CEN, 1994; EMEA, 1996) with slight theoretical reservations. Due to its carcinogenic and mutagenic effect, sterilisation with ethylene oxide was discontinued in Germany (Bundesgesundheitsamt, 1986). Recent investigations, however, have documented the suitability of the procedure if performed in combination with thermal treatment, centrifugation, ultrasonic treatment and washing steps, in the case of human femoral heads (Lomas et al., 2000). A possible reduction of the osteoinductive potential by sterilisation with ethylene oxide is discussed controversially (Thoren and Aspenberg, 1995; Aspenberg and Lindqvist, 1998; Zhang et al, 1997). The use of beta-propiolactone (Lo Grippo,

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1987) and formaldehyde also have their limitations and, in addition, clearly reduce the osteoinductive effect (Munting et al, 1988). Since the 1980s, ethanol and peracetic acid (Sprossig and Mucke, 1969; Wutzler and Sauerbrei, 2001) have increasingly been used for tissue sterilisation (von Versen et al, 1992; Pruss et al, 1999; Pruss et al., 2001a). A prerequisite for effective sterilisation of spongiosa bone tissue is the preceding defatting step (Thoren et al., 1995) as well as to observe a maximal transplant thickness of 15 mm (Pruss et al., 2001a). No significant reduction of osteoinductivity or biomechanical properties after PES sterilisation were reported by Haynert (1990) and Thielicke et al. (1990). So far, only incomplete data existed for validating the various methods regarding their inactivating efficiency against clinically relevant pathogens. Considering the demands of clinicians and patients for maximal protection from infections, the following procedures were to be investigated scientifically, in accordance with the guidelines currently in force: • Peracetic acid-ethanol sterilisation (PES model). • Gamma irradiation (irradiation model). • Disinfection by moist heat with the Lobator sd-2 system (thermal treatment model). The panel of pathogens investigated included the majority of potentially possible in-vivo infections of bone donors. The results presented are based on investigations which have already been published in part (Pruss et al, 1999; 2001b; 2002). 2. Material and Methods 2.1. Selection of bone tissue donors Potential bone tissue donors underwent clinical examination for a variety of infectious diseases: virus hepatitis, tuberculosis, syphilis, septicaemia, systemic viral disease, and mycosis, demonstrable at time of death. Excluded were donors with the above-mentioned diseases; those with malignoma; those who

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had received human growth factor preparations or dura mater transplants; and those with other exclusion criteria by the European Association of Tissue Banks (EATB, 1999). Every bone tissue donor was tested ante or post mortem for hepatitis B virus surface antigen (HBsAg) and for antibodies against HIV-1/-2/ -gO, HCV, and Treponema pallidum. Only individuals negative for these markers were considered as donors. 2.2. PES model 2.2.1. Spongiosa cube specimen As source material for these transplants, spongiosa tissue was collected under sterile conditions from the columna vertebralis and from the epiphyses of femur and tibia, respectively. Fat and connective tissue were carefully removed under aseptic conditions, using scalpel and surgical tweezers. The processchallenge devices (15 x 15 x 15 mm) were cut on the belt saw (Bizerba, Balingen/Germany). Then they were rinsed for 30 minutes using sterile water at 37°C under high pressure to remove the blood completely from the bone tissue. Any remaining fat was removed by placing the tissue into a defatting mixture of chloroform-methanol (two volumes of chloroform and one volume of methanol) under constant agitation (laboratory shaker THYS 2, [MLW, Leipzig/Germany]) over a period of two hours (change of defatting medium after every 30 minutes). Subsequently, the tissues were flushed with methanol eight times (a 15-minute ultrasonic bath treatment) to completely remove any residual chloroform. Methanol was removed by flushing the tissues twice with sterile deionised water. 2.2.2. Virus contamination, sterilisation and homogenisation The defatted and air-dried spongiosa cuboid was placed in a sterile 50 mL Falcon polyethylene cell culture tube (BectonDickinson, Heidelberg/Germany) with a screw-type cap, and covered in 15 mL of the virus suspension. The tube was closed

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Fig. 1. Peracetic acid-ethanol sterilisation method.

with a cap that had been perforated eight times to allow equilibration of pressure, placed in an exsikkator, and subjected to negative pressure (200 mbr) by means of a vacuum pump (Meintrup Labortechnik, Lahden-Holte/ Germany), see Fig. 1. After 15 minutes the cuboids were completely penetrated. The remaining suspension was decanted and the tube overlaid with 15 mL of the respective sterilisation solution or, as control, with 15 mL medium. The following three test assays were set up per virus — sample I (SI): contaminated spongiosa cuboid +15 mL peracetic acid-ethanol mixture; sample II (S2): contaminated spongiosa cuboid + 15 mL medium inside the exsikkator; sample III (S3): contaminated spongiosa cuboid + 15 mL medium outside the exsikkator. The sample groups were placed in an exsikkator with a vacuum of 200 mbr, and incubated under continuous agitation at room temperature for four hours. Afterwards, the virus titres in the supernatants (neutralised with sodium thiosulfate 1%; 1/1, v/v) of all three samples were determined. The spongiosa cuboids (volume 2 ml) were transferred to sterile steel beakers of an Omni-Mixer (type OM, Ivan Sorvall

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Inc., Norwalk, CT, USA), and after addition of 10 mL of 1% sodium thiosulfate, the cuboids were homogenised under cooling in a water ice bath at 1,500 rpm at 4°C for two minutes. Homogenates were centrifuged at 3,000 rpm for 10 minutes at 4°C to collect all virus in the supernatant. Virus titres in the supernatant of the homogenates were determined. Penetration of the bone tissue up into the centre of the cuboid had been verified in preliminary tests. Here, 1 /im flow cytometer (FACS, Becton Dickinson) calibration particles were used as virus analogue. The size of the particles was definitely larger than that of the virus species investigated (PRV 170 nm, HIV about 100 nm, BVDV about 50 nm, HAV about 30 nm, FV-1 about 30 nm, PPV/BPV about 20 nm; Modrow and Falke, 1998). Under the terms of the assay described here, the calibration particles were brought in contact with the cuboid subjected to negative pressure. Subsequently, the central part of the cuboid (a cylinder which was earlier punched from the cuboid, with the upper and lower sections removed, the middle part of the central cylinder repositioned, and the cavities filled and sealed with bone wax [Ethic-R bone wax™]) was collected and centrifuged. From the supernatant, the recovery rate for the particles in the centre of the bone cuboid was determined by flow cytometric analysis and calculations of the concentration. 2.3. Irradiation model 2.3.1. Femoral diaphyses specimen, virus contamination The manufacturing process for diaphysis transplants included the following steps: preparation of diaphyses from human femurs after removing all attached muscles and connective tissue from the bone surface; sawing of diaphyses into segments of 75 mm length (belt saw, Bizerba); washing of the bone marrow canal several times with physiological salt solution (0.9% NaCl, B-Braun, Melsungen/Germany) in order to remove blood from the tissue.

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It is known that irradiation at low temperatures positively affects the biomedical properties of implants (Ascherl et al, 1986). To use an experimental design as close as possible to that of the production process, all inactivation experiments by irradiation were therefore performed in a Styrofoam box filled with dry ice. Temperature validation (frozen diaphysis on dry ice with medium outside the irradiation facility) showed that the virus suspension inside the diaphysis reached a temperature of - 3 0 ± 5 ° C during irradiation. For the inactivation experiments, the lower open end of each diaphysis was tightly sealed with bone cement (Palacos R™, Heraeus Kulzer, Wehrheim/Germany). The exterior surface of the diatheses was coated with a film of Ethic-R bone wax (Ethicon; thickness approx. 0.1 mm) in order to close any small canals or holes on the bone surface. That the bone was watertight was confirmed by filling the bone marrow canal with 5 ml aqua ad iniectahilia for 24 hours. After removing the water, the diaphyses were transferred into a plastic vessel containing 0.251 of water, fixed with styroflex in an upright position and frozen at -21°C. Then 5 ml of a suspension of cell-free virus were pipetted into the cavity of the diaphyses; the open end was

1^ ^ ^ Fig. 2. Irradiation model (femoral diaphyses).

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sealed with bone wax, and the diaphyses stored at -21°C (Fig. 2). All steps were carried out under sterile conditions and safety precautions in a biological safety cabinet. The virus-contaminated diaphyses were transported to the gamma irradiation facility of Gamma Service Produktbestrahlung GmbH (Radeberg/Germany), irradiated (see 2.3.2.) with doses ranging from 30.6-35.4 kGy deduced from the D 10 values of the most resistant viruses (BPV, HIV-2, PV-1). One contaminated but not irradiated diaphysis per virus investigated was retained as control to monitor storing and transportation conditions. The titre of viruses in the control sample was used as reference for calculating the experimentally determined inactivation factors. After irradiation all samples were returned to the virological laboratory under the cooling conditions described above. After thawing of the samples the virus titres were determined. 2.3.2. Determination of gamma ray dose distribution, irradiation procedure For technical reasons, it is difficult to measure the X-ray dose directly in specimens, i.e., in the experimentally contaminated diaphyses. Therefore, a dose-distribution study within and on the surfaces of the box used in the irradiation experiments, was performed in the gamma irradiation facility, using a cobalt source 60 Co. Validation took place in a Styrofoam box (length: 34 cm, width: 25 cm, height: 34 cm, weight: 1.46 kg, sample load: 6 plastic flasks with diaphyses or paraffin phantoms) at room temperature and a dose of 1 kGy/h. The following measure points were chosen to determine the dose distribution: two diaphyses and four paraffin phantoms corresponding in size and shape to diaphyses, were used to study the dose distribution inside the box and the influence of mass variation. Dosimeters were placed (i) inside the diaphyses or phantoms (an alanine pellet dosimeter at each end); (ii) on the surface of samples (four alanine foil dosimeters for each sample); (iii) on the inner surface of the box (four alanine foil dosimeters); and (iv) on the outer

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surface of the box (four alanine foil dosimeters). All procedures followed EN 552 (Deutsches Institut fur Normung, 2001) without major changes. 2.3.3. Determination of virus inactivation kinetics and the decimal reduction value (D 10 value) Eight 15 mL Nunc tubes were filled with 5 mL of cell-free virus suspension each, transferred into a plastic vessel, fixed with styroflex, and placed into a Styrofoam box containing dry ice, which was transported to the irradiation facility in a transport container according to EN 829 (Deutsches Institut fur Normung, 1996). One tube was removed from each group to be used as control, and placed into a plastic vessel on dry ice. The remaining seven vials in the box were exposed to the 60 Co source. At defined intervals (1 kGy/h), one vial each was removed from the box and stored in a plastic vessel on dry ice (temperature in the virus suspension -30±5°C) until virus titration, which took place in the virological laboratory after transportation and thawing as described above. The results of the inactivation kinetics of the different viruses showed a linear relationship between the logarithm of the infectivity titres and the radiation dose applied. To evaluate the results of irradiation, the following equation was used: N(D) = N 0 xlO("D/Dio)" N 0 is the virus titre (log) before and N(D) after irradiation with the dose D; D 10 is the dose necessary to reduce the titre of the infectious agent by a factor of 1 logio; and n is the correction factor introduced to avoid a systematic error (Fritz-Niggli, 1997). 2.4. Thermal treatment model The investigations have not been completed yet (experiments with HIV-2 to be performed yet, control experiments at 4°C). Therefore preliminary results are represented.

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2.4.1. Femoral head specimen Human femoral heads ( 0 55 ± 1 mm) from donors were used, all of whom had been tested serologically, and whose medical history did not feature exclusion criteria. Subsequently, cartilage was removed from femoral heads by means of a cartilage milling cutter (Aesculap, Tuttlingen/Germany). A central cylinder ( 0 11 mm) was then drilled by means of a keyhole saw from the sagittal plane of the femoral head. Into this defect an exactly matching polypropylene tube (Falcon tube 15 ml) was positioned. The lower end of the tube was equipped with one of the 5 mm corticospongiod cylinder segments, tightly closed with a rubber plug, and sealed with Palacos-R bone cement (Heraeus Kulzer, Wehrheim/Germany). The central cylinder was provided with a 6 mm drillhole, divided into four segments, indicated by the dotted lines (see Fig. 3), and placed into the polypropylene tube. Subsequently, 1 mL virus suspension was pipetted into the central drilling channel. Then the upper corticospongious cylinder segment (5 mm) was fitted-in properly; a cover was screwed onto the tube and fixed with Palacos. The femoral head thus prepared for the experiments was placed into the sterile transplant container and treated thermally according to the manufacturer's instructions (see below) in Ringer solution (B. Braun, Melsungen/Germany). After the cooling phase the incubation fluid was removed under sterile conditions. After the automatic thermal disinfection procedure with the Lobator sd-2 was completed, the central cylinder, the slices of 5 mm spongiosa with corticalis polypropylene tube

-^-^Y

~*V-^——.

central cylinder with drilled hole and virus suspension (segm.) cap

rubber plug/sealing (Palacos) sealing (Palacos)

Fig. 3. Sketch of process-challenge device in diagram form.

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167

corticalis, as well as the suspension still in the tube, were removed, and 9 mL of cell culture medium were added (1:10 predilution). Bone material and medium were homogenised in an autoclaved stainless steel container of an Omni mixer (type OM, Sorvall) in the ice bath at 1.500 U/min, and afterwards centrifuged (4°C, 3,000 x g). The supernatant obtained, which is considered to be a 1:10 starting dilution, was titrated. In order to adequately verify that a core temperature of at least 82.5°C was maintained for at least 15 minutes, femoral heads were used in three series for this investigation: native femoral heads, femoral heads with the cartilage removed, and the virus model (femoral head with cartilage removed plus tube). For safety reasons, and to protect the investigator, no parallel temperature measurements were taken in the virus experiments, so that the measurement of comparative core temperatures served to validate the above-mentioned virus model. A central drill hole (0.5 x 27.5 mm) was made at the onset of the Ligamentum capitis femoris into the centre of the femoral head (native or decartilaged), into which the sensor was fitted properly. The temperature sensor (Therm 2281-8, AMR, Holzkirchen/Germany) was run hermetically through the screw-type cap of the disinfection container, and fixed in the bone at the position 27.5 mm with Palacos-R bone cement (Heraeus Kulzer, Germany). Because the sensor takes only measures at its end point, the central temperature gradient was recorded. The starting temperature within the femoral head ranged between 24°C and 26°C. Measurements (temperature/ time) were taken every minute and entered in a computer program (off-line version 4.32/DOS-7.10, DEMA-soft GmbH, Holzkirchen/Germany). The result logs were saved, printed out and evaluated. 2.4.2. "Lobator sd-2" thermodisinfection system (Marburg bone bank system) The lobator sd-2 (see Fig. 4) was developed by the company telos H+V GmbH (Marburg/Germany) for thermal disinfection

A. Pruss, M. Kao & G. Pauli

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Fig. 4. Marburg bone bank system (Lobator sd-2).

of allogeneic femoral head bone grafts for clinical application. At present, the procedure has been implemented in Germany exclusively in bone banks affiliated with hospitals. This thermophysical disinfection system guarantees a temperature of 82.5°C for at least 15 minutes, in the centre of femoral heads with a diameter of < 56 mm. The temperature gradients (heating phase, plateau, cooling phase; total duration: 94 minutes) are predetermined by programming of the device, and are not alterable. 2.5. Viruses The viruses investigated were obtained from stocks by the Robert Koch-Institut Berlin, and are registered there and documented. 2.5.1. Enveloped viruses • Human immunodeficiency virus Type 2 (HIV-2), ssRNA, retroviridae genus lentivirus, isolate SBL6669, only tested in PES and irradiation model.

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• Bovine Virus Diarrhoe Virus (BVDV), ssRNA, flaviviridae genus pestivirus, strain Ug 59/Denmark as model virus for hepatitis C virus (HCV). • Pseudorabies virus (PRV; Aujeszky's disease virus), dsDNA, herpesviridae genus varicellovirus, strain Bartha.

2.5.2. Non-enveloped viruses • Hepatitis A virus (HAV), ssRNA, picornaviridae genus hepatovirus, strain HM 175cyt. Table 1. Overview of the cell lines used, and parameters of cell cultivation. Virus

Cell line

Cell cultivation

HIV-2

Lymphoma cells (Molt 4 clone 8)

Cell culture medium: RPMI 1640 reading: 10-14 days pi.*

BVDV

Calf lung cells

Cell culture medium: DMEM f with anti-BVDV-free serum reading: 5 days p.i.

PRV

Mink lung cells

Cell culture medium: DMEM reading: 3 days p.i.

HAV

Embryonal rhesus monkey kidney CRL 1688

Cell culture medium: DMEM reading: 10-14 days p.i.

PV-1

Fetal lung cells

Cell culture medium: DMEM reading: 5 days p.i.

PPV

Fetal porcine testis cells CRL 1746

Cell culture medium: DMEM reading: 7 days p.i.

BPV

Calf lung cells

Cell culture medium: DMEM reading: 7 days p.i.

*p.i. = post infection tDulbecco's Modified Eagle Medium high glucose, fetal calf serum 5%, glutamine (0.5 mg/ml), Penicillin (40 E/ml), Streptomycin (0.04 mg/ml).

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• Poliomyelitis virus type 1 (PV-1), ssRNA, picornaviridae genus enterovirus, vaccine strain PI 18. • Porcine parvovirus (PPV)/Bovine parvovirus (BPV), ssDNA, parvoviridae genus parvovirus. 2.6. Cell cultures/cell lines The cell lines investigated were obtained from stocks by the Robert Koch-Institut Berlin and are registered there and documented (Table 1). The respective viruses were obtained from the supernatants of cultivated infected cells, following procedures reported elsewhere (Scheidler et ah, 1998). The cell debris was discarded after centrifugation, and the viruses obtained were frozen in aliquots at -70°C. 2.7. Virus titrations, virus titre calculation 2.7.1. Preparation of the micro titre plates After preparing 10-fold dilutions of the virus suspensions (supernatants/homogenates) with cell culture medium, 100 jA of each dilution were pipetted into each of four or eight wells of a 96-well micro titre plate. Each of these contained 100 |il medium with 1-5 x 104 cells suitable for the cultivation of the respective virus (Table 1). The micro titre plate was covered, and incubated at 37°C until the virus control showed a cytopathogenic effect (CPE). 2.7.2. Virus titration, CPE, reduction factor Determination of the virus content was done by end-point titration (micro titre plate, four- or eightfold preparation). Regarding the procedure of titration or the methodology of the reduction factor calculation, see the specifications of the publication "Requirements of validation studies as evidence of the virus

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Table 2. Criteria for the determination of the cytopathogenic effect (CPE). Virus

CPE

HIV-2 BVDV PRV HAV PV-1 PPV, BPV

Syncytia formation Formation of vacuoles, granulation of the cytoplasm Giant cells, polycaryocytes Formation of vacuoles, cell degeneration, no cell lysis Rounding off of cells, decentralisation of the nucleus Plaque formation, cell lysis

safety of drugs from human blood or plasma" (Paul-EhrlichInstitute and Bundesinstitut filr Arzneimittel und Medizinprodukte, 1994). Viruses can cause different forms of the CPE (cell lysis, cell fusion, inclusion bodies, syncytia formation, transformation, etc.). Table 2 gives an overview of the CPEs relevant to the present study. The cytopathogenic effects were observed over several days by means of inverse transmitted light microscopy, always from the same investigator, while a second person read and confirmed them before the concluding evaluation. When toxic effects occurred in the supernatant examined, the appropriate suspension dilution was assessed as "inactivated" in the sense of a detection limit. A quantitative measurement of the virus reduction, using the decrease of the virus genome in the experimental preparation, is problematic due to false positive results which are to be expected (cross-reactivity with genome from blood cells or bone tissue cells) as well as the missing information regarding infectiosity (Willkommen, 2001). The titre reduction indicates the degree of virus inactivation. The titre was given as the virus dilution when 50% of the cell culture showed a cytopathogenic effect (TCID50 = tissue culture infectious dose 50%). The titre was calculated according to Reed and Munch (1938) a n d / o r Spearman and Karber (1974).

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3. Results 3.1. PES m o d e l 3.1.1. Suspension test (kinetics) Using the PES treatment in the suspension tests, it could be shown that most viruses (PV-1, BVDV, HIV-2, PRV) were inactivated under the level of detection as early as after an exposure time of five minutes. In the experimental design described, the reduction for these viruses was associated with a depletion by more than 4 logio gradates. Due to the high toxicity of PES on the HIV-2 cell system, as well as the relatively low starting titre, the exact reduction factor (R,) could not be determined accurately. HAV and PPV showed higher resistance against PES, and even after four hours HAV was not completely inactivated. Here the inactivating kinetics showed a biphasic course, i.e., a rapid initial inactivation by approx. 2.6 log 10 after the first five minutes was followed by a depletion by around 1 logio gradates over the following four hours. Therefore the reduction factor was R = 3.4 logio after 1 hour, and R = 3.7 logio after four hours. The kinetics for PPV also showed a rapid Table 3. Inactivation kinetics of the viruses in the suspension test. Virus concentration (log TCID.50/ml)

Exposure time (min)

PV-1

BVDV

HIV-2

PRV

HAV

PPV

0 5 10 20 30 60 120 240

7.76 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9

6.25 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9

4.44 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9

6.38 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9 <1.9

6.5 3.9 3.7 3.3 3.3 3.1 2.7 2.8

6.2 3.5 3.5 3.5 3.3 3.1 2.12 <1.9

5.86

4.35

2.54

4.48

3.7

4.3

R{

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inactivation after five minutes, but achieved a reduction of more than 4 logio gradates after four hours. The results for all virus suspension experiments are represented in Table 3. 3.1.2. Carrier test (spongiosa cuboids) — Supernatants In correlation with the suspension experiments the results show that the PES-ethanol procedure causes an efficient depletion of the viruses investigated in the supernatants (SI-S3). With the exception of HAV (Rf. 3.22 log10) all other viruses were inactivated under the level of detection. Because of the high toxicity of peracetic acid, this level was very high (< 2.11 logio) for PRV, PV-1, PPV and BVDV, and was also not sufficiently reduced by adding sodium thiosulfate (1/1 v/v). Virus titres below this value were not detectable. Nevertheless, for the viruses mentioned, a depletion could be achieved by more than 4 log 10 steps. In the cell systems for HIV-2 the PES toxicity was even higher (HIV-2: 3.55 logio), which led, together with

Table 4. Virus titres in the supernatants of contaminated spongiosa cuboids. Virus titre of s u p e r n a t a n t s

(TCID 5 o/ml)

Virus

SI

S2

S3

R (logio)

PRV PV-1 PPV HAV BVDV HIV-2

<2.11 £2.11 <2.11 2.83 <2.11 <3.55

6.17* 8.38* 6.32* 6.05* 6.25* 2.81

6.23 8.53 6.36 6.41 6.25 3.72

>4.06 >6.27 >4.21 3.22 >4.14 —

SI: PES treatment, negative pressure. S2: medium, negative pressure. S3: medium, normal pressure. *These values were used for the determination of the reduction factor Rj.

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spontaneous inactivation of this virus, to the fact that no reduction factor could be indicated. An overview of the results of virus titration in the supernatants is shown in Table 4. The effect of the negative pressure on the positive controls (S2, S3) was not significant. 3.1.3. Carrier test (spongiosa cuboids) — Homogenates After decanting the supernatants, the PES- or medium-treated cuboids were homogenised under addition of 10 mL sodium thiosulfate 1%. Then, the supernatants of the centrifuged homogenates were titrated. In these experiments no active virus was detected any more, with the exception of HAV. For HAV the reduction factor was 2.87 logi 0 ; in all other cases about 4 logio or more were achieved. HIV-2 was also completely inactivated; however, spontaneous inactivation of the positive controls (H2, H3) led to a severely lowered reference titre. Thus no reduction factor could be indicated here either. The toxic effect of PES on the cells could be clearly reduced by adding sodium thiosulfate. The recovery rate for the FACS calibration particles (as virus Table 5. Virus titres of the spongiosa homogenates.

Virus

Titre of virus stocks

PRV PV-1 PPV HAV BVDV HIV-2

6.38 7.76 6.14 6.53 6.25 4.44

Virus titre of spongiosa homog enates (TCID 50 /ml) HI

H2

H3

Ri (logio)

<0.69 <0.69 <0.69 3.00 <0.69 <0.69

4.87* 6.38 6.17 5.87* 4.80* 2.38

5.65 6.26* 5.65* 6.14 4.80* 2.27

<4.19 <5.57 <4.96 2.87 <4.11 -

HI: PES treatment, negative pressure. H2: medium, negative pressure. H3: medium, normal pressure. 'These values were used for the determination of the reduction factor R;.

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models) in the centre of the bone cuboid (penetration model) was determined by flow cytometric analysis and calculations of the concentration. This rate amounted to 79.6 % (Pruss et ah, 2001). An overview of the virus titres in the homogenates is shown in Table 5. 3.2. Irradiation m o d e l 3.2.1. Gamma ray dose distribution Validation of the dose distribution in the Styrofoam box used for irradiation in the production process of diaphyses, showed that the values in and on the phantoms, as well as in and on the box, varied within a range of approximately 3% (Table 6). From the Table 6. Dose distribution in and on the Styrofoam box used for irradiation experiments.

Sample number

Mass of sample (g)

Phantom 1 Phantom 2 Phantom 3 Phantom 5 Diaphysis 4 Diaphysis 6 Mean value

152 151 100 147 185 170

Sample centre

Sample surface

30.7 30.1 31.1 30.2 n.t. n.t. 30.5

30.9 30.9 32.6 31.2 25.8" 19.7** 31.4

Dose on the surface of the plastic vessel: mean value 31.6 kGy. Dose on the box surface: mean value 31.1 kGy (98% of the mean value of the sample centre). 'Given are the mean values of the measurements at the different measure points. **The values determined at the surface of the diaphyses showed a lower dose than expected from the values measured at the surface of the phantoms, which could be a result of solving of alanine from the thin-film dosimeter (water evaporated from the diaphyses). n.t., not tested

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results obtained by the dose distribution experiments it was possible to calculate the effective dose in the internal cavity of the diaphyses to be approximately 98% of that on the surface of the box. This value was within the range of variation determined at the different measuring points. From these results it seemed feasible to determine the irradiation dose during the inactivation experiments on the surface of the box via dosimeter. 3.2.2. Suspension test (kinetics) The virus inactivation kinetics were performed in frozen suspensions (-30 ± 5°C), using an experimental design as close as possible to that used in the production process. The titre of the virus suspension used as transport and incubation control served as reference. As expected, a linear relationship between the reduction factor and the irradiation dose was observed. From the regression curve the Dw values for the different viruses were calculated (Table 7). BVDV showed the highest sensitivity (D10 value < 3 kGy) to irradiation, whereas BPV was the most resistant virus (D10 value 7.3 kGy). From the Dw values the doses necessary to reduce virus titres by 4 logio and 6 logio were calculated (Table 7).

Table 7. Dw values and calculated doses to reduce virus titres by 4 logio or 6 logio, respectively.

Virus

Dio-value* (kGy)

4 logio Reduction (kGy)

6 logio Reduction (kGy)

BVDV PRV HAV HIV-2 PV-1 BPV

<3.0 5.3 5.3 7.1 7.1 7.3

<12.0 21.2 21.2 28.4 28.4 29.2

<18.0 31.8 31.8 42.6 42.6 43.8

T h e D 10 values were calculated for frozen virus suspensions in plastic tubes.

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3.2.3. Carrier test (diaphyses) Data of inactivation kinetics studies with frozen virus suspensions, suggested that for most of the viruses investigated, a reduction factor of > 4 logio can be achieved using an irradiation dose of approximately 30 kGy. The reduction factors obtained for virus suspensions were verified in a model system using viruscontaminated diaphyses. At least two independent experiments were performed for each of the viruses (Table 8). In general, the reduction factors obtained, corresponded to those calculated from the D 10 values (Table 8). With the model system of contaminated Table 8. Comparison of experimental data for the inactivation of different viruses in experimentally contaminated diaphyses with the calculated Dw values. Reduction factor (logio) determined Virus

Dose (kGy)

experimentally

Reduction factor (logio) calculated from D 10 '

BPV BPV BPV BPV BPV BPV

33.7 35.4 31.3 33.9 31.3 33.9

3.7 4.8 3.1 4.1 3.7 4.1

4.7 4.9 4.4 4.7 4.4 4.7

PRV PRV

30.6 35.4

>4.4 >5.5

5.9 6.8

PV-1 PV-1

30.6 35.4

5.9 >8.1

5.8 6.8

BVDV BVDV

33.7 35.4

>6.5 >5.6

>11.2 >11.8

HAV HAV

33.7 35.4

>7.2 >7.7

7.2 7.5

HIV-2 HIV-2

33.7 35.4

>4.1 >4.0

6.3 6.7

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diaphyses it was also shown that parvoviruses were the most resistant viruses regarding irradiation, with reduction factors of approx. 4 log 10 when a dose of 34 kGy was used. 3.3. Thermal treatment model 3.3.1. Core temperature measurements It could be demonstrated using decartilaged femoral heads with a diameter of up to 56 mm, that the temperature of 82.5°C necessary for virus inactivation could be achieved without difficulty. For femoral heads of a diameter of 60 mm or larger, a sufficient duration (> 15 minutes) of exposure to the effective temperature is no longer given. Since the virus-inactivating investigations were performed with femoral heads with a diameter of 55 + 1 mm, the results of the core temperature measurements are also valid in the model. As expected, the polypropylene tube used in the virus model had a reducing effect on the level and duration of the maximum temperature (1-3°C) compared to the real-life conditions. However, in all virus model experiments with all femoral heads examined (diameter ranging between 51 and 56 mm) the specification of 82.5°C over at least 15 minutes, was also achieved. To that extent the reduction factors determined in the virus model correspond to the real-life conditions of the thermal disinfection of femoral heads. 3.3.2. Carrier test (femoral heads) For each experiment two femoral heads with an identical diameter (54-56 mm) were prepared. Both were spiked with a given virus suspension, then one femoral head was submitted to the disinfection procedure in the Lobator, and the other was incubated at room temperature (RT) and at 4°C, respectively, as controls to determine the influence of the bone material and storage conditions on the virus inactivation. Three independent experiments were performed for each virus. In two experiments

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Table 9. Virus inactivation e x p e r i m e n t s in the Lobator sd-2 (Lsd-2) w i t h a control at r o o m t e m p e r a t u r e (RT), values are given as logi 0 .

Virus BPV BPV BPV PRV PRV PV-1 PV-1 HIV-2 HIV-2 BVDV BVDV HAV HAV HAV

Reduction Reduction Starting Virus titer Virus titer factor factor titer 94 min/RT 94 min/Lsd-2 94 min/RT 94 min/Lsd-2 (TCIDso/ml) (TCIDso/ml) (TCID 50 /ml) (TCID 50 /ml) (TCID 50 /ml) 8.49 8.25 7.49 8.00 8.37 10.38 9.87 5.55 5.00 6.74 6.62 7.40 6.50 7.90

4.74 5.74 3.74 4.49 6.25 7.87 7.37 <1.49 <1.49 5.49 5.25 4.88 5.50 6.50

>2.49 >2.49 >1.49 >2.49 >1.49 n.e.p. n.e.p. <1.49 <1.49 >2.49 >2.49 >1.49 >2.49 >2.49

3.75 2.51 3.75 3.51 2.12 2.51 2.50 >4.06 >3.51 1.25 1.37 2.52 1.00 1.40

>6.00 >5.76 >6.00 >5.51 >6.88 n.e.p. n.e.p. >4.06 >3.51 >4.25 >4.13 >5.91 >4.01 >5.41

n.e.p.: no evaluation possible TCID5[)/ml was calculated according to Spearman and Karber.

Table 10. Virus inactivation e x p e r i m e n t s in the Lobator sd-2 (Lsd-2) w i t h a control at 4°C, v a l u e s are given as logm.

Virus

Starting titer (TCIDso/ml)

BPV HIV-2 PRV PV-1 BVDV HAV

5.75 7.00 8.50 8.00 6.00 8.00

Reduction Reduction factor Virus titer Virus titer factor 94 min/4°C 94 min/Lsd-2 94 min/4°C 94 min/Lsd-2 (TCIDso/ml) (TCIDso/ml) (TCIDso/ml) (TCIDso/ml) 6.00 5.75 7.50 8.00 6.25 7.49

<1.49 <1.49 <1.49 <1.49 <1.49 <1.49

None 1.25 1.00 None None 0.51

>4.26 >5.51 >7.01 >6.51 >4.51 >6.51

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the incubation of the controls was performed at room temperature (20-25°C, see Table 9). In a third experiment the incubation of the controls was done at 4°C (see Table 10). The inactivation studies showed that in the disinfection device all viruses were inactivated below the level of detection, i.e., the application of the Lobator sd-2 program led to a virus reduction of more than 4 logi 0 in the core of human femoral heads. The controls incubated in parallel at room temperature showed very high spontaneous inactivation, particularly for HIV-2. When the controls were incubated at 4°C, no or only a slight effect on the virus titers were observed. In some of the experiments — probably depending on the biological and structural properties of the femoral head used — toxic effects of the suspension on the indicator cells were observed after incubation for 94 min in the Lobator sd-2 system. As summarised in Table 9, this resulted in a higher detection limit (TCID50 given as log 10 ), i.e., < 2.49 (BPV, PRV, BVDV, HAV), respectively. Furthermore, in the PV-1 inactivation experiments, the virus suspensions as well as the homogenates of the bone cylinders had a toxic effect on cells, and in these suspensions virus titers could not be determined (Table 9). In no other experiment in the Lobator sd-2 were toxic effects observed. The level of virus detection could be calculated as low as < 1.49 TCID50. Considering the results obtained in the three independent experiments, it can be concluded that the incubation temperature of the controls (RT versus 4°C) has a significant effect on the spontaneous loss of infectivity. Furthermore, toxic effects observed with individual bone preparations, hamper the determination of the reduction factor. 4. Discussion In the studies presented, we verified the inactivating capacity of the internationally most frequently used sterilisation or disinfection procedures (gamma irradiation, thermodisinfection,

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peracetic acid-ethanol treatment) of human bone transplants which were contaminated with clinically relevant viruses or their model viruses. It could be documented in suspension experiments that a number of viruses (PRV, PV-1, BVDV) are inactivated within a very short time by the peracetic acid-ethanol mixture. Here a reduction factor of > 4 log 10 is already achieved after five minutes of treatment. HIV-2 is also inactivated within five minutes under the level of detection; however no actual reduction factor could be determined because of the low control titres (spontaneous inactivation of the virus). For PPV the reduction titre of > 4 log 10 was only achieved much later. The data reported could be confirmed in the carrier test in every regard. The relatively high resistance of HAV against peracetic acid was unexpected. The subsequent validation of a further process step, the chloroform/ methanol defatting of spongiosa cuboids which had been contaminated with HAV-infected cells, demonstrated a reduction of the HAV titre by 7.0 log 10 (Pruss et al., 1999) which has to be attributed in particular to the inprocess-flushings (8 methanol flushings in an ultrasonic bath for 15 min. each). Limited and controversial results have been reported for the irradiation dose required to effectively inactivate viruses in bone tissue. The relatively broad spectrum of Dw values determined for HIV-1 (4-8.8 kGy) (Conway et al, 1991; Campbell and Li, 1999) and poliovirus (1.9-5 kGy) (Mahnel et al, 1980) raised the question of whether environmental factors such as protein concentration or ions, might influence the sensitivity of viruses to gamma irradiation. In the inactivation kinetics studies as well as in the model experiment using contaminated diaphyses, bovine parvovirus showed the highest resistance to irradiation (D10 of 7.3 kGy). It was unexpected that BVDV as a model for HCV revealed the highest sensitivity with respect to gamma irradiation (D10 of < 3 kGy). All other viruses including HIV-2 showed Dio values around 5-7 kGy.

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Combining the results of the virus-inactivation kinetics and of the diaphysis model system, a reduction factor of 4 logio is obtained for parvovirus using a dose of approximately 34 kGy. Parvovirus was revealed to be the virus with the lowest sensitivity to gamma irradiation (small single-stranded DNA genome); all other viruses showed a lower D 10 value, indicating a higher sensitivity to gamma irradiation. Estimations on the doses of gamma irradiation necessary to inactivate HIV and HCV in bone tissue, are in agreement with the results obtained in our investigations. Conrad et al. (1995) calculated that a dose of 17 kGy is sufficient to completely inactivate Hepatitis C virus in allografts. From our own and from published results, a dose of 34 kGy can be recommended for the sterilisation of bone allografts to achieve a high level of virus safety. This recommendation is only given for temperatures of approximately -30°C, because virus infectivity is significantly influenced by the temperature prevalent during irradiation. With increased irradiation temperature, a lower dose is necessary for the same log reduction; that is, the same dose leads to a higher inactivation factor (Hernigou et al., 2000). Furthermore, Tosello (1995) showed that doses between 25 and 50 kGy did not significantly alter the biomechanical characteristics of bone. Thermal treatment of femoral head grafts with the lobator sd-2, which has also been called "Marburg bone bank system" was examined in detail by Knaepler et al. (1994). The temporal and spatial expansion of femoral heads of different sizes and density served as a basis for the practical application of the thermal treatment, which took place in a water bath that was heated up within 30 minutes to 80°C. Also, the exposure time was determined, which was necessary to achieve this temperature for at least 10 minutes in the core of very large femoral heads. Current findings from investigations by the CLB (Central Laboratory of the Netherlands Transfusion Service, Department Clinical Viro-Immunology) regarding BVDV and CPV (canine

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parvovirus) inactivation led to an increase in the safety factor when defining the effective temperature/time function (82.5°C/ 15 minutes; CLB, 1996a; 1996b). Various authors were concerned with the question of thermal instability of the human immunodeficiency virus. This virus is considered as relevant for the safety of bone transplantation, and was classified as weakly resistant to physicochemical treatment. It is well known that HIV is unstable under the influence of heat, and is completely inactivated at 60°C (Einarsson et al, 1989; Evengard et al, 1985; Gleeson et al, 1990). The validation of the Lobator system regarding a sufficient inactivation of HIV, was published in a preceding study (von Garrel et al, 1997). A validation of a virusinactivating procedure ought to guarantee a direct contact of the viruses investigated with the core of the femoral head, since in this way it is possible to approximate the natural conditions of a contaminated femoral head. In addition, it must be mentioned that the application of moist heat (as in the lobator system) has advantages for virus inactivation (Brauniger et al., 1994; 2000). The results of the investigations reported here can be said to document a sufficient depletion (4 logio gradates) of clinically relevant viruses in the thermal disinfection treatment with the lsd-2 procedure. In consideration of physiological fluctuations in morphology and fat content, a transverse diameter (of the femoral head to be treated) of < 56 mm is recommended. All three sterilisation methods investigated are recommended for bone graft sterilisation, provided that additional safety measures are taken, like general and specific anamnestic information (donor criteria by EATB/EAMST and AATB); clinical examination of the donor; infectious serology (Anti-HIV-l/2/gO, HBsAg, Anti-HBc, Anti-HCV, TPHA) and, in the case of multiorgan donors, tests for HIV, HBV and HCV genome (PCR test). The recommendations apply only when the following prerequisites are fulfilled: • Peracetic acid-ethanol treatment: maximal transplant thickness 15 mm, spongiosa defatted.

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PRUSS, A. (b), HANSEN, A., KAO, M., GURTLER, L., PAULI, G., VERSEN, R. VON (2001). Comparison of the Efficacy of Virus Inactivation Methods in Allogeneic Avital Bone Tissue Transplants, Cell Tissue Banking 2, 201-215 REED, L.J. and MUNCH, H. (1938). A simple method for estimating fifty percent endpoints, Am. J. Hyg. 27, 493-497. ROCK, M.G. (1991). Biomechanics of allografts. In: Orthopedic Allograft Survey, A.A. Czitrom and H. Winkler, eds., Springer Wien, New York, pp. 29-37. SAUTIN, E.N. (1963). Sterilisation of bony tissue by Co 60 gamma rays, Radiobiology 3, 621-625. SCHEIDLER, A., ROKOS, K., REUTER, T., EBERMANN, R. and PAULI, G. (1998). Inactivation of viruses by beta-propiolactone in human cryo-poor plasma and IgG concentrates, Biologicals 26, 135-144. SPEARMAN, A. and KARBER, G. (1974). In: Biometrie. Grundzuge biologisch-medizinischer Statistik, L. Cavalli-Sforza, ed., Gustav Fischer-Verlag, Stuttgart, pp. 171-173. SPROSSIG, M. and MUCKE, H. (1969). Die Virusdesinfektion durch Peressigsaure in Gegenwart von Alkoholen, Wiss. Z. Humboldt. Univ. Math. Nat. R 18, 1171-1173. STARKE, R. and VERSEN, R. VON (1984). Experimentelle Untersuchungen zur Entkeimung von Transplantationsmaterial mit Peressigsaure, Z. Exp. Chir. Transplant Kiinstl. Organe. 17, 254258. THIELICKE, U., THIELICKE, B., VERSEN, R. VON and DENNER, K. (1990). Klinische Studie zum Einsatz von demineralisierter Knochenmatrix (DBM) in der Chirurgischen Stomatologie. [Clinical study on the application of demineralised bone matrix (DBM) in surgical orthodontics], Beitr. Orthop. Traumatol. 37, 461-465.

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THOREN, K. and ASPENBERG, P. (1995). Ethylene oxide impairs allograft incorporation in a conduction chamber, Clin. Orthop. 318, 259-264. THOREN, K., ASPENBERG, P. and THORNGREN, K.G. (1995). Lipid extracted bank bone: Bone conductive and mechanical properties, Clin. Orthop. 311, 232-246. TOSELLO, A. (1995). Conditions optimales d'irradiation de type gamma en vue de l'inactivation du VIH present dans les fragments osseux. Consequences sur la resistance biomecaniques du tissu osseux. [Optimal conditions of gamma type irradiation for inactivating HIV in bone fragments. Consequences in biomechanical resistance of the bone tissue.] Chirurgie 120, 104-106. VON GARREL, T., KNAEPLER, H. and GURTLER, L. (1997). Untersuchungen zur Inaktivierung von HIV-1 in humanen Femurkopfen durch Verwendung eines thermischen Desinfektionssystems Lobator SD-1, Unfallchirurg 100, 375-381. VON VERSEN, R., HEIDER, H., KLEEMANN, I., STARKE, R. (1992). Chemische Sterilisation biologischer Implantate mit einer Kombinationsmethode. In: Osteologie Aktuell, H.J. Pesch, H. Stofi and B. Kummer (Hrsg.), eds., Springer-Verlag, VII, Suppl., pp. 380-386. WILLKOMMEN, H. (2001). Erfahrungen mit der Praxis von Infektionsassays und Modellviren fur die Beurteilung der Sicherheit von Blutprodukten fur den Patienten. In: Nosokomiale Virusinfektion — Erkennung und Bekampfung, H.F. Rabenau, O. Thraenhart and H.W. Doerr (Hrsg.), eds., Pabst Science Publishers, Lengerich, pp. 166-173. WUTZLER, P. and SAUERBREI, A. (2001). Peressigsaure-Ethanol — Ein potentielles viruzides Handedesinfektionsmittel. In: Nosokomiale Virusinfektionen — Erkennung und Bekampfung, H.F.

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Rabenau, O. Thraenhart and H.W. Doerr (Hrsg.), eds., Pabst Science Publishers, Lengerich, pp. 92-100. ZHANG, Q., CORNU, O. and DELLOYE, C. (1997). Ethylene oxide does not extinguish the osteoinductive capacity of demineralised bone. A reappraisal in rats, Acta Orthop. Scand. 68, 104-108.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

7 METHODS OF CULTURING, PROBLEMS ASSOCIATED WITH BACTERIOSTASIS AND RADIATION STERILISATION OPTIONS

MARTELL WINTERS Bioburden Section Leader, Nelson Laboratories, Inc. 6280 South R e d w o o d Road Salt Lake City, UT 82123

1. Introduction There is a growing concern regarding the sterility or microbiological quality of tissue used for transplantation in the US and other nations. The discussion here is to review the existing culturing methods in this industry, and offer information which may be useful, using a background in radiation sterilisation of medical devices and pharmaceuticals. 2. Topics of D i s c u s s i o n 2.1. Culturing methods Currently many methods are being used in this industry to screen for micro-organisms. Some examples of these methods are direct sterility testing of the tissue (results are positive or negative for growth), direct bioburden testing of the tissue (enumeration of 193

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organisms from non-sterile product), and swabbing followed by either sterility testing or enumeration. 2.2. Sterility assurance Many tissue products are being released for transplantation with little or no assurance of the degree of sterility when compared to devices or pharmaceuticals. 2.3. Tissue bank standards — Radiation sterilisation Currently for sterilisation, the AATB standards recommend either a method from the Association for the Advancement of Medical Instrumentation (AAMI) guidelines, or 10% destructive sterility testing, or 100% swab testing. They add that if the 10% destructive sterility test option is chosen, it must also include at least 2 Bis of Bacillus pumilis at 103 colony forming units (CFU) in the irradiation load in the most challenged area. Lastly, the dose used for sterilisation must be at least 15 kGy. 2.4. Food and drug administration (FDA) The FDA is demonstrating greater concern regarding the sterility assurance of tissue products, although no standard methods have been published to demonstrate sterility assurance of tissue. Tissue banks should expect heightened inspections from the FDA related to sterilisation and sterility assurance of their tissue. This increases the need for appropriate guidance from both standards-writing bodies and government agencies. 3. Discussion on Culturing Methods A great deal of effort and concern are placed into the general microbiological screening of tissue. The primary methods used are swabbing and destructive sterility testing (direct immersion).

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There are two variables associated with bioburden or sterility determination using the swabbing technique: 1) The ability of the swab to remove organisms from the product; and 2) The ability to remove the organisms from the swab for testing. These two variables can drastically impact the results of a test, so it is critical that they be accounted for. Both variables are related to the ability to remove organisms from a product, so they will be addressed together. 3.1. R e m o v i n g organisms from a product One cannot assume that a single swab or rinse of a product removes 100% of the organisms present. To obtain accurate results, a validation must be performed to determine the method's efficiency in removing organisms from the product. The accuracy of a bioburden or sterility test, therefore, hinges completely on this demonstrated efficiency. The national and international standards regarding determination of bioburden on a product (ANSI/A AMI/ISO 11737-11995) describe what is called a recovery efficiency. The recovery efficiency is a correction factor which is used to adjust the bioburden to account for the fact that not all of the organisms are being removed during the test. The recovery efficiency is determined using one of two methods: 1) exhaustive rinse; and 2) inoculated product. 3.2. Exhaustive rinse recovery efficiency validation In the exhaustive rinse method the organism removal process (the extraction) is performed on the same product unit several times, usually four to six times, and bioburden is determined for each extraction. A recovery efficiency ratio is determined by dividing the results of the first extraction by the total results of all of the extractions. Note that this method uses the bioburden which is already on the product, not an artificially added bioburden. An example is shown below:

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First extraction bioburden count: Second extraction bioburden count: Third extraction bioburden count: Fourth extraction bioburden count:

25 CFU 9 CFU 2 CFU 0 CFU

Recovery Efficiency:

25/36 = 0.69 or 69%

This data indicates that bioburden results determined using a single extraction must be adjusted to account for the fact that only 69% of the organisms are removed on the first extraction. Thus, with a bioburden count average of 20 CFU, the bioburden is adjusted using the following calculation: 20 CFU/ 0.69 = 29 CFU In this case, the adjusted bioburden on the product is actually 29 CFU, not 20. Obviously this is not a great increase in count, but in cases of lower recovery efficiency percentages, it will make a greater difference. In fact, the results of the recovery efficiency validation may indicate that the extraction method is completely invalid due to the inability of the method to effectively remove organisms from the product. In these cases, another extraction method should be used. For a recovery efficiency validation, at least three to five non-sterile product units are tested, and the average recovery efficiency percentage of the group is used as the validated recovery efficiency. It is often appropriate that the products which have been used for this validation are placed into nutrient agar or soy broth after the final extraction, to determine if there are still organisms present which were not removed. In the example above, the count of 0 on the last extraction does not indicate that there are no longer any organisms on the product — just that the extraction method is no longer removing organisms. This final step of immersing the product in agar or broth can verify the absence of organisms on the product after the final extraction. The benefit of using this method to determine the recovery efficiency is that since the bioburden on the product is natural,

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not artificial, it is more representative of a typical product bioburden. The disadvantage is that products with naturally low bioburden (e.g. 1 or 2 CFU per product) do not give good results. 3.3. Inoculated product recovery efficiency validation The second method of determining the recovery efficiency is the inoculated product method. This method is used in cases where there is little or no natural bioburden on the product. Organisms are artificially added to the product, allowed to dry or absorb, then tested using the extraction method. Usually only one extraction is performed (not multiple extractions as in Sec. 3.2) and the results are compared to the number of organisms which were added to the product. An example is shown below: Number of organisms inoculated onto product: 150 Number of organisms recovered on the first extraction: 115 Recovery efficiency:

115/150 = 0.77 or 77%

This method works best when the product is inoculated with spores rather than vegetative organisms, to eliminate the variable of the organisms dying as they dry (which some vegetative organisms are prone to do). Here also, three to five product units are used for the validation. The bioburden is corrected with this recovery efficiency just as is done with the exhaustive rinse recovery efficiency described in the previous section. The benefit of the inoculated product method is that there is always a high number of organisms on the product because the laboratory has control over how much inoculum is used. In cases of low natural bioburden, there is often no choice but to use this method. The disadvantage is that the organisms are artificially added, so it is likely not representative of the location, environment, adherence factors, and types of naturally-occurring organisms on the product.

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3.4. Important areas of concern — S w a b b i n g to determine bioburden counts COUNTS: This is common practice in the tissue industry. A product is swabbed, and the swab is placed into a vial of solution. The vial containing the solution and swab is agitated to remove the organisms (or sometimes there is no agitation, it is left on a table for a certain period of time), then the solution is tested and the swab discarded. In these situations, two recovery efficiencies should be determined and used to correct the results because there is an uncertainty associated with both removing the organisms from the product, and removing the organisms from the swab. In these cases, failure to adjust bioburden counts with recovery efficiencies could easily result in the bioburden counts being grossly underestimated (underestimations of greater than one log are often seen in these situations). There are swabs available which do not dissolve in water, but which do dissolve in 1% sodium citrate. After the swabbing, the swab is immersed in 1% sodium citrate and agitated until the swab dissolves; then the sodium citrate is assayed. Using these swabs can eliminate some of the underestimation which occurs. 3.5. Important areas of concern — S w a b b i n g to determine product sterility STERILITY: This is also a common practice in the tissue industry. The same uncertainties which apply to bioburden determination also apply here, but with much greater concern. A sterility test must be a very sensitive test if it is meant to verify the sterility of a product. In a bioburden test, not detecting one organism which is present on the product causes little concern. However, in a sterility test, not detecting one organism on a product (a false negative test) can mean the difference between passing and failing, or life and death in some cases. This problem is

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magnified when, as mentioned above, the swab is extracted and the extract tested for sterility rather than the swab itself. In any case, when swabbing is used for product sterility testing, the uncertainties associated with having correction factors (recovery efficiencies) will very often completely invalidate the sterility test results due to the lack of sensitivity. Recovery efficiency data would demonstrate that, using this method, either more product units have to be tested, or the product contamination levels have to be very high before they can be detected. These concerns are reduced when using the dissolving swabs, but only slightly. Following is an example of this situation. With a recovery efficiency value of 20% for removing the organisms from the product (which can be common), and a value of 40% for removing the organisms from the swab, the cumulative recovery efficiency is 8%. This means that there is an 8% chance of finding an organism which is on a product. Obviously, this is not acceptable for a sterility test. These concerns can be greatly reduced in both bioburden and sterility testing by using the methods below. These methods will work in most situations, but may not work in all. 3.5.1. Recommendations: Bioburden — Immersion and extraction In this method the products are immersed in a sterile fluid which usually contains a surfactant and a buffer or peptone. An extraction method is used — usually a manual or mechanical shake (orbital shaking or stomaching) — and the fluid is tested, usually by filtration. This method still requires the determination of a recovery efficiency, but by direct immersion rather than swabbing, the sensitivity is increased. This method is best for solid tissues such as bone, or tissues which are still in large pieces. It may also be possible to perform a validation which would allow for transplantation of the product after testing, just as is done when swabbing is used.

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3.5.2. Recommendations: Bioburden — Most Probable Number (MPN) In this method the non-sterile products are immersed in a sterile nutrient broth and incubated. A simple calculation is performed which is based on the number of product units which demonstrate growth after incubation. It is identical to a sterility test, but is performed on a non-sterile product. The product bioburden must be low enough that occasionally there are no viable microorganisms on a product after processing. Since most tissue products tend to be very low in bioburden, this method works well, and is much improved over swabs or extraction, due to the increased sensitivity. When the MPN method is used for bioburden, no recovery efficiency validation needs to be performed, because there is no need to remove the organisms from the product for testing. One disadvantage is that the product is unusable after testing. 3.5.3. Recommendations: Sterility — Direct immersion This method is less frequently used because the product is unusable after testing. However, there can be no doubt that it is the most sensitive of the sterility tests, and is the only method which can truly demonstrate sterility (or at least as close as possible). Following are some testing/validation options which may make using direct immersion sterility testing a more plausible option due to the reduced quantity of product units needed. 3.6. Discussion on sterility assurance / tissue bank standards and radiation sterilisation As mentioned above, some options for demonstrating sterility are related to performing either swab testing or direct immersion sterility testing after irradiation. Unfortunately, a 10% destructive sterility test after irradiation says nothing regarding the sterility

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of the other 90% unless a validation is in place which can justify that link. Similarly, a 100% swab test, with its recovery efficiency downfalls, cannot demonstrate sterility. The following statement applies here and can be demonstrated to be true: there is never a scientific basis for routine post-sterilisation product sterility testing. In defence of this comment is the following explanation. The degree of sterility of a particular product is usually described in terms of sterility assurance level (SAL). The SAL describes the likelihood of having a non-sterile product. Most medical devices are sterilised to an SAL of 10~6, which is a 1 in 1,000,000 chance of having a non-sterile product. If a sterility test is performed on 10% of a batch, and it proves to be sterile at 15 kGy, how sterile is it and what does it mean for the rest of the batch? If the 10 product units were non-sterile until they received 14.9 kGy, and were finally sterile at 15.0 kGy, it is likely that, of the next 10 product units tested, there will be one positive or one non-sterile unit. This corresponds to an SAL of about 10 _1 , or 1 in 10 non-sterile. However, if your 10 product units became sterile at 2 kGy (which is entirely possible), it is likely that you have a 13 log overkill, which is an SAL of 10"13 or 1 in 10,000,000,000,000 non-sterile. This means that a 15 kGy dose is often much more radiation than needed to provide an acceptable SAL. However, without data to indicate an SAL, the results of a sterility test of a few product units cannot be applied to others which are not tested. In summary, a sterilisation dose chosen because everyone else uses it, and 'validating' the appropriateness of the sterilisation dose by post-irradiation sterility testing, demonstrates nothing. Initial bioburden count and the resistance of that bioburden to radiation (D-value or Dw) is paramount in determining the sterility assurance of a product. Most D-values obtained in medical device testing range between 0.5 and 2.0 kGy. There is no reason to believe that it should be very different for the tissue industry. One of the organisms which causes great concern in the tissue industry is

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Clostridium. Tales have been told of its incredible radiation resistance which are not supported in the literature. Reported D-values from the Parenteral Drug Association (Geoffrey, 1994) range from 0.8 to 4.6 kGy, most of them in the 1 to 2 kGy range. In Disinfection, Sterilisation and Preservation (Block, 2001), ranges between 0.8 and 4.2 kGy are reported, again with most of them in the 1 to 2 kGy range. Studies performed at Nelson Laboratories confirm these reports. Using this general D-value information, a 15 kGy dose can provide from a 3 to a 15 log kill of Clostridium sporogenes. Mention is also made in the AATB standard, of testing two Bis of 103 Bacillus pumilis after the 15 kGy dose. Bacillus pumilis typically has a D-value of about 1.5 kGy. Simple math demonstrates that a BI of 103 Bacillus pumilis will be sterile at about 4.5 kGy (3 logs of kill times 1.5 kGy D-value). The requirement that the Bis be placed in the most-difficult-to-sterilise location is nullified by the fact that a contract irradiator will perform a dose map to assure that even the most-difficult-to-sterilise location will receive at least 15 kGy. With this in mind, every BI tested after 15 kGy has received an approximate 10 kGy overkill. This results in useless information related to the sterility of the product, unless a limit is set for product bioburden counts or radiation resistance. 3.7. The AAMtylSO guidelines The numbers used to derive the tables in the AAMI/ISO guidelines for setting sterilisation doses of radiation (e.g. 111371995 Method 1 or TIR 27 VDmax) are based on a group of organisms called Population C. This population of organisms came from cotton gauze in the 1970s. In the early 1980s, it was decided by those involved in the medical-device sterilisation industry that Population C was a good benchmark population which could be used for comparison. They believed that the bioburden of most products would demonstrate either similar resistance to radiation, or be less resistant to radiation. Thus,

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dosimetry tables were derived using this information, which is still present in all commonly used AAMI/ISO radiation methods. This population has been used in the medical device industry for about 20 years and has proven to be a good benchmark. One problem with the AAMI/ISO guidelines from a tissue bank perspective is that they frequently require too many product units, or they result in radiation doses which are too high. Some of the options below will address these issues. 4.

Options/Recommendations

Following are some options which may be helpful in the tissue industry. Most options are not discussed in great detail, but they are mentioned briefly for consideration; or to initiate discussion. Some options are already commonly used in the medical device and pharmaceutical industry, and some are new methods or variations of the existing methods. Keep in mind that every option will not apply or function for every tissue bank. 4.1. Process validation Process validation is sometimes performed in the tissue industry, but routine process monitoring seems to be more common. Routine process monitoring refers to the testing of a certain number of product units or percentage of every batch of product, regardless of the previous results. In process validation, the individual steps of the process are monitored, and their impact on the final product determined. Once a link has been validated between the process and the final product, routine process monitoring is performed in lieu of routine product monitoring. An occasional test may be performed on the product to substantiate continuing the link to the process monitoring. As more acceptable data from the process is accumulated, monitoring on a smaller scale can be substantiated. In the end, process validation demonstrates the effectiveness of

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the sterilisation process, and certain controls are monitored, but no post-sterilisation product sterility testing is performed. There is one variable in tissue banking that is likely not to be an issue for medical device or pharmaceutical companies: the consistency of the microbiological quality of the "raw material". Medical device or pharmaceutical companies can usually plan on the raw material being processed and originating from a consistent source, which is not the case with tissue banks. Although tissue may not come from a consistent source, many recovery sites are reliable in their removal, cleaning and monitoring of tissue which they release, which means that some degree of consistency can be determined. Sterilisation science is not meant to be an all-inclusive science that will always produce sterile product regardless of the amount of control in a system. It is dependant on some degree of control of the process and raw materials. With this fact in mind, process validation is possible in the tissue industry, and could be beneficial. In addition, tissue banks are concerned about how much product is used for testing verses transplantation. Certainly there is a moral obligation not to "waste" tissue, and to use as much as possible. Many of the following proposed methods will decrease the wasted tissue, lower the radiation sterilisation doses being used, increase the sensitivity of much of the testing and improve the sterility assurance level. Herein are radiation sterilisation options which could be used to assist in validating the process to the effect that testing of product may only need to be performed once every quarter. These options are merely ideas which can be very flexible if need be, and the total number of other options or similar options is limitless. 4.2. ANS1/AAM1/ISO 11137b: Method 1 This validation consists of a bioburden test of 10 product units from three batches (30 total), and a sterility test of 100 product

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units which have been dosed at a verification dose. The verification dose is based on the bioburden count, and is substantially lower than the sterilisation dose. The advantage of this method is that sterilisation doses as low as 14 kGy can be validated (at 10~6 SAL). The disadvantage is that 130 product units are used in testing. Upon completion of this validation, a dose audit should be performed every quarter to verify continued release of product at the current sterilisation dose. 4.3. Modified Method 1 This is similar to the Method 1 described above, but with some modification so that the sterility test is performed on 10 product units rather than 100. The table used in Method 1 can easily be adjusted to set a dose for 10 product units. This validation consists of a bioburden test of 10 product units from three batches (30 total), and a sterility test of 10 product units which have been dosed at a verification dose. The verification dose is based on the bioburden count, and is substantially lower than the sterilisation dose. The same advantage applies as with the Method 1, and the disadvantage is that this method is not yet fully developed for use. Upon completion of this validation, a dose audit should be performed every quarter to verify continued release of product at the current sterilisation dose. 4.4. ANS1/AAM1/ISO 11137b: Method 2A This validation does not require bioburden testing to establish the sterilisation dose. Instead, 20 product units from three batches (60 total for each dose) are irradiated at nine incremental doses starting at 2 kGy and ending at 18 kGy (increments of 2 kGy and a total of 540 product units). The product units are tested for sterility, and the results are used to indicate a verification dose which is applied to 100 product units which are also tested for sterility. The results of the second sterility test indicate the sterilisation dose. The advantage of this method is

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that sterilisation doses as low as 11 kGy can be validated (at 10"6 SAL), which is generally the lowest possible sterilisation dose that can be determined using natural bioburden. The disadvantage is that 640 product units are used in testing. Upon completion of this validation, a dose audit should be performed every quarter to verify continued release of product at the current sterilisation dose. 4.5. Modified Method 2 Considering the usual low bioburden counts on tissue, the Method 2 mentioned above could be scaled down to use only the lower doses in the initial testing (e.g. only testing at 2, 4, 6, and 8 kGy), then a 10 sample test used for the verification dose. The same advantage applies as with Method 2. The disadvantage is that 250 product units are used in testing, and that this method is not yet fully developed for use. Upon completion of this validation, a dose audit should be performed every quarter to verify continued release of product at the current sterilisation dose. 4.6. AAMI TIR 27 — V D M A X This validation consists of a bioburden test of 10 product units from three batches (30 total), and a sterility test of 10 product units which have been dosed at a verification dose. The verification dose is based on the bioburden count, and is substantially lower than the sterilisation dose. The advantage of this method is that only 40 product units are used in testing. The disadvantage is that the sterilisation dose is always 25 kGy (at 10" 6 SAL) regardless of the bioburden count. 4.7. Modified V D M A X This is similar to Method 1 described above, but with some modification so that the sterilisation doses are lower (e.g., 12, 15,

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18, and 20 kGy). An initial bioburden test of 10 product units determines which sterilisation dose is applicable. Ten product units are irradiated at a corresponding verification dose, then tested for sterility. The results of the sterility test indicate which sterilisation dose is applicable. This method is not yet fully developed for use. 4.8. Inoculated product sterilisation validation A worst-case organism could be determined, or a "benchmark organism" chosen (perhaps Clostridium) to be used in this type of validation. The desired SAL is chosen which would determine the initial titer to be used to inoculate the products. Based on the initial titer and the organism chosen, one or more doses are applied to 10 product units, which are then tested for sterility. Based on the results, a predetermined sterilisation dose would be applicable. The advantage of this method is that a consistent challenge organism is being used. The disadvantage is that it is not performed using the natural bioburden, so the sterilisation dose may be slightly higher than really needed. Also, this method is not yet fully developed for use. 4.9. Sterility assurance level requirements A new AAMI standard — ST 67 — will soon be published, which provides guidance on selecting an SAL for a product. This standard explains that if a product cannot withstand the sterilisation doses required to provide an SAL of 10"6, a less-stringent SAL can be used (i.e. 10"5, 10~4, or 10"3). Porcine heart valves are mentioned specifically as an example of a product which would apply to this situation. Considering that an SAL of 10"3 still provides at least a 1 in 1,000 chance of a living organism, it is better than nothing in cases where the product is radiation sensitive.

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4.10. Gamma verses electron beam There is data to support the possibility that some materials are less damaged due to oxidation or cross-linking with electron beam radiation, than they are with gamma. Also, since the electron beam dose is delivered in a matter of seconds rather than hours, it is easier to maintain products at lower temperatures during the entire irradiation process. This option may be a favorable one to consider if material degradation or temperature is critical. 4.11. Use of companion, rejected, or scrap product for testing Some tissue banks use these kinds of product for testing, rather than using an actual product in destructive tests. Even though a product may have been rejected, it still may be microbiologically representative of the other products, and thus be usable for some sterilisation-related testing. This is common practice in medicaldevice testing and is specifically allowed in the standard. 5. Problems Associated w i t h Bacteriostasis Relatively few problems with bacteriostasis have been demonstrated with tissue itself. It is possible that residual cleaning solutions or antibiotics can exhibit some bacteriostatic effects if present. The concern regarding bacteriostasis is that bioburden or sterility tests may show that the organism counts are low, when in reality they are high; but the organisms cannot grow due to some inhibitory agent on or in the product. Generally, bacteriostasis is determined using the standard United States Pharmacopeia method (e.g. USP 25, 2002), or something very similar to it. USP suggests inoculating the extract fluid from the product, or the growth medium in which the product is placed, with less than 100 CFU of three to six different organisms, depending on the option used. These

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organisms usually represent a variety of types based on kingdom, Gram stain, and colony morphology. After incubation, the filter or medium is observed for growth. Similar results must be demonstrated when comparing the sample growth to the control to verify the absence of bacteriostasis or fungistasis. In the tissue industry methods similar to, or exactly like these, are being employed. Some use different organisms than those suggested in USP, which may be appropriate considering the situation. 6. Summary As more becomes required of tissue banks, further guidance is necessary in the near future to ensure the success of tissue banks around the nation and world. This guidance can be drawn from similar industries which have already developed internationally accepted standards and guidelines, but most must be developed in AATB, FDA, or other bodies with jurisdiction. Regardless of its origin, this guidance is critical to the future success of the tissue bank industry. The author offers his time and resources to this development and will also be able to obtain additional help from sterilisation experts found in the AAMI ranks should the assistance be desired. This paper identifies several areas where initial improvement is needed in the standards to make the required testing more meaningful. Also, the included suggestions could be expounded into full methods or procedures. The author welcomes any questions, concerns or discussion related to anything mentioned herein. 7. References AAMI TIR 27-2001. Sterilisation of Health Care Products — Radiation Sterilisation Substantiation of 25 kGy as a Sterilisation Dose — Method VD-Max. Association for the Advancement of Medical Instrumentation, Arlington, VA.

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ANSI/AAMI/ISO 11137-1994. Sterilisation of Health Care Products — Requirements for Validation and Routine ControlRadiation Sterilisation. Association for the Advancement of Medical Instrumentation, Arlington, VA. ANSI/AAMI/ISO 11737-1-1995. Sterilisation of Medical Devices — Microbiological Methods, Part 1: Estimation of Population of Microorganisms on Products. Association for the Advancement of Medical Instrumentation, Arlington, VA. BLOCK, S.S. (2001). Disinfection, Sterilisation, and Preservation, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA, pp. 734. JACOBS, G.P. (1994). A Report of D 10 Values for Gamma and Electron Irradiated Microorganisms. Parenteral Drug Association, Bethesda, MD, Jerusalem Israel, pp. 15-16. UNITED STATES PHARMACOPEIA 25 & NATIONAL FORMULARY 20. (2002). United States Pharmacopeial Convention, Inc., Rockville, MD, pp. 1873-1878.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

8 IAEA CODE OF PRACTICE FOR THE RADIATION STERILISATION OF TISSUE ALLOGRAFTS: REQUIREMENTS FOR VALIDATION AND ROUTINE CONTROL

A N IAEA CONSULTATION D O C U M E N T

1. Introduction This code of practice for the radiation sterilisation of tissue allografts adopts the principles which the International Standards Organisation (ISO) applied to the radiation sterilisation of health care products. The approach has been adapted to take into account the special features associated with human tissues, and the features which distinguish them from industrially produced sterile health care products. The code, as described here, is not applicable if viral contamination is identified. Thus, it is emphasised that the human donors of the tissues must be medically and serologically screened. To further support this screening, it is recommended that autopsy reports are also reviewed if available. This adaptation of established ISO methods can thus only be applied for sterilisation of tissue allografts if the radiation sterilisation described here is the terminal stage of a careful detailed, documented sequence of procedures, involving: • donor selection; • tissue retrieval; 211

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tissue banking general procedures; specific processing procedures; labelling; and distribution;

all of which are conducted according to the IAEA International Standards for Tissue Banks. It shall not be used outside this context. The methods proposed here for the establishment of a sterilisation dose are based on statistical approaches used for the sterilisation of health care products (ISO 11137:1995, ISO 13409:1996, ISO 15844:1998, AAMI TIR 27:2001) and modified appropriately for the low numbers of tissue allograft samples typically available. For a standard distribution of resistance (SDR), the tissue bank may elect to substantiate a sterilisation dose of 25 kGy for microbial levels up to 1,000 colony forming units (cfu) per allograft product. Alternatively, for the SDR and other microbial distribution, specific sterilisation doses may be validated depending on the bioburden levels and radiation resistances (Dio values) of the constituent microorganisms. International standards have been established for the radiation sterilisation of health care products which include medical devices, medicinal products (pharmaceuticals and biologies) and in vitro diagnostic products (ISO 11137:1995 (E); ISO 11737-1: 1995; ISO 11737-2:1998; ISO/TR 13409:1996, ISO/TR 15844:1998 and AAMI TIR 27:2001). Following intensive studies of the effects of ionising radiation on chemical, physical and biological properties of tissue allografts and their components, these are now radiation sterilised using a variety of methods and practices. Through its radiation and tissue banking programme, the International Atomic Energy Agency has sought during the period 2001-2002 to establish a code of practice for the radiation sterilisation of tissue allografts and its requirement for validation and routine control of the sterilisation of tissues.

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Annex A describes the methods for selecting a sterilisation dose. Annex B provides three worked examples applying these methods. Annex C gives tables which contain microbial survival data relating to Standard Distribution of Resistances. Annex D gives a bibliography of key references for the sterilisation of tissues by ionising radiation. This code sets out the requirements of a process, in order to ensure that the radiation sterilisation of tissues produces standardized sterile tissue allografts suitable for safe clinical use. Although the principles adopted here are similar to those used for the sterilisation of health care products, there are substantial differences in practice arising from the physical and biological characteristics of tissues. For health care products, the items for sterilisation come usually from large production batches. For example, syringes are uniform in size and have bacterial contamination arising from the production process, usually at low levels. It is the reduction of the microbial bioburden to acceptable low levels which is the purpose of the sterilisation process, where such levels are defined by the sterility assurance level (SAL). The inactivation of microorganisms by physical and chemical means follows an exponential law and so the probability of a surviving microorganism can be calculated if the number and type of microorganisms are known and if the lethality of the sterilisation process is also known. Two methods are used in ISO 11137:1995 to establish the radiation doses required to achieve low SAL values. Method 1 of ISO 11137:1995 relies on knowing the bioburden (assuming a Standard Distribution of Resistances) before irradiation and uses this data to establish a verification dose, which will indicate the dose needed for a SAL of 10~2. The method involves a statistical approach to setting the dose based on three batches and hence relatively large numbers of samples are required for both establishing the initial bioburden and the verification dose, both per product batch. A further adaptation of method 1 for

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a single production batch has also been developed (ISO/TR 15844-1998). In Method 2 of ISO 11137:1995, the bioburden levels are measured after giving a series of incremental doses to the samples, these doses being well below the dose required for a SAL of 10"6. In this method, 280 samples are required to determine the dose to produce a SAL value of 10"2, from which the dose needed to yield a SAL value of 10~6 may be extrapolated. No assumptions are made in method 2 about the distribution of microorganisms and their resistances. In a later ISO/TR 13409:1996, Method 1 was adapted to allow the use of as few as 10 samples to determine the verification dose. In this modification, the dose needed for a SAL value of 10 _1 is used to establish the dose required for a SAL value of 10"6. The sole purpose, however, of this modification is to substantiate whether 25 kGy is an appropriate dose to achieve a SAL value of 10"6. In AAMI TIR 27:2001, another method to substantiate the sterilisation dose of 25 kGy was developed. 1.1. Sterilisation of tissue allografts Tissues used as allografts comprise a wide range of materials and bioburden levels such that the above quality assurance methods developed for health care products cannot be applied without careful and due consideration given to the differences between health care products and tissue allografts. Tissues which are sterilised currently include: bone, cartilage, ligaments, tendons, fascias, dura mater, heart valves, vessels, skin and amnion. Unlike health care products, the variability in types and levels of bioburden in tissues is much greater than that found for health care products where the levels of microbial contamination are usually low and relatively uniform in type and level. In addition, tissue allografts are not products of commercial production processes involving large numbers of samples. These

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differences mean that extra attention must be given to the following: (a) uniformity of sample physical characteristics (shape and density); (b) uniformity of bioburden in sample; (c) donor screening for viral contamination; and (d) whether low numbers of samples can be used for sterilisation dose setting purposes. 2. Objective The objective of this code is to provide the necessary guidance in the use of ionising radiation to sterilise tissue allografts in order to ensure their safe clinical use. 3. Scope This code specifies requirements for validation, process control and routine monitoring of the selection of donors, tissue processing, preservation, storage and the radiation sterilisation of tissue allografts. They apply to continuous and batch type gamma irradiators using the radioisotopes 60Co and 137 Cs, electron beam accelerators and X-rays. The principles adopted here are similar to those elucidated in ISO 11137:1995 in that statistical approaches to establishing doses to assure sterility of the tissue products are proposed. 4. References The following standards contain provisions which are relevant to this code: ISO 9001:2000 Quality management systems — Requirements. ISO 11137:1995 Sterilisation of health care products — Requirements for validation and routine control Radiation — sterilisation.

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ISO 11737-1: 1995 Sterilisation of medical devices — Microbiological methods — Part 1. ISO 11737-2:1998 Sterilisation of medical devices — Microbiological methods — Part 2. ISO/TR 13409:1996 Sterilisation of health care products — Radiation sterilisation — Substantiation of 25 kGy as a sterilisation dose for small or infrequent production batches. ISO/TR 15844:1998 Sterilisation of health care products — Radiation sterilisation — Selection of sterilisation dose for a single production batch. AAMI Technical Information Report (TIR 27):2001 — Sterilisation of health care products — Radiation sterilisation-Substantiation of 25 kGy as sterilisation dose — Method VD max . ISO/ASTM 51261 (2002) Guide for Selection and Calibration of Dosimetry Systems for Radiation Processing. IAEA (May, 2002) International Standards for Tissue Banks. 5.

Definitions

The majority of the definitions relating to the sterilisation process are given in ISO 11137:1995. The following definitions are particularly useful for this code and are given below. Allograft: A graft transplanted between two different individuals of the same species. Allograft product: An allograft or a collection of allografts within a primary package. Absorbed dose: The quantity of radiation energy imparted per unit mass of matter. The unit of absorbed dose is the gray (Gy), where 1 gray is equivalent to the absorption of 1 joule per kilogram (1 Gy = 100 rad). Batch (irradiation): Quantity of final product irradiated at the same cycle in a qualified facility.

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Batch (production): Defined quantity of finished tissue product from a single donor that is intended to be uniform in character and quality, and which has been produced during a same single cycle of processing. Bioburden: Population of viable microorganisms on tissue allograft and package prior to the sterilisation process. Distribution: Transportation and delivery of tissues for storage or use in recipient. Dose mapping: An exercise conducted within an irradiation facility to determine the distribution of the radiation dose throughout a load of tissue allograft or simulated items of specified bulk density, arranged in irradiation containers in a defined configuration. Dosimeter: A device having a reproducible measurable response to radiation, which can be used to measure the absorbed dose in a given material. Dosimetry system: System used for determining absorbed dose, consisting of dosimeters, measuring instrumentation and procedures for the system's use. Dw: Radiation dose required to inactivate 90 per cent of the homogeneous microbial population where it is assumed that the death of microbes follows first-order kinetics. Good tissue banking practice (GTBP): Practice that meets accepted standards as defined by relevant government or professional organisations. Irradiator: Assembly that permits safe and reliable sterilisation processing, including the source of radiation, conveyor and source mechanisms, safety devices and shield. Positive test of sterility: A test of sterility which exhibits detectable microbial growth after incubation in a suitable culture medium.

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Qualification: Obtaining and documenting evidence concerning the processes and products involved in tissue donor selection, tissue retrieval, processing, preservation and radiation sterilisation that will produce acceptable tissue allografts. Recovery efficiency: Measure of the ability of a specified technique to remove microorganisms from a tissue allograft. Reference standard dosimeter: Dosimeter, of high metrological quality, used as standard to provide measurements traceable to and consistent with measurements made using primary standard dosimeters. Routine dosimeter: A dosimeter calibrated against a primary or reference dosimeter and used routinely to make dosimetric measurements. Sample item -portion (SIP): Defined standardized portion of a tissue allograft that is tested. Sterile: Free of viable micro-organisms. Sterility assurance level (SAL): Probability of a viable microorganism being present on a tissue allograft after sterilisation. Sterilisation: A validated process to destroy, inactivate, or reduce microorganisms to a sterility assurance level (SAL) of 10" 6 . (Sterility is expressed by several national legislations and international standards as a SAL of 10"6.) Sterilisation dose: Minimum absorbed dose required to achieve the specified sterility assurance level (SAL). Test of sterility: Test performed to establish the presence or absence of viable microorganisms on tissue allograft, or portions thereof, when subjected to defined culture conditions. Tissue bank: An entity that provides or engages in one or more services involving tissue from living or cadaveric individuals for transplantation purposes. These services include assessing

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donor suitability, tissue recovery, tissue processing, sterilisation, storage, labeling and distribution. Validation: Refers to establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. A process is validated to evaluate the performance of a system with regard to its effectiveness based on intended use. Verification dose: Dose of radiation to which tissue allograft, or portions thereof are nominally exposed in the verification dose experiment with the intention of achieving a predetermined sterility assurance level (SAL). 6. Personnel Responsibility for the validation and routine control for sterilisation by irradiation including tissue donor selection, tissue retrieval, processing, preservation, sterilisation and storage shall be assigned to qualified personnel in accordance with subclauses 6.2.1 and 6.2.2 of ISO 9001:2000, whichever is applicable. 7. Validation of Pre-sterilisation Processes 7.1. General An essential step in the overall radiation sterilisation of tissues is rigorous donor selection to eliminate specific contaminants. Full details about donor selection, tissue retrieval, tissue banking general procedures, specific processing procedures, labelling and distribution are given in IAEA international standards for tissue banks. Such tissue donor selection, retrieval, processing and preservation are processes which determine the characteristics of the tissue allograft prior to the radiation sterilisation process. The most important characteristics are those relating to use of

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the tissues as allografts, namely, their physical, chemical and biological properties, the latter including the levels and types of microbial contamination. Validation of these processes shall include the following: (a) (b) (c) (d)

qualification of the tissue bank facilities; qualification of the tissue donors; qualification of the tissue processing and preservation; certification procedure to review and approve documentation of (a), (b) and (c); (e) maintenance of validation; and (f) process specification. 7.2. Qualification of the tissue bank facilities Tissue banks shall have facilities to receive procured tissues and to prepare tissue allograft material for sterilisation. Such facilities are expected to include laboratories for the processing, preservation and storage of tissues prior to sterilisation. These laboratories and the equipment contained therein shall meet international standards enunciated by the various tissue bank professional associations and now combined in the IAEA International standards for tissue banks. A regularly documented system should be established which demonstrates that these standards are maintained, with special emphasis on the minimisation of contamination by microorganisms throughout the tissue retrieval, transportation, processing, preservation and storage stages to bioburden levels which comply with the IAEA international standards for tissue banks. Tissue banks shall also have access to qualified microbiological laboratories to measure the levels of microorganisms on the tissue allografts at various stages in their preparation for the purposes of assessing both the levels of contamination at each stage and also typical bioburden levels of the pre-irradiated tissue allografts. The standards expected of such laboratories are specified in: ISO 11737-1:1995 and ISO 11737-2:1998.

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The overall purpose of the above facilities contained within tissue banks is to demonstrate that they are capable of producing preserved tissue allografts which have acceptably low levels of microorganisms in the preserved product prior to their sterilisation by radiation. 7.3. Qualification of tissue donors The main aim of the tissue donor selection process carried out prior to processing, preservation, storage and sterilisation is to produce tissue allografts which are free from transmissible infectious diseases. Such a selection process in order to produce acceptable tissues shall include the following minimal information: (a) time of retrieval of tissue after death of donor, conditions of body storage; (b) age of donor; (c) medical, social and sexual history of donor; (d) physical examination of the body; (e) serological (including molecular biology) tests; and (f) analysis of autopsy as required by law. Such information shall be used to screen donors to minimise the risk of infectious disease transmission from tissue donors to the recipients of the allografts. The information so collected shall be comprehensive, verifiable and auditable following good practice on tissue banking, as specified in the IAEA international standards for tissue banks. The following serological tests shall be carried out as a minimum on each donor: (a) antibodies to human immunodeficiency virus 1 and 2 (HIV 1, 2); (b) antibodies to hepatitis C virus (HCV); (c) hepatitis B surface antigen (HBs-Ag); and (d) syphilis: non-specific (e.g. VDRL) or preferably specific (e.g. TPHA).

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Other tests may be required by statutory regulations or when specific infections are indicated as specified in the IAEA international standard for tissue banks. 7.4. Qualification of tissue processing and preservation The processing of tissue allograft materials such as bone, cartilage, ligaments, fascias, tendons, dura mater, heart valves and vessels, skin and amnion comprises various stages such as removal of bone marrow, defatting, pasteurisation, antibiotic treatment, percolation and treatment with disinfectants such as hypochlorite, ethyl alcohol and glycerol. The inclusion of any or all of these stages will depend on a number of factors including: (a) the preferred practice of the tissue bank; (b) the nature of the tissue (and its anticipated use in the clinic); and (c) the degree of contamination of the procured tissue. The preservation of the processed tissue allografts include: (a) (b) (c) (d) (e)

may

freeze drying; deep freezing; air drying; heat drying; and chemical treatment.

An important function of these processes in Sees. 7.2 to 7.4 is to produce tissue allografts which have low levels of microbial contamination and in particular less than 1,000 cfu per allograft product when it is desired to substantiate a sterilisation dose of 25 kGy. In the latter case, for a bioburden of 1,000 cfu per allograft product, a 25 kGy dose is sufficient to achieve a SAL of 10"6 for a standard distribution of resistances. The capacity of all of the tissue processing and preservation procedures

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to remove microorganisms should be checked periodically and documented. 7.5. Maintenance of validation For each of the qualifications detailed above in Sees. 7.2-7.4, a validation process should be specified, which will demonstrate that the standards expected will be maintained. As a minimum, these validation processes shall include: (a) an audit of the origin and history of the procured tissues with reference to 7.3 (a) to (d); (b) a random, statistically significant sampling of procured tissues (that is, prior to processing and preservation) followed by a laboratory-based screening for viruses and infectious agents (see Sec. 7.3); (c) measures of particle count and microbial contamination in the environment of each of the separate facilities of the tissue bank; (d) random, statistically-significant sampling of tissue allografts prior to and after tissue processing and preservation for measurements of bioburden levels; and (e) determination of the ability of the tissue processing and preservation procedures to both reduce the levels of microorganisms and to produce the levels of bioburden required for the radiation sterilisation process. This should ensure a microbial contamination level of 1,000 cfu per allograft product or less when it is required to substantiate a sterilisation dose of 25 kGy. 7.6. Process specification A process specification shall be established for each tissue allograft type. The specification shall include: (a) the tissue allograft type covered by the specification; (b) the parameters covering the selection of tissue for processing;

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(c) details of the tissue processing and preservation carried out prior to irradiation as appropriate to each tissue type; (d) details of the equipment, laboratory and storage facilities required for each of the processing and preservation stages, particularly with regard to acceptable contamination levels; (e) details of the routine preventative maintenance programme; and (f) process documentation identifying every processed tissue, including details of its origin (see Sec. 7.3), its processing and preservation, dates of performing all processes, details of process interruptions, details of any deviations from the adopted processing and preservation procedures. 8. Validation of the Serilisation Process 8.1. General The guidance given here is based on the procedures specified in previous documents (ISO 11137:1995, ISO/TR 13409:1996, ISO/ TR 15844:1998 and AAMI TIR 27:2001) for the sterilisation of health care products. More emphasis is given here, however, on the factors which affect the ability of the sterilisation process to demonstrate that an appropriate sterility assurance level (SAL) can be achieved with low numbers of tissue allografts, which may have more variability in the types and levels of microbial contamination than is found in health care products and which may also be more variable in size and shape. More specifically, several approaches to establishing a sterilisation dose are proposed for the small numbers of tissue allografts typically processed. Emphasis is placed on the need to take into account both the variability of bioburden from one tissue donor to another, as well as the variability of size and shape of tissue allografts, which can affect both the accuracy of product dose mapping (and hence the sterilisation dose itself) and also the applicability of using Sample Item Portions (SIP) of a tissue allograft product.

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Validation of the sterilisation process shall include the following elements: (a) qualification of the tissue allografts and their packaging for sterilisation; (b) qualification of the irradiation facility; (c) process qualification using a specified tissue allografts or simulated products in qualified equipment; (d) a certification procedure to review and approve documentation of (a), (b) and (c); and (e) activities performed to support maintenance of validation. 8.2. Qualification of the tissue allografts for sterilisation 8.2.1. Evaluation of the tissue allograft and packaging Prior to using radiation sterilisation for a tissue allograft, the effect that radiation will have on the tissue allograft and its components shall be considered. The key references given in Annex D contain information on this aspect. Similarly, the effect of radiation on the packaging shall also be considered. Guidance on the latter is given in Annex A of ISO 11137:1995. Using such information, a maximum acceptable dose shall be established for each tissue allograft and its packaging. 8.2.2. Sterilisation dose selection A knowledge of the number and resistance to radiation of the microorganism population as it occurs on the tissue allografts shall be obtained and used for determination of the sterilisation dose. For the sterilisation of health care products, a reference microbial resistance distribution was adopted in ISO 111371:1995 for microorganisms found typically on medical devices. Studies should be carried out to establish the types of microorganisms that are normally found on the tissue types to be

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sterilised as well as their numbers and resistance to radiation. Such studies should take account of the distribution of the microorganisms within the tissue allograft itself since this may not be uniform. This should be determined by taking sample item portions (SIP) of the tissue and demonstrating that there are no significant statistical variations in distribution from SIP to SIP. If such studies show a consistent distribution of microoranisms from one tissue allograft to another, and one which is less resistant than the standard distribution of resistances (SDR) (see Table 1), then a table similar to B24 in ISO 11137:1995 giving a distribution of resistances appropriate to the allografts may be constructed for the purpose of sterilisation dose setting. This would allow the use of appropriate and perhaps lower sterilisation doses than would be the case if method 1 in ISO 11137:1995, based on the SDR in Table 1, were used. In the absence of such studies, the SDR may be used to establish sterilisation doses. To establish a sterilisation dose which will give a sterility assurance level (SAL) of 10~6, the methods based on those in ISO 11137:1995, ISO/TR 15844:1998, ISO/TR 13409:1996 and AAMI TIR 27:2001 should be used. A summary of these approaches as they apply to tissue allografts is given in Annex A. 8.2.3. Technical requirements The technical requirements to generate the information required for selection of the sterilisation dose shall be: (a) access to qualified microbiological and dosimetric laboratory services; (b) Microbiological testing performed in accordance with ISO 11737-1:1995 and ISO 11737-2:1998; and (c) access to a 60Co or 137Cs radiation source, or electron beam or X-ray irradiators.

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8.2.4. Transfer of sterilisation dose The conditions for transferring the sterilisation dose between two irradiation facilities are the same as those given in ISO 11137:1995 (Sec. 6.2.3) and apply equally to tissue allografts. 8.3. Qualification of the irradiation facility The principles covering the documentation of the irradiation system, its testing, calibration and dose mapping are covered in ISO 11137:1995 (Sec. 6.3) and apply equally to tissue allografts. 8.4. Qualification of the irradiation process 8.4.1. Determination of the product-loading pattern The principles given in ISO 11137:1995 (Sec. 6.4.1) covering this shall also apply for the sterilisation of tissue allografts. 8.4.2. Product dose mapping In general, the guidelines given in ISO 11137:1995 (Sec. 6.4.2) apply also to tissue allografts. However, it should be recognised that the product dose mapping of relatively uniform (i.e. in shape, size, composition and density) health care products is a more straight-forward task than the product dose mapping of tissue allografts, which by their nature are more variable in their physical characteristics. In particular, the density of tissue allografts may vary depending on their water content. In addition, some tissue allografts may be heterogeneous in their distribution of density within the product, requiring an appropriate number of dosimeters for the dose mapping exercise. A consideration of these factors affecting the actual absorbed dose in tissue allografts must be undertaken so that the level of accuracy in delivering a dose to a particular tissue can be determined.

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The acceptability of the accuracy of delivering a dose to tissue allografts will depend on the dose delivered in the verification dose experiments. If, for example, the actual dose delivered at its lowest possible accuracy limit is less than 90% of the verification dose, then the verification test must be repeated at a higher dose. Similarly, the minimum absorbed dose administered for sterilisation should take into account the likely variation in dose delivered so that sterilisation can be assured. As a guideline, uncertainties in the delivered dose should be within +10%. 8.5. Maintenance of validation The guidelines covering calibration of equipment and dosimetric systems, irradiator requalification and sterilisation dose auditing are the same as given in ISO 11137:1995 (Sec. 6.6) and apply equally to tissue allografts. 8.6. Routine sterilisation process control The guidelines covering process specification, tissue allograft handling and packing in the irradiation container, sterilisation process documentation are similar to those given in ISO 11137: 1995 (Sec. 7) and apply equally to tissue allografts. 9. Quality, Safety and Clinical Application of the Tissue Allograft A programme to demonstrate the quality, safety and clinical application of the tissue allograft throughout its shelf life shall be performed. Sampling procedures appropriate to the tissue type should be devised for this purpose. 10. Documentation and Certification Procedures Information gathered or produced while conducting the qualification and validation of the tissue allografts, tissue bank facilities

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and tissue processing, preservation and radiation sterilisation procedures shall be documented and reviewed for acceptability by a designated individual or group and retained in accordance with ISO 9001:2000 and the IAEA international standard for tissue banks or revision thereof, whichever is applicable. 11. Management and Control Control of the procedures involved in the selection of tissue donors, tissue processing and preservation prior to sterilisation by radiation and the radiation sterilisation process itself, shall be fully documented and managed in accordance with ISO 9001:2000 and IAEA International Standard for Tissue Banks, whichever is applicable. Annex A. Establishing a Sterilisation D o s e A.l.

Scope

This annex describes the practices and procedures for determining the bioburden levels of the tissue allografts and the application of this information to establish the radiation sterilisation dose. It must to be emphasised hat such samples must be the end results of the series of validated donor screening and subsequent procedures as are described in the IAEA international standards for tissue banks. A.2. Selection of tissue allograft products Tissue allografts can be prepared from a wide range of tissues such as skin, amnion, bone, cartilage tendons and ligaments. If samples can be prepared from these tissues, which are reasonably reproducible in shape, size and composition and also in sufficient numbers for statistical purposes, then the usual sampling procedures apply, as given, for example, in ISO 11137 and ISO/ TR 13409. However, if allograft products are both few in number

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(less than 10) and cannot be considered as identical products then it may be necessary to take multiple sample item portions of a single tissue allograft product for both bioburden analysis prior to sterilisation and also for the purpose of establishing a sterilisation dose. In such instances, it is important to have confidence in the distribution of microorganisms throughout the sample, obtained, for example, by periodic monitoring of such products. A.3. Sample item portion (SIP) The SIP shall validly represent the microbial challenge presented to the sterilisation process. SIPs may be used both to verify that microorganisms are distributed evenly, bioburden estimation and for establishing a sterilisation dose. It is important to ascertain that the SIPs are representative, not only in shape size and composition but also in bioburden. Statistical tests should be applied to establish this. At least 20 SIPs should be used (10 for bioburden testing and 10 for the verification dose experiments). A.4. Bioburden determination Bioburden determination could include the count of aerobic bacteria, spores, yeasts, molds and anaerobic bacteria. Many factors determine the choice of the tests most appropriate for the tissue allograft. At a minimum, the aerobic bacteria and fungi should be counted. The objective of the bioburden determination is to: (a) determine the total number of viable microorganisms within or on a tissue allograft and the packaging after completion of all processing steps before sterilisation; (b) act as an early warning system for possible production problems; and (c) calculate the dose necessary for effective radiation sterilisation.

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The validation of the bioburden estimation requires the determination of the effectiveness and reproducibility of the test method. The steps to estimate bioburden are the shown in the following flow chart and full details can be found in ISO 117371:1995. Sample collection For large production batches, randomly select units or SIPs of tissue allografts. For small production batches, take either sample item portions (SIPs) or whole sample from tissues allografts. For a single large piece of allografts, collect the total volume of the eluent solution from the last washing of the tissue allograft processing. Transport of the sample to the laboratory During transportation, tissue samples for bioburden estimation should be kept under the same conditions as for the whole production batch. Removal of micro-organisms from the sample Stomaching: This method is particularly suitable for skin, amnion and other soft tissue-like films or in the form of a tube. The test item and a known volume of eluent should be enclosed in a sterile stomacher bag. Reciprocating paddles operate the bag and force the eluent through and around the item. The time of treatment should be recorded. Shaking with or without glass beads: The test item is immersed in a known volume of eluent within a suitable vessel and shaken using a mechanical shaker (reciprocating, orbital, vortex mixing or wrist action). Glass beads of a defined size may be added to increase surface abrasion and thereby recovery efficiency. The time and frequency of shaking should be recorded.

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Ultrasonication: The test item is immersed in a known volume of eluent within a suitable vessel. The time and ultrasonic intensity of the treatment should be recorded. Flushing: The test item is flushed with a known volume of eluent and the resulting solution is collected. Transfer to culture medium and incubation A number of transferring methods can be employed, including: membrane filtration, pour plating, spread plates, most probable number (MPN). Enumeration For tissue bioburden determination, the total microbial count should be carried out. Characterization For contaminants that are commonly found and those suspected to be most radiation resistant should be isolated and characterized. A.5. Verification dose experiments In ISO 11137, the concept of establishing a verification dose for a SAL value which is much higher than 10 " 6 , for example, for a SAL value of 10~2 was proposed as an experimental method of establishing the sterilisation dose corresponding to a SAL of 10~6. For such verification dose experiments, samples of tissue allografts should be taken from production batches and irradiated at the calculated verification dose. In these experiments it is assumed (and should be demonstrated statistically) that the tissue allograft products are reasonably uniform in shape, size, composition and bioburden distribution. For single batch sizes up to 999, the numbers of sample required may be obtained from

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Table 1 of ISO/TR 13409. For minimum batch sizes of 20-79, for example, 10 samples are required for the bioburden determination and 10 for the verification dose experiment. In general, the number of samples required for the bioburden determination and verification dose experiments will depend on the number of batches and the number of samples in each batch. For each circumstance, the number of positive sterility tests allowed in the verification dose experiment should be calculated statistically using an acceptable range of values of probability for 0, 1, 2, 3 etc. positive tests of sterility. For the 100 samples used in method 1 of ISO 11137, for example, there is a 92% chance of there being 1% positives when up to 2 positives are detected and a 10% chance of accepting a batch with 5.23% positives (W.A. Taylor and J.M. Hansen, Alternative Sample Sizes for Verification Dose Experiments and Dose Audits, Radiation Physics and Chemistry (1999) 54, 65-75). For the 10 samples taken in ISO/TR 13409:1996 from a batch of 20, up to one positive test of sterility is proposed. For 30 or more, up to 2 positive tests of sterility are proposed (ISO/TR 13409:1996). It should be noted here that these latter statistical tests do not offer the same degree of protection as obtained when accepting up to two positive tests of sterility for a sample size of 100. For example, when accepting up to one positive test of sterility in a sample size of ten, there is a 95% chance of accepting a batch with 3.68% positives and a 10% chance of accepting a batch with 33.6% positives. Alternative sampling strategies are now available [see Taylor and Hansen (1999) above] which include for example, double sampling plans which can minimise sample sizes and yet offer similar protection. For single batches of low sample sizes, protection levels similar to those of the 100 sample approach in ISO 11137 can only be obtained by accepting a small number (possibly even zero) of positive sterility tests. For example, accepting up to one positive for a sample size of 50 offers similar protection. Hence, in ISO/TR 13409:1996 the verification dose for 10 samples taken from a batch of 20 is that which is required to

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produce a SAL of 10"1 (the reciprocal of the number of SIPs used) and is that dose which will yield not more than one positive test of sterility from the ten irradiated SIPs. In order to calculate the verification doses as well as the doses required to produce a SAL value of 10" 6 , one of several approaches may be taken to establish an appropriate verification dose for low sample numbers (up to 100 but typically much less). The methods proposed here for the establishment of a sterilisation dose are based on statistical approaches used previously for the sterilisation of health care products (ISO 11137: 1995, ISO 13409:1996, ISO 15844:1998, AAMI TIR 27:2001) and modified appropriately for the typical low numbers of tissue allografts samples available. For a standard distribution of resistance (SDR), the tissue bank may elect to substantiate a sterilisation dose of 25 kGy for microbial levels up to 1,000 cfu per unit. Alternatively, for the SDR and other microbial distribution, specific sterilisation doses may be validated depending on the bioburden levels and radiation resistances (D10 values) of the constituent microorganisms. (a) For establishing specific sterilisation doses for standard distribution of resistance and other microbial distribution for samples sizes between 10 and 100 an adaptation of method 1 of ISO 11137:1995 may be used. Method 1 of ISO 11137 is normally used for multiple batches containing a large number of samples per batch. For batches of 100 samples for example, verification dose experiments are carried out for a SAL of 10"2. A successful experiment (up to 2 positive tests of sterility) will then enable the dose required to achieve a SAL value of 10" 6 to be calculated from the survival curve of a standard distribution of resistances (SDR). In this code, an extension of Table 1 of ISO 11137 is given so that verification doses for SAL values between 10~2 and 10" 1 may be found for bioburden levels up to 1,000 cfu per allograft product. These SAL values correspond to relativelow sample sizes of 10-100. This allows method 1 to be used for typical tissue allografts where relatively low numbers of samples are available and also where the distribution of microbial radiation

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resistances is known and different to the SDR. The worked example given later uses this approach and, in addition, applies it (with appropriate statistical sampling, see above) to a microbial population which has a different distribution of radiation resistances than the SDR. However, for low bioburden levels combined with low sample numbers, it may be anticipated that there is an increased probability using this adaptation of method 1 that the verification dose experiment may fail. In the case of failure, the methods outlined in (b) a n d / o r (c) may be used. (b) For substantiation of a 25 kGy sterilisation dose, the method in ISO/TR 13409:1996 may be used to calculate the verification dose. This is an accredited method and is essentially a modification of the method in (a) above and applies only to a standard distribution of resistances. In this method, the verification dose for a given SAL is approximated to the initial bioburden by a series of linear relationships. Each linear equation is valid for a particular ten-fold domain of bioburden level, e.g., 1-10 cfu. The method in ISO/TR 13409:1996 can only be used to substantiate a dose of 25 kGy. It should be noted that the statistical approach allowing up to one positive test for sample sizes up to 30 and up to 2 positive tests for sample sizes above 30 does not offer the same level of protection as for the 100 samples in ISO 11137 until the sample size reaches 100. Alternative sampling strategies may be employed (Taylor and Hansen, 1999) for all the verification dose methods proposed here. (c) For substantiation of a 25 kGy sterilisation dose, an alternative and more recent method in A AMI TIR 27 may be used. The modification takes into account how the verification dose varies with bioburden level for a given SAL (and sample size) on the assumption that an SAL of 10" 6 is to be achieved at 25 kGy. Depending on the actual bioburden levels to be used (1-50 or 51-1,000 cfu per allograft product), a linear extrapolation of the appropriate SDR survival curve is made from either (log NQ, 0 kGy) or (log 10"2) to (log 10" 6 , 25 kGy) for 1-50 cfu and 5 1 1,000 cfu, respectively. For bioburden levels less than 1,000 cfu per allograft unit, these constructed survival curves represent a

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more radiation resistant bioburden than would otherwise be the case. The validity of this approach arises from the purpose of the method which is to validate a sterilisation dose of 25 kGy. For all bioburden levels below 1,000 cfu per allograft product, this means that for the reference microbial resistance distribution given in Table B24 of ISO 11137:1995 for medical devices, a more conservative approach to the calculation of a verification dose is taken. Hence, this modification allows the use of greater verification doses than would be allowed using the formula given in either method 1 of ISO 11137 or in ISO/TR 13409:1996. The result is that there are fewer unexpected and unwarranted failures relative to verification doses experiments carried out using the method in ISO/TR 13409:1996. At a bioburden level of exactly 1,000 cfu per allograft product (the maximum in both methods), there is no difference in the outcome of the methods, i.e., the calculated verification doses are identical. A.6. Procedures (a) Establish test sample sizes Select at least 10 allograft products or SIPs, as appropriate, for the determination of the initial bioburden. The number of allograft products or SIPs should be sufficient to represent validly the bioburden on the allograft product(s) to be sterilised. Select between 10 and 100 allograft products (or SIPs) for the verification dose experiments and record the corresponding verification dose SAL (= 1/n, where n is the number of allograft products or SIPs used). For 20-79 allograft products in a single batch, 10 allograft products may be used for both the bioburden determination and the verification dose experiment. (b) Determine the average bioburden Using methods such as those in ISO 11737-1:1995 and as described above (Bioburden estimation), determine the average

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bioburden of at least 10 allograft products or SIPs (the number will depend on the number of batches and the number of samples in the batches). For SIP values less than unity, the bioburden level for the whole product should be calculated and should be less than 1,000 cfu per allograft product for verification dose experiments carried out to substantiate a 25 kGy sterilisation dose. (c) Establish the verification dose The appropriate verification dose depends on the number of samples (allograft products or SIPs) to be used in the experiment (= 1/number of samples). The verification dose calculation depends on which of the three methods above is being used, as follows: (i) For establishing specific sterilisation doses for standard distribution of resistance and other microbial distribution for samples sizes between 10 and 100: an adaptation of method 1 of ISO 11137:1995. Calculate the dose required to achieve the required SAL from a knowledge of the initial bioburden level and from the microbial distribution and associated radiation resistances. This may be calculated from the equation, Ntot =N 0(1) 10-( D / D i) + N 0(2) 10-( D / D 2) + --- + N 0( „ ) 10-( D / D «), where Niot, represents the numbers of survivors; N0^ represents the initial numbers of the various microbial strains i (where z' = l - n); and D\, D 2 , ..., D(„) represent the Dio values of the various microbial strains. D represents the radiation dose and n the number of terms in the equation for a standard distribution of resistances (n = 10). For the reference standard distribution of resistances (Davis, K.W., Strawderman, W.E. and Whitby, J.L. (1984). /. Appl. Bacteriol. 57, 31-50) used in ISO 11137:1995 for medical devices (see Table 1), this equation will produce data similar to Table B.l of ISO 11137:1995 but for SAL values

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between 10" 2 and 10" 1 instead. By equating N tot to the selected SAL value and by using the appropriate D 10 values for each microbial type together with their numbers prior to irradiation, the verification dose, D, for SAL values between 10~~2 and 10 _1 can be calculated. These values are set out in Table 2(a). The same calculation can be used to find the sterilisation dose for the desired SAL of 10" 6 or reference can be made to Table B.l of ISO 11137:1995. In this method, the sterilisation dose is calculated using the bioburden level of the whole product. Alternatively, approximate values of the verification doses to achieve the same SAL values may be calculated using the equation given in ISO/ TR 13409:1996 (see next paragraph). (ii) For substantiation of a 25 kGy sterilisation dose, method ISO/TR 13409:1996: From a knowledge of the average bioburden and the number of samples or SIPs to be used in the verification experiment, the verification dose for a standard distribution of resistances is approximated by the equation: Verification dose at a the selected SAL = I + [S x log (bioburden)] where I and S are given in Annex C, Table 3 of this code. (iii) For substantiation of a 25 kGy sterilisation dose, AAMI TIR 27:2001: The calculation of the verification dose follows the procedures by Kowalski and Tallentire, 1999 (Radiat. Phys. Chetn. 54, 55-64) where the bioburden levels refer to either the SIP or whole product whichever is being used in the verification dose experiment: For bioburden levels of 1 to 50 cfu per allograft product or SIPs Step 1: D Iin = 25 kGy/(6 + log N 0 ), Step 2: Verification dose = D lin (log N0 - log SAL V D)/ where Diin represents the D 10 dose for a hypothetical survival curve which is linear between the coordinates (log N 0 , 0 kGy) and (log 10 " 6 , 25 kGy) for initial bioburden levels, N0, up to 1,000 cfu per allograft product. This linear plot therefore represents a constructed survival curve in which there is 1 out of

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106 probability of a survivor at 25 kGy. The method is valid therefore only for the substantiation of a 25 kGy sterilisation dose regardless of whether a lower dose could in fact be validated. For bioburden levels of 51 to 1,000 cfu per allograft product or SIPs Step 1: For a particular value of bioburden, use Table B.l of ISO 11137:1995 to identify the doses (kGy) corresponding to SAL values of 10" 2 [D(10~2)] and lO" 6 [D(1CT6)]. From these values, calculate TD10 from the following equation: TD10 = (Dose" 6 kGy - Dose" 2 k G y ) / 4 , where TDW represents the hypothetical D 10 value for a survival curve for a standard distribution of resistances which has been modified such that it is linear between log 10 - 2 and log 10 - 6 (log SAL values) when plotted against dose, with the log 10~6 value being set at 25 kGy. Essentially, this produces a survival curve which is more resistant to radiation than the SDR (for bioburden levels less than 1,000 cfu per allograft product) and one which is appropriate to substantiation of a 25 kGy sterilisation dose only. Step 2: Verification dose = 25 kGy - [TDW (log S A L V D + 6)], where SALVD is the sterility assurance level at which the verification dose experiment is to be performed. (d) Perform verification dose experiment Irradiate the tissue allografts or SIPs thereof at the verification dose. Irradiation conditions of the samples for verification of the substerilisation dose should be the same as the whole batch which is to be sterilised. For example, if the produced tissue batch is irradiated in frozen condition, the samples for the substerilisation dose verification studies should be irradiated in the same condition and the frozen condition should be kept during the whole irradiation process.

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The defined test sample size (SIP < 1), according to the SAL and batch size, is exposed to radiation at the verification dose. The dose delivered should not be less than 90% of the calculated verification dose. Test the tissue allografts for sterility using the methods in ISO 11737-2:1998 and record the number of positive tests of sterility. The irradiated SIPs, of all types of tissue allografts, are transferred to a growth medium and incubated for at least 14 days at an appropriated temperatures. Positive and negative sterility tests results should be registered. For bone and skin allografts, an additional test is recommended to detect anaerobic bacteria. (e) Interpretation of results For a verification dose experiment performed with up to 30 allograft products or SIPs, statistical verification is accepted if there is no more than one positive test of sterility observed. For 30 to 100 products or SIPs, statistical verification is accepted if there are no more than two positive tests of sterility observed (ISO/TR 13409:1996). Where the verification dose experiment is successful, the dose required to produce a SAL of 10" 6 for the whole allograft product should be calculated for procedure c(i) as indicated above and calculated in Annex C, Table 2(b). For procedures c(ii) and c(iii), a successful verification dose experiment substantiates the use of 25 kGy as a sterilisation dose. A.7. Routine use of sterilisation doses The routine use of a sterilisation dose calculated in procedure c(i) or of 25 kGy as substantiated by either procedure c(ii) or c(iii) shall only be valid if the tissue selection and tissue processing procedures have been demonstrated to produce tissues allografts with consistent bioburden levels. It should be demonstrated that the level of variation in bioburden, is consistent with the

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sterilisation dose to be used routinely. In such cases, sterilisation dose audits should be carried out at regular intervals, at least every three months. Annex B. Sterilisation of Tissue Allografts (Examples of Sterilisation Procedures) B.l. Limited number of amnion samples w i t h l o w bioburden and l o w bacterial resistance u s i n g method 1 of ISO 11137:1995 to calculate the verification dose B.l.l.

Introduction

This method uses method 1 of ISO 11137:1995 but applies it to sample sizes of less than 100 in a single production batch. The example chosen consists of a single batch of 20 amnion membranes ( 5 x 5 cm) from which 10 are used for the bioburden determination and 10 are used for the verification dose experiment. The data used in the example are consistent with data on bioburden levels, bacterial types and distribution found in Hilmy et al. (2000). /. Cell Tissue Banking 1, 143-147. In that study, the most radiation resistant microbes were assumed to have a D 10 value of 1.8 kGy, i.e., a distribution which differs from the reference microbial resistance distribution in that there are no microbes with a D 10 value higher than 1.8 kGy. Furthermore, the tissue processing and preservation procedures have produced tissue allografts which are much lower than 1,000 cfu per allograft product. For such samples, a sterilisation dose which is significantly less than 25 kGy is confirmed from the verification dose experiment. B.1.2. Procured tissue qualification (a) Tissue type: ... Amnion samples of 5 x 5 cm (b) Screening of tissue for transmission of disease: ...

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Age of donor: ... 25 ... Medical, social and sexual history: ... None to suggest risk of transmissible disease Serological tests: ... HIV (HIV-1,2 Ab) ... negative; Hepatitis C (HCV-Ab) ... negative; Hepatitis B (HBs-Ag) ... negative; Syphillis (VDRL) ... negative. B.1.3. Tissue processing and preservation qualification (a) Description of processing technique ... hypochlorite, (b) Description of preservation technique ... lyophilization (c) Typical microbial levels of procured tissue before processing ... in the range of 5,000-10,000 cfu per tissue ... (d) Typical bioburden levels of processed and preserved tissues ... 57 cfu per allograft product (Note 1) It is noted from the study of Hilmy et al. (see above) that the bioburden levels of the processed tissue (i.e. before sterilisation by irradiation) decreased from about 1,400 cfu to 120 cfu during the study period 1994 to 1997, with 1998 data showing an average of 57 cfu per allograft product (range 12-160 cfu). Clearly, good processing techniques can have a dramatic effect on the bioburden levels of the tissue being prepared for sterilisation by irradiation. The level of reduction used in this example is probably therefore a conservative estimate of the degree of elimination of bacteria B.1.4. Qualification of tissue allografts for sterilisation Typical bioburden distribution: The distribution of bacterial resistances given below is assumed to consist entirely of bacteria with a D 10 value of 1.8 kGy and represents a distribution which is similar but not identical to the standard distribution of resistances, i.e.: D 10 (kGy) 1.8; Frequency 1.0.

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B.1.5. Calculation of the sterilisation dose Stage Stage 1 Production batch size

Value 40

Comments 5 x 5 cm amnion samples.

Test sample size for bioburden determination

10

Test sample size for the verification dose experiment

10

Verification dose required for SAL 10" 1 (= 1/10).

20

10 for bioburden; 10 for verification dose experiment.

Stage 2 Obtain samples

The whole allograft product is used.

Stage 3 SIP Average bioburden Stage 4 Verification dose calculation

57

Bioburden results of 15, 91, 99, 30, 30, 99, 8, 84, 91, 23.

3.2 kGy

Using the bacterial resistance distribution given above (and not the SDR), the survival equation is constructed (see Annex A) and used to calculate the verification dose (D) for a N(tot) value of 0.1 (equivalent to a SAL value of 0.1, the reciprocal of the number of samples used) and where the total initial number of microorganisms (Continued)

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Value

Comments per product (SIP = 1) is equal to 57. The survival equation is: Ntot = 57 x 10-( D / 18 ) From this data, the verification dose is calculated as 3.2 kGy.

Stage 5 Verification dose experiments

B.1.6.

3.3 kGy (delivered dose) 1 positive/10 samples

The sterility test yielded one positive test out of ten and therefore the verification dose experiment was successful (but note that the level of protection is significantly less than allowing up to 2 positives for a sample size of 100, see Annex A) and the sterilisation dose for SAL = 10~6 can be calculated from the survival equation given above (= 14.0 kGy). Note: In the case that a SIP < 1 was taken instead, the bioburden for the whole product should be used to calculate the sterilisation dose.

Conclusion

This e x a m p l e s h o w s h o w the c o m b i n a t i o n of g o o d tissue p r o c e s s i n g a n d p r e s e r v a t i o n a n d sterilisation b y ionising r a d i a t i o n , for s a m p l e s w h i c h are k n o w n to h a v e bacterial contamination relatively susceptible to radiation, can allow the u s e of a sterilisation dose w h i c h is m u c h less t h a n 25 kGy.

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B.2. Limited number of amnion samples requiring only substantiation of 25 k G y as a sterilisation dose B.2.1.

Introduction

In this example, it is assumed that there is a standard distribution of resistances which defines the bacterial contamination of the tissue allografts. The example chosen consists of a single batch of 40 amnion membranes ( 5 x 5 cm) from which 10 are used for the bioburden determination and 10 are used for the verification dose experiment. The data used in the example are consistent with data on bioburden levels, bacterial types and distribution found in Hilmy et al. (2000). /. Cell Tissue Banking 1, 143-147. Furthermore, for the limited number of samples to be tested, it is required only to establish that a 25 kGy dose may be used to achieve an SAL of 10" 6 . It is shown below that when the method in ISO 13409:1996 is applied for 20 samples (10 for the bioburden determination and 10 for the verification dose experiment), from a batch size of 40, the samples fail the verification dose experiment. To increase the probability of a successful verification dose experiment, whilst at the same time substantiating a sterilisation dose of 25 kGy, the method of Tallentire and Kowalski is applied (see Annex A). This allows the use of a higher verification dose and it is then found that the samples pass this test, substantiating the use of a 25 kGy sterilisation dose. B.2.2. Procured tissue qualification (a) Tissue type ... Amnion ( 5 x 5 cm) (b) Screening of tissue for transmission of disease Age of donor ... 25 Medical, social and sexual history ... None to suggest risk of transmissible disease Serological tests: HIV (HIV-1,2 Ab) ... negative; Hepatitis C (HCV-Ab) ... negative; Hepatitis B (HBs-Ag) ... negative; Syphillis (VDRL) ... negative.

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B.2.3. Tissue processing and preservation qualification (a) Description of processing technique ... hypochlorite (b) Description of preservation technique ... lyophilization (c) Typical microbial levels of procured tissue before processing ... in the range of 5,000-10,000 cfu per tissue ... (d) Typical bioburden levels of processed and preserved tissues ... 57 cfu per allograft product (Note 1). B.2.4. Qualification of tissue allografts for sterilisation Typical bioburden distribution (it is assumed that the standard distribution of resistances, see Annex A, is valid). Stage Stage 1 Production batch size

Value 40

Comments 5 x 5 cm amnion samples.

Test sample size for bioburden determination

10

Test sample size for the verification dose experiment

10

Verification dose required for SAL 10"1 (= 1/10).

20

10 for bioburden; 10 for verification dose experiment.

Stage 2 Obtain samples Stage 3 SIP

The whole allograft product is used. (Continued)

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(Continued) Stage Average bioburden

Value

Comments

57

Bioburden results of 15, 91, 99, 30, 30, 99, 8, 84, 91, 23. Average bioburden for whole product 57 cfu. (This is less than 1,000 cfu and therefore the method may be used.) Note: If a SIP < 1 was taken, then the bioburden of the whole product should be calculated and should be less than 1,000 cfu per allograft product for this method to be valid.

Stage 4 Verification dose 4.6 kGy The verification dose is calculated calculation (1) using the method in ISO/TR 13409: 1996. In this method (applicable to a standard distribution of resistances only), the verification dose for a given SAL is approximated to the initial bioburden by a series of linear relationships using the parameters I and S (see below). Each linear equation is valid for a particular ten-fold domain of bioburden level, e.g. 10-100 cfu. For a bioburden of 57 and sample size of 10, I and S values of 0.67 and 2.23 respectively are obtained from ISO/TR 13409:1996 and are given here in Annex C, Table 3. The verification dose is given by: Dose = I + [ S x log (average SIP bioburden)] = 0.67 + (2.23 x log x 57) = 4.6 kGy. (Continued)

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(Continued) Stage Stage 5 Verification dose experiments (1)

Verification dose calculation (2)

Value

Comments

4.5 kGy (delivered dose) 2 positives/ 10 samples

The sterility test yielded two positive tests out of ten and therefore the verification dose experiment was not successful and a sterilisation dose of 25 kGy could not be substantiated.

8.6 kGy

A new verification dose was calculated using the method of Tallentire and Kowalski (see Annex A). This method takes into account how the verification dose for a standard distribution of resistances (reference microbial resistance distribution) varies with bioburden level for a given SAL (and sample size) on the assumption that an SAL of 10~6 is to be achieved at 25 kGy. Application of method 1 of ISO 11137:1995 for bioburden levels of less than 1,000 cfu would yield sterilisation doses of less than 25 kGy. The method of Tallentire and Kowalski assumes instead that only substantiation of a 25 kGy sterilisation dose is required regardless of the bioburden level. Extrapolation of the reference distribution to produce an SAL of 10" 6 at 25 kGy for bioburden levels of less than 1,000 cfu allows the use of higher verification doses (Continued)

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(Continued) Stage

Value

Comments than would be predicted by method 1 of ISO 11137:1995 and hence a greater probability of a successful verification dose experiment. For a bioburden level of 120 (i.e. between 51 and 1,000), the doses corresponding to this bioburden for SAL values of 10 - 6 and 10~2 are found from Table 1 of ISOH137 and are designated Dose - 6 and Dose" 2 respectively, from which the TDW is calculated as follows: TDW = (Dose" 6 kGy - Dose" 2 kGy)/4 = (20.4 - 7.3)/4 = 3.27 kGy, where TDm represents the hypothetical Dio value for a survival curve for a standard distribution of resistances which has been modified such that it is linear between log 10 - 2 and log 10~6 (log SAL values) when plotted against dose, with the log 10 - 6 value being set at 25 kGy. Essentially, this produces a survival curve which is more resistant to radiation than the SDR (for bioburden levels less than 1,000 cfu per allograft product) and one which is appropriate to substantiation of a 25 kGy sterilisation dose only. Note: Table 1 of ISO 11137:1995 does not have a value corresponding to a (Continued)

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Value

Comments bioburden of 57 and so the next highest value of 59.2 is used. The verification dose, VD, is then calculated, as follows: VD = 25 kGy - [TDW (log SAL VD + 6)] = 25 - [3.27 (log 0.1 + 6)] = 8.6 kGy, where SAL V D is the sterility assurance level at which the verification dose experiment is to be performed, (= the reciprocal of the number of samples), in this case, 0.1.

Verification dose experiments (2)

The 10 samples are irradiated at this 8.5 kGy 1 positive/ verification dose and tested for 10 samples sterility. The sterility tests yielded one positive test out of ten and therefore the use of 25 kGy as a sterilisation dose (SAL = 10~6) could be substantiated (note however that this result does not offer the same level of protection when allowing up to 2 positives in a sample size of 100, see above).

B.2.5. C o n c l u s i o n A l t h o u g h the tissue processing a n d preservation p r o d u c e d tissues w i t h relatively l o w b i o b u r d e n for w h i c h sterilisation doses substantially less t h a n 25 k G y c o u l d h a v e b e e n u s e d (see example above), the tissue b a n k r e q u i r e d only a m e t h o d to substantiate a sterilisation dose of 25 kGy. The application of the m e t h o d s of ISO 13409:1996 a n d of Tallentire a n d Kowalski,

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which are particularly suitable for bioburden levels much less than 1,000 cfu per allograft product, allowed the use of relatively high verification doses and, hence a greater probability of success. In the example chosen, the method in ISO 13409:1996 failed and hence the method of Tallentire and Kowalski was used as well. For tissue banks which prefer to use a standard 25 kGy sterilisation dose, this latter method will be more efficient in that fewer verification dose experiments will fail. B.3. Limited number of bone samples w i t h very l o w bioburden and SDR u s i n g ISO/TR 13409:1996 to calculate the verification dose (SIP < 1) B.3.1. Introduction This method uses ISO/TR 13409:1996 an applies it to a sample of 40 small pieces of bone. Typically, very low bioburden levels are found after processing. In this example, very low SIP values are used so that most of the allograft product can be retained for use. B.3.2. Procured tissue qualification (a) Tissue type: ... bone cut into 40 small pieces (chips) (b) Screening of tissues donor Age of donor ... 36 ... Medical, social and sexual history: None to suggest of transmissible disease Serological tests: HIV (HIV-1,2 Ab) ... negative; Hepatitis C (HCV-Ab) ... negative; Hepatitis B (HBs-Ag) ... negative; Syphillis (VDRL) ... negative. B.3.3. Tissue processing and preservation qualification (a) Description of processing technique ... cut into standardised small pieces.

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(b) Description of preservation technique ... frozen. (c) Typical b i o b u r d e n levels of processed a n d p r e s e r v e d tissues ... 40 cfu per allograft p r o d u c t . B.3.4. Qualification of t i s s u e allografts for sterilisation Stage Stage 1 Production batch size

Value

Comments

5

Bone cut into 40 small pieces (1 cc each) packed in flask, produced from one donor in one processing batch.

Test sample size for bioburden determination

10

According ISO/TR 13409:1996, Table 1.

Test sample size for verification dose experiment

10

According ISO/TR 13409:1996, Table 1.

20

A random sample of 20 standardised product portions of 1 cc each was obtained from the production batch.

Stage 2 Obtained samples

Stage 3 SIP SIP bioburden

Average bioburden

0.025

Calculated from 1/40.

1

Bioburden results of 1, 0, 2, 0, 1, 2, 1, 1, 1, 1 were observed from the 10 SIP tested, for an average bioburden of 1.

40

The average bioburden for the product tested was calculated as follow: 1/0.025 = 40. This is (Continued)

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Value

Comments less than 1,000 cfu per allograft product and therefore this method is valid.

Stage 4 Verification dose calculation

1.3

Stage 5 Verification dose 1.3 kGy experiment (delivered dose) 0 positive/ 10 samples

Stage 6 Interpretation of results

B.3.5.

Verification dose formula: I + (S x log (average SIP bioburden) kGy. According ISO/TR 13409: 1996, Table 2, the I and S values are 1.25 and 1.65 respectively: = 1.25 + (1.65 x log 1) = 1.25 kGy = 1.3 kGy

The test sterility yielded 0 positive from the 10 SIPs tested. Therefore, the verification experiment was successful and no further action was necessary.

The test of sterility result was acceptable, the sterilisation dose of 25 kGy was confirmed.

Conclusion

A l t h o u g h a lower sterilisation dose could be justified if the a d a p t a t i o n of m e t h o d 1 of ISO 11137:1995 w a s applied, the tissue b a n k elected to u s e I S O / T R 13409:1996 to substantiate a 25 kGy sterilisation dose only.

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Annex C. Tables 1, 2 and 3 Table 1. Microbial standard distribution of resistance (SDR) (Davis, K.W., Strawderman, W.E. and Whitby, J.L. (1984). The rationale and computer evaluation of a gamma sterilisation dose determination method for medical devices using a substerilisation incremental dose sterility test protocol, J. Appl. Bad. 57, 31-50). Dio (kGy) 1.0 1.5 2.0 2.5 2.8 3.1 3.4 3.7 4.0 4.2 % 65.487 22.493 6.302 3.179 1.213 0.786 0.350 0.111 0.072 0.007

Table 2(a). Radiation dose (kGy) required to achieve given SAL for different bioburden (cfu) having standard distribution of resistances. Sample size (n) SAL (1/n) Bioburden 0.06 0.08 0.09 0.10 0.12 0.14 0.17 0.19 0.22 0.26 0.29 0.34 0.39 0.44 0.50 0.57 0.65 0.73 0.83 0.93 1.0 1.2 1.4 1.6 1.8 2.0

10 1/10

1.0 1.1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.7

15 1/15

1.0 1.1 1.2 1.3 1.3 1.4 1.5 1.5 1.7 1.8 1.9 1.9 2.0

20 1/20

1.0 1.1 1.2 1.3 1.4 1.4 1.5 1.6 1.7 1.7 1.9 2.0 2.1 2.2 2.2

25 30 1/25 1/30

1.0 1.1 1.2 1.3 1.3 1.4 1.5 1.6 1.7 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4

1.0 1.1 1.2 1.3 1.4 1.5 1.5 1.6 1.7 1.8 1.9 2.0 2.0 2.1 2.3 2.4 2.5 2.5

35 1/35

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.6 1.7 1.8 1.9 2.0 2.1 2.1 2.3 2.4 2.5 2.6 2.7

40 1/40

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.2 2.4 2.5 2.6 2.7 2.8

45 50 1/45 1/50

1.0 1.1 1.2 1.3 1.4 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.3 2.5 2.6 2.7 2.8 2.9

1.0 1.1 1.2 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.1 2.2 2.3 2.4 2.5 2.7 2.8 2.9 3.0

60 1/60

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.5 2.7 2.8 2.9 3.0 3.1

70 1/70

90 80 1/80 1/90

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.2 3.3

1.0 1.1 1.1 1.2 1.3 1.5 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.1 3.2 3.3 3.4

1.1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.2 3.3 3.4 3.5

100 1/100

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.4 3.5 3.6

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255

Table 2(a). (Continued) Sample size (n) SAL(l/n)

2.2 2.6 3.0 3.2 4.0 4.4 5.0 5.4 6.0 7.0 8.0 9.0 10 11 12 13 14 15 16 17 18 19 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 150 200 250 300 350

10 15 20 25 30 35 40 45 50 60 70 80 90 100 1/10 1/15 1/20 1/25 1/30 1/35 1/40 1/45 1/50 1/60 1/70 1/80 1/90 1/100

1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.5 2.7 2.8 2.9 3.0 3.0 3.1 3.2 3.3 3.3 3.4 3.4 3.5 3.5 3.6 3.8 4.0 4.1 4.3 4.4 4.5 4.6 4.7 4.8 4.8 4.9 5.0 5.0 5.1 5.2 5.2 5.7 6.0 6.2 6.5 6.6

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.4 3.5 3.6 3.7 3.7 3.8 3.8 3.9 4.0 4.0 4.1 4.3 4.5 4.6 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.4 5.5 5.6 5.6 5.7 5.8 6.2 6.6 6.8 7.0 7.2

2.3 2.4 2.5 2.6 2.8 2.9 3.0 3.0 3.1 3.3 3.4 3.5 3.6 3.7 3.7 3.8 3.9 4.0 4.0 4.1 4.1 4.2 4.2 4.5 4.6 4.8 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.6 5.7 5.8 5.8 5.9 5.9 6.4 6.8 7.0 7.2 7.4

2.5 2.6 2.7 2.8 3.0 3.0 3.2 3.2 3.3 3.5 3.6 3.7 3.8 3.9 4.0 4.0 4.1 4.2 4.2 4.3 4.3 4.4 4.5 4.7 4.9 5.0 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.9 6.0 6.1 6.1 6.2 6.7 7.0 7.3 7.5 7.7

2.6 2.7 2.9 2.9 3.1 3.2 3.3 3.4 3.5 3.6 3.8 3.9 4.1 4.1 4.2 4.3 4.4 4.4 4.5 4.6 4.6 4.7 4.7 5.0 5.2 5.3 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.2 6.3 6.4 6.4 6.5 7.0 7.3 7.6 7.8 8.0

2.8 2.9 3.0 3.1 3.3 3.4 3.5 3.6 3.6 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.4 4.5 4.6 4.6 4.7 4.7 4.8 5.0 5.2 5.4 5.6 5.7 5.9 6.0 6.1 6.2 6.2 6.3 6.4 6.5 6.5 6.6 6.7 7.1 7.5 7.8 8.0 8.2

3.0 3.1 3.2 3.3 3.5 3.6 3.7 3.8 3.9 4.0 4.2 4.3 4.4 4.5 4.6 4.7 4.7 4.8 4.9 4.9 5.0 5.1 5.1 5.4 5.6 5.7 5.9 6.0 6.1 6.3 6.4 6.4 6.5 6.6 6.7 6.8 6.8 6.9 7.0 7.5 7.7 7.8 7.9 8.1 8.2 8.3 8.4 8.5

2.9 3.0 3.1 3.2 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.2 4.3 4.4 4.4 4.5 4.6 4.7 4.7 4.8 4.9 4.9 5.0 5.2 5.4 5.6 5.7 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.6 6.7 6.8 6.8 7.3

3.0 3.2 3.3 3.4 3.6 3.7 3.8 3.9 4.0 4.1 4.3 4.4 4.4 4.5 4.6 4.7 4.8 4.9 4.9 5.0 5.0 5.1 5.1 5.4 5.6 5.7 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.9 7.0 7.0 7.1 7.6 7.9 8.2 8.5 8.7

3.2 3.4 3.5 3.5 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.6 4.7 4.8 4.9 5.0 5.0 5.1 5.2 5.3 5.3 5.4 5.4 5.7 5.9 6.1 6.2 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.0 7.1 7.2 7.3 7.3 7.8 8.2 8.5 8.7 8.9

3.3 3.5 3.6 3.7 3.9 4.0 4.1 4.2 4.3 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.3 5.4 5.5 5.5 5.6 5.9 6.1 6.2 6.4 6.5 6.7 6.8 6.9 7.0 7.1 7.2 7.2 7.3 7.4 7.4 7.5 8.0 8.4 8.7 8.9 9.1

3.5 3.6 3.8 3.8 4.0 4.1 4.2 4.3 4.4 4.6 4.7 4.9 5.0 5.1 5.2 5.3 5.3 5.4 5.5 5.6 5.6 5.7 5.7 6.0 6.2 6.4 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.5 7.6 7.7 8.2 8.5 8.8 9.1 9.3

3.6 3.7 3.9 3.9 4.1 4.2 4.4 4.5 4.6 4.7 4.8 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.6 5.7 5.8 5.8 5.9 6.1 6.3 6.5 6.7 6.8 7.0 7.1 7.2 7.3 7.4 7.5 7.5 7.6 7.7 7.8 7.8 8.3 8.7 9.0 9.2 9.4

3.7 3.8 4.0 4.0 4.2 4.3 4.5 4.6 4.7 4.8 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.7 5.8 5.9 5.9 6.0 6.3 6.5 6.6 6.8 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.7

7.8 7.9 7.9 8.5 8.8 9.1 9.4 9.5

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Table 2(a). (Continued) Sample size (n) 10 15 20 25 30 35 40 45 50 1/10 1/15 1/20 1/25 1/30 1/35 1/40 1/45 1/50 SAL (1/n) 400 450 500 550 600 650 700 750 800 850 900 950 1,000

6.7 6.9 7.1 7.2 7.3 7.4 7.5 7.6 7.6 7.7 7.8 7.9 7.9

7.4 7.5 7.7 7.8 7.9 8.0 8.1 8.2 8.2 8.3 8.4 8.5 8.5

7.6 7.7 7.8 8.0 8.1 8.2 8.3 8.4 8.5 8.5 8.6 8.7 8.7

7.9 8.0 8.1 8.2 8.4 8.5 8.5 8.6 8.7 8.8 8.9 8.9 9.0

8.2 8.3 8.5 8.6 8.7 8.8 8.9 9.0 9.0 9.1 9.2 9.3 9.3

8.4 8.5 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.3 9.4 9.5 9.6

8.5 8.7 8.8 9.0 9.1 9.2 9.3 9.4 9.4 9.5 9.6 9.7 9.7

8.7 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.8 9.9

8.8 9.0 9.1 9.2 9.3 9.5 9.6 9.7 9.7 9.8 9.9 10.0 10.0

60 70 80 90 1/60 1/70 1/80 1/90

100 1/100

9.6 9.8 9.9 10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.8

9.7 9.9 10.0 10.2 10.3 10.4 10.5 10.6 10.6 10.7 10.8 10.9 11.0

9.1 9.2 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.1 10.2 10.3

9.3 9.4 9.6 9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.3 10.4 10.5

9.4 9.6 9.7 9.9 10.0 10.1 10.2 10.3 10.4 10.4 10.5 10.6 10.7

Table 2(b). Radiation dose (kGy) required to achieve an SAL of 10 different bioburdens having standard distribution of resistances. Bioburden Dose 0.06 0.08 0.09 0.10 0.12 0.14 0.17 0.19 0.22 0.26 0.29 0.34 0.39 0.44 0.50 0.57 0.65 0.73 0.83

10.4 10.6 10.8 11.0 11.3 11.5 11.7 11.9 12.1 12.3 12.5 12.7 12.9 13.1 13.3 13.5 13.6 13.8 14.0

Bioburden Dose 0.93 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.6 3.0 3.2 4.0 4.4 5.0 5.4 6.0 7.0 8.0 8.8

14.2 14.2 14.3 14.6 14.8 14.9 15.2 15.3 15.5 15.8 16.0 16.2 16.3 16.5 16.6 16.8 17.0 17.2 17.3

Bioburden Dose 9.0 10 11 12 13 14 15 16 17 18 19 20 30 40 50 60 70 80 90

17.4 17.6 17.7 17.9 18.0 18.1 18.2 18.3 18.4 18.5 18.6 18.7 19.3 19.7 20.1 20.3 20.6 20.8 21.0

6

for

Bioburden Dose 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1,000

21.1 21.8 22.2 22.6 22.9 23.1 23.3 23.5 23.7 23.8 24.0 24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8

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Table 3. I and S for calculation of verification dose for test sample size and bioburden level (ISO/TR 13409:1996). Verification dose at a given SAL = I + (S x log (Avergare SIP bioburden)). I = intercept; S = slope. Bioburden 1 to 10

Test sample size

10 20 30 40 50 60 70 80 90

Bioburden 11 to 100

Bioburden 101 to 1,000

I

S

I

S

I

S

1.25 1.71 2.00 2.21 2.38 2.52 2.65 2.76 2.86

1.65 1.82 1.93 2.01 2.07 2.12 2.16 2.19 2.22

0.67 1.14 1.46 1.69 1.88 2.03 2.16 2.30 2.39

2.23 2.41 2.49 2.55 2.59 2.63 2.66 2.67 2.70

-0.26 0.35 0.71 1.00 1.21 1.40 1.55 1.67 1.80

2.71 2.81 2.87 2.90 2.93 2.95 2.97 2.99 3.00

Annex D . Key References for the Sterilisation of Tissues by Ionising Radiation D . l . Bone AKKUS, O. and RIMNAC, C M . (2001). Fracture resistance of gamma radiation sterilised cortical bone allografts, /. Orthop. Res. 19, 927-934. CORNU, O., BANSE, X., DOCQUIER, P.L., LUYCKX, S. and DELLOYE, C. (2000). Effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone, /. Orthop. Res. 18, 426-431. MOREAU, M.F., GALLOIS, Y., BASLE, M.F. and CHAPPARD, D. (2000). Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblastlike cells, Biomaterials 21, 369-376. SILBERMAN, F. and KAIRIYAMA, E. (2000). Radiation sterilisation and the surgical use of bone allografts in Argentina, Advances in Tissue Banking 4, 27-38.

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ARAM, N., MYOUI, A., KURATSU, S., HASHIMOTO, N., INOUE, T., KUDAWARA, I., UEDA, T., YOSHIKAWA, H., MASAKI, N. and UCHIDA, A. (1999). Intraoperative extracorporeal autogenous irradiated bone grafts in tumour surgery, Clin. Orthop. 368, 196-206. RUSSELL, J.L. and BLOCK, J.E. (1999). Clinical utility of demineralised bone matrix for osseous defects, arthrodesis and reconstruction: impact of processing techniques and stud methodology, Orthopedics 22, 524-531. MARCZYNSKI, W., TYLMAN, D. and KOMENDER, J. (1997). Long-term follow up after transplantation of frozen and radiation sterilise bone grafts, Ann. Transplant. 2, 64-66. RUSSELL, J., SCARBOROUGH, N. and CHESMEL, K. (1997). Re: Ability of commercial demineralised freeze-dried bone allograft to induce new bone formation, /. Peridontol. 68, 804-806. ZHANG, Q., CORNU, O. and DELLOYE, C. (1997). Ethylene oxide does not extinguish the osteoinductive capacity of demineralised bone. A reappraisal in rats, Acta Orthop. Scand. 68, 104-108. FIDELER, B.M., VANGSNESS, C.T. Jr., LU, B., ORLANDO, C. and MOORE, T. (1995). Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts, Am. J. Sports Med. 23, 643-646. GOERTZEN, M.J., CLAHSEN, H., BURRIG, K.F. and SCHULITZ, K.P. (1995). Sterilisatation of canine anterior cruciate allografts by gamma irradiation in argon. Mechanical and neurohistological properties retained one year after transplantation, /. Bone Joint Surg. Br. 77, 205-212, retracted publication. WHITE, J.M., GOODIS, H.E., MARSHALL, S.J. and MARSHALL, G.W. (1994). Sterilisation of teeth by gamma radiation, /. Dent. Res. 73, 1560-1567.

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LOTY, B., TOMENO, B., EVRARD, J. and POSTEL, M. (1994). Infection in massive bone allografts sterilised by radiation, Int. Orthop. 18, 164-171. YAHIA, L.H., DROUIIN, G. and ZUKOR, D. (1993). The irradiation effect on the initial mechanical properties of meniscal grafts, Biomed. Mater. Eng. 3, 211-221. ZASACKI, W. (1991). The efficacy of application of lyophilized, radiation-sterilised bone graft in orthopedic surgery, Clin. Orthop. 272, 82-87. KOMENDER, J., MALCZEWSKA, H. and KOMENDER, A. (1991). Therapeutic effects of transplantation of lyophilized and radiation-sterilised, allogeneic bone, Clin. Orthop. 272, 38-49. DZIEDZIC-GOCLAWSKA, A., OSTROWSKI, K., STACHOWICZ, W., MICHALIK, J. and GRZESIK, W. (1991). Effect of radiation sterilisation on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing, Clin. Orthop. 272, 30-37. ANGERMANN, P. and JEPSEN, O.B. (1991). Procurement, banking and decontamination of bone and collagenous tissue allografts: guidelines for infection control, /. Hosp. Infect. 17, 159-169. LOTY, B., COURPIED, J.P., TOMENO, B., POSTEL, M., FOREST, M. and ABELANET, R. (1990). Bone allografts sterilised by irradiation. Biological properties, procurement and results of 150 massive allografts, Inst. Orthop. 14, 237-242. WEINTROUB, S. and REDDI, A.H. (1988). Influence of irradiation on the osteoinductive potential of demineralised bone matrix, Calcif. Tissue Int. 42, 255-260. MACDOWELL, S. (1988). Irradiated cartilage, Plast. Surg. Nurs. 8, 14-15.

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WANGERIN, K., EWERS, R. and BUMANN, A. (1987). Behaviour of differently sterilised allogenic lyophilized cartilage implants in dogs, /. Oral Maxillofac. Surg. 45, 236-242. LINBERG, J.V., ANDERSON, R.L., EDWARDS, J.J., PANJE, W.R. and BARDACH, J. (1980). Preserved irradiated homolgous cartilage for orbital reconstruction, Opthalmic Surg. 11, 4 5 7 462. HOROWITZ, M. (1979). Sterilisation of homograft ossicles by gamma radiation, /. Laryngol. Otol. 93, 1087-1089. KOMENDER, J., MALCZEWSKA, H. and LESIAK-CYGANOWSKA, E. (1978). Preserved bone in clinical transplantation, Arch. Immunol. Ther. Exp. (Warz) 26, 1071-1073. KOMENDER, J. (1978). Evaluation of radiation-sterilised bone and clinical use, Acta Med. Pol. 19, 277-281. BURWELL, R.G. (1976). The fate of freeze-dried bone allograft, Transplant. Proc. 8, 95-111. DEXTER, F. (1976). Tissue banking in England, Transplant. Proc. 8, 43-48. KOMENDER, J., KOMENDER, A., DZIEDZIC-GOCLAWSKA, A. and OSTROWSKI, K. (1976). Radiation-sterilised bone grafts evaluated by electron spin resonance technique and mechanical tests, Transplant. Proc. 8, 25-37. URIST, M.R. and HERNANDEZ, A. (1974). Excitation transfer in bone. Deleterious effects of cobalt 60 radiation-sterilisation of bank bone, Arch. Surg. 109, 586-593. IMAMALIEV, A.S. and GASIMOV, R.R. (1974). Biological properties of bone tissue conserved in plastic material and sterilised with gamma rays (clinico-experimental study), Acta Chir. Plast. 16, 129-135.

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OSTROWSKI, K., DZIEDZIC-GOCLAWSKA, A., STACHOWICZ, W., MICHALIK, J., TARSOLY, E. and KOMENDER, A. (1971). Application of the electron spin resonance technique for quantitative evaluation of the resorption rate of irradiated bone grafts, Calcif. Tissue Res. 7, 58-66. TARSOLY, E., OSTROWSKI, K., MOSKALEWSKI, S., LOJEK, T., KURNATOWSKI, W. and KROMPECHER, S. (1969). Incorporation of lyophilized and radiosterilised perforated and unperforated bone grafts in dogs, Acta Chir. Acad. Sci. Hung. 10, 55-63. OSTROWSKI, K., KECKI, Z., DZIEDZIC-GOCLAWSKA, A., STACHOWICZ, W. and KOMENDER, A. (1969). Free radicals in bone grafts sterilised by ionizing radiation, Sb. Ved. Pr. Lek. Fak. Karlovy Univerzity Hradci Kralove Suppl.: 561-563. MARQUIT, B. (1967). Radiated homogenous cartilage in rhinoplasty, Arch. Otolaryngol. 85, 78-80.

D.2. HIV SMITH, R.A., INGELS, J., LOCHEMES, J.J., DUTKOWSKY, J.P. and PIFER, L.L. (2001). Gamma irradiation of HIV-1, /. Orthop. Res. 19, 815-819. HERNIGOU, P., GRAS, G., MARINELLO, G. and DORMONT, D. (2000). Inactivation of HIV by application of heat and radiation: implication in bone banking with irradiated allograft bone, Acta Orthop. Scand. 71, 508-512. CAMPBELL, D.G. and LI, P. (1999). Sterilisation of HIV with irradiation: relevance to infected bone allografts, Aust. N. Z. J. Surg. Jul 69, 517-521. SALAI, M., VONSOVER, A., PRITCH, M., VON VERSEN, R. and HOROSZOWSKI, H. (1997). Human immunodeficiency virus

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(HIV) inactivation of banked bone by gamma irradiation, Ann. Transplant. 2, 55-56. FIDELER, B.M., VANGNESS, C.T. Jr., MOORE. T., LI, Z. and RASHEED, S. (1994). Effects of gamma irradiation on the human immunodeficiency virus. A study in frozen human bonepatelar ligament-bone grafts obtained from infected cadavera, J. Bone Joint Surg. Am. 76, 1032-1035. CAMPBELL, D.G., LI, P., STEPHENSON, A.J. and OAKESHOTT, R.D. (1994). Sterilisation of HIV by gamma irradiation. A bone allograft model, Int. Orthop. 18, 172-176. BEDROSSIAN, E.H. Jr. (1991). HIV and banked fascia lata, Ophthal. Plast. Reconstr. Surg. 7, 284-288.

D.3. Biomaterials HOLY, C.E., CHENG, C , DA VIES, J.E. and SHOICHET, M.S. (2001). Optimizing the sterilisation of PLGA scaffolds for use in tissue engineering, Biomaterials 22, 25-31. ANDRIANO, K.P., CHANDRASHEKAR, B., MCENERY, K., DUNN, R.L., MOYER, K., BALLIU, CM., HOLLAND, K.M., GARRETT, S. and HUFFER, W.E. (2000). Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix, /. Biomed. Mater. Res. 53, 36-43. CHEUNG, D.T., PERELMAN, N., TONG, D. and NIMNI, M.E. (1990). The effect of gamma-irradiation on collagen molecules, isolated alpha-chains and cross linked native fibers, /. Biomed Mater. Res. 24, 581-589. BRUCK, S.D. and MUELLER, E.P. (1988). Radiation sterilisation of polymeric implant materials, /. Biomed. Mater. Res. 22, 133144.

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SCHWARZ, N., REDL, H., SCHIESSER, A., SCHLAG, G., THURNHER, M., LINTNER, F. and DINGES, H.P. (1988). Irradiation-sterilisation of rat bone matrix gelatin, Acta Orthop. Scand. 59, 165-167. PHILLIPS, G.O. (1984). Chemical processes induced during radiation sterilisation of cellulose, Anselme Payen Award Symposium at American Chemical Society, 188th National Meeting (Philadelphia). NAKAMURA, Y., OGIWARA, Y. and PHILLIPS, G.O. (1985). Free Radical Formation and Degradation of Cellulose by Ionising Radiations, Polymer Photochemistry 6, 135-159. PHILLIPS, G.O. (1985). Radiation Degradation of Cellulosic Systems. In: Proc. Int. Symp. Fiber Science and Technology (Hakone, Japan), 88-90. WOZNIAK-PARNOWSKA, W. and NAJER, A. (1978). Studies on the sterilisation of pharmaceutical base materials with ionizing radiation and ethylene oxide, Acta Microbiol. Pol. 27, 161-168.

B.4. Soft tissues TYSZKIEWICZ, J.T., UHRYNOWSKA-TYSZKIEWICZ, LA., KAMINSKI, A. and DZIEDZIC-GOCLAWSKA, A. (1999). Amnion allografts prepared in the Central Tissue Bank in Warsaw, Ann. Transplant. 4, 85-90. MARTINEZ PARDO, M.E., REYES FRIAS, M.L., RAMOS DURON, L.E., GUTIERREZ SALGADO, E., GOMEZ, J.C., MARIN, M.A. and LUNA ZARAGOZA, D. (1999). Clinical application of amniotic membranes on a patient with epidermolysis bullosa, Ann. Transplant. 4, 69-73. JOHNSON, K.A., ROGERS, G.J., ROE, S.C., HOWLETT, C.R., CLAYTON, M.K., MILTHORPE, B.K. and SCHINDHELM, K.

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(1999). Nitrous acid pretreatment of tendon xenografts crosslinked with glutaraldehyde and sterilised with gamma irradiation, Biomaterials 20, 1003-1015. MAEDA, A., INOUE, M., SHINO, K., NAKATA, K., NAKAMURA, H., TANAKA, M., SEGUCHI, Y. and ONO, K. (1993). Effects of solvent preservation with or without gamma irradiation on the material properties of canine tendon allografts, /. Orthop. Res. 11, 181-189. HINTON, R., JINNAH, R.H., JOHNSON, C , WARDEN, K. and CLARKE, H.J. (1992). A biomechanical analysis of solventdehydrated and freeze-dried human fascia lata allografts. A preliminary report, Am. J. Sports. Med. 20, 607-612. BUMANN, A., KOPP, S., EICKBOHM, J.E. and EWERS, R. (1989). Rehydration of lyophilised cartilage grafts sterilised by different methods, Int. J. Oral. Maxillofacial Surg. 18, 370-372. CANTORE, G., GUIDETTI, B. and DELFINI, R. (1987). Neurosurgical use of human dura mater sterilised by 7 rays and stored in alcohol: long term results, /. Neurosurg. 66, 93-95. ARMAND, G., BAUGH, P.J., BALAZS, E.A. and PHILLIPS, G.O. (1975). Radiation protection of hyaluronic acid in the solid state, Radial Res. 64, 573-580. HALL, A.N., PHILLIPS, G.O. and RASSOL, S. (1978). Action of ionizing radiations on a hyaluronate tetrasaccharide, Carbohydrate Res. 62, 373-376. MOORE, J.S., PHILLIPS, G.O. and RHYS, D. (1973). Chemical effects of ?-irradiation of aqueous solutions of chondroitin-4sulphate, Int. J. Radial Biol. 23(2), 113-119. LITWIN, S.B., COHEN, J. and FINE, S. (1973). Effects of sterilisation and preservation on the rupture force and tensile strength of canine aortic tissue, /. Surg. Res. 15, 198-206.

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DONNELLY, R.J., APARICIO, S.R., DEXTER, E, DEVERALL, P.B. and WATSON, D.A. (1973). Gamma-radiation of heart valves at 4 degrees C; a comparative study using techniques of histochemistry and electron and light microscopy, Thorax 28, 95-101. MANDELCORN, M.S. and CRAWFORD, J.S. (1972). Feasibility of a bank for storage of human fascia lata sutures, Arch. Opthalmol. 87, 535-537. KORLOF, B., SIMONI, E., BARYD, I., LAMKE, L.O. and ERIKSSON, G. (1972). Radiation-sterilisation split skin: a new type of biological wound dressing. Preliminary report, Scand. J. Plast. Reconstr. Surg. 6, 126-131. RITTENHOUSE, E.A., SANDS, M.P., MOHRI, H. and MEERENDINO, K.A. (1970). Sterilisation of aortic valve grafts for transplantation, Arch. Surg. 101, 1-5. WELCH, W. (1969). A comparative study of different methods of processing aortic homograft, Thorax 24, 746-749. MALM, J.R., BOWMAN, F.O. Jr., HARRIS. P.D., KAISER. G.A. and KOVALIK, A.T. (1969). Results of aortic valve replacement utilizing irradiated valve homografts, Ann. N.Y. Acad. Sci. 30, 740-747. BALAZS, E.A., DAVIES, J.V., PHILLIPS, G.O. and YOUNG, M. (1967). Transient intermediates in the radiolysis of hyaluronic acid, Radiat. Res. 31, 243-255.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

9 IAEA INTERNATIONAL STANDARDS FOR TISSUE BANKS

A N IAEA CONSULTATION D O C U M E N T

1. Introduction The document is divided in two parts. Part I contains international standards for tissue banks. The standards include two sections. Section 1.1 deals with general and organisational policies. Section 1.2 deals with the implementation of these policies. Part II is a guide for legal and regulatory control. The guidelines in Part II advise regulatory bodies on the aspects which must be considered in setting up a system and evaluating compliance with that system. In Part I, international standards for tissue banks have been established by the IAEA that should be used as a starting point for good tissue banking practices. These standards describe the safety and quality dimensions of human tissue for transplantation, quality management, processing method and tissue sterilisation and validation. These standards apply to all types of tissues, including corneas and cells (see definitions). Part II is a guide for legal and regulatory control. In order for a tissue banking programme to be successfully implemented, there is need for a variety of laws and regulations to be legislated and enforced. These laws and regulations should cover the safety of the tissue to recipients as well as ethical concerns such as maintaining the dignity of the donor and his/her family and 267

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respect the gratuity of the donation. Regulations should be based on standards adopted by the country, individual tissue banks or associations representing tissue banks in the specific country/ area. An international or intergovernmental approach to the development of laws and regulations is suggested for those areas of the world that have common legal systems, eliminating redundant or conflicting regulations. In the international atomic energy agency's view, this guide for legal and regulatory control shall present requirements in a form that can be used for establishing tissue banks and determining whether a tissue bank complies with current good tissue banking practices. It shall also serve as an aid for interpreting and clarifying the standards. It is also intended to support the harmonisation of inspection and internal audit procedures. The reasons for the justification of this guide for legal and regulatory controls are clear: there is need to protect the health and well-being of the citizens, encourage cost-effective and improved healthcare, promote social programmes that work for the well being of the community, prohibit unethical practices, avoid health hazards associated with the distribution and transplantation of tissues and to protect against tissue banks that refuse to adhere to acceptable practices. I. International Standards for Tissue Banks 1.1. General and organisational policies 1.1.1.

Introduction

The international standards for tissue banks apply to human tissues used for therapeutic purpose, excluding reproductive and genetically modified tissues. They do not apply to animal tissues. The purpose of the international standards for tissue banks is to bring together the current State of the Art practice on selection of donors, tissue retrieval, testing, processing, storage, labeling and distribution of finished tissue, in order to provide safe tissue of reliable quality while respecting the ethical rules.

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The therapeutic use of tissues raises ethical and safety concerns. The safety of tissues includes the following aspects: avoiding transmission of communicable diseases including bacteria, parasites, viruses, prions and of tumours; avoiding adverse events due to additives and residues from chemical or physical methods of processing; preserving efficient biological qualities and assuming reproducibility and traceability. Besides bacterial and parasitic infection, several cases of viral disease* and Creutzfeldt-Jakob** disease transmission have been reported in the literature. These events should be compared with the thousands of patients that have received tissues successfully, but imply the need for preventive measures. Not only the risks, but also the risk — benefit balance has to be considered. Risks include known risks, which imply preventive measures and unknown risks, which call for precautionary measures. On the other side, the benefit and the existence or absence of alternative treatments should be appreciated. The factors of clinical safety are well known and include donor selection, retrieval conditions, processing protocol and controls, distribution protocol, traceability and record keeping, including proper indication, surgical technique and postoperative care. See Annex 1.1 for definitions. 1.1.2. Ethical and legal rules In each country, the applicable inter-governmental, national, regional and local law or regulation governing consent and retrieval of tissues from living or cadaver donors shall be followed. Recommendations about the ethical aspects of the use of human tissues for therapeutic purpose have been published by Two cases of HIV, three of hepatitis B and two of hepatitis C. Three cases through corneas and more than sixty through non-viable freedried dura-mater.

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the World Health Organisation (WHA 44.25, May 1991) and Council of Europe (78-29, May 1978). The Council of Europe has also adopted a convention on the human rights and biomedicine (Oviedo, April 1997) and is preparing an additional protocol to the convention on transplantation of organs and tissues of human origin. Recommendations about the safety aspects of tissue banking have also been adopted by the Council of Europe [Recommendation No. R (94) 1 on Human Tissue Banks] and by the European Group on Ethics in Science and New Technologies to the European Commission (Opinion on Ethical Aspects of Human Tissue Banking, adopted on 21 July 1998). 1.1.2.1. Permission for tissue retrieval Permission for tissue retrieval is governed by the following principles if there is no applicable inter-governmental, national, regional and local law or regulation: Cases of living donor consent comprise voluntary donation of tissue and collection of surgical residues. In the case of voluntary donation of tissue, appropriate medical investigation shall be made to evaluate and reduce the risk to the health of donor and recipient. The donor must be given appropriate information before the removal about the possible consequences of this removal, in particular medical, social and psychological, as well as the importance of the donation for the recipient. An informed consent in writing shall be obtained from the living donor. Consent before an official body may be necessary according to applicable inter-governmental, national, regional and local law or regulation. In the case of a minor or otherwise legally incapacitated person, informed consent shall be obtained from his legal representative, if the donor does not object to it. The appropriate authority shall be consulted in accordance to applicable inter-governmental, national, regional and local law or regulation. The donation of substances, which cannot regenerate, is usually confined to transplantation

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between family related persons and restricted to major and capable persons. Surgical residues are collected during a surgical procedure where the material is collected for therapeutic purpose other than to obtain tissue (e.g. femoral head, skin and amnion). Informed consent shall be obtained from the donor according to applicable regulation. Non-living donor consent is governed by the following guidelines. No removal of tissue will take place when there was an open or presumed objection on the part of the deceased. Permission or confirmation of the absence of objection for tissue donation shall be obtained from the next of kin. In case of a minor or legally incapacitated person, the consent of his legal representative is required. Removal of tissue can be effected if it does not interfere with a forensic examination or autopsy as required by law. Consent for tissue donation shall be documented. The consent form shall specify whether there is a general permission for organs a n d / o r tissues or permission for specified organs a n d / o r tissues only. 1.1.2.2. Monetary inducement for donation Monetary inducement for donation is subject to the following restrictions. Payment to the donor is prohibited. Monetary payment or advantages for the donation shall not be made to living donors, cadaver donor's next of kin or any donor-related party. As regards compensation for donation-related expenses, donors or their family shall not be financially responsible for expenses related to retrieval of tissues. 1.1.2.3.

Anonymity

Anonymity between donor and unrelated recipient shall be strictly preserved. Anonymity between donor and recipient shall allow tracking of tissues, through anonymous identification numbers.

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1.1.3. Organisation of a tissue bank 1.1.3.1. Institutional identity and authorisation The purpose and institutional identity of a tissue bank shall be clearly established and documented. The tissue bank shall state whether it is a free standing entity or part of an institution. As regards authorisation, licensing or registration, the tissue bank shall comply with all applicable inter-governmental, national, regional and local law or regulation for authorisation, licensing or registration. Collaboration with other organisations can have the form of a written agreement or an on-site audit. Each tissue bank shall have written agreements or contracts with all other organisations, which perform donor screening services, tissue retrieval, processing or distribution for the tissue bank. Tissue banks which contract for laboratory services shall verify the laboratory licensing or accreditation, according to applicable intergovernmental, national, regional and local law or regulation. The tissue bank shall maintain documentation, which is auditspecific for the services performed for the tissue bank. Such documentation shall itemise all operational systems, which were audited to determine compliance with standards or applicable regulation. 1.1.3.2. Personnel The personnel of a tissue bank includes a medical director, an administrative director and staff. The medical director shall be qualified by training and experiences for the scope of activities being pursued in accordance with applicable inter-governmental, national, regional and local law or regulation. The medical director shall be responsible for medical operations, including compliance with these standards. His/her responsibilities include determining what tissues are to be collected, define donor screening policies and prescribe technically acceptable means for their processing, quality assurance, storage and distribution. The medical director

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shall be responsible for policies and procedures regarding donor suitability and adverse events. It is recommended that a tissue bank set up a medical advisory board to provide medico-technical and scientific advice (external from the tissue bank). The administrative director, when applicable, shall be responsible for administration, management, and other general activities. The administrative director shall not be responsible for medical activities. The tissue bank shall have sufficient qualified personnel for pursuing the various tasks. The tissue bank staff must possess the educational background, experience and training, sufficient to assure assigned tasks are performed in accordance with the tissue banks established procedures. The technical staff shall be responsible for implementation of policies and procedures as established by the medical director. The duties of each staff member shall be described in a written job description. Staff must demonstrate competency in operations to which they are assigned. The scope of activities, specific staff responsibilities and reporting structure shall be established by the medical director. The medical director shall ensure that all staff members have adequate training to perform their duties safely and competently. The medical director shall be responsible for ensuring that technical staff maintain their competency by participation in training courses and technical meetings or other educational programmes. All staff shall review applicable institutional policies and procedures annually and when changes are made. 1.1.3.3. Quality management system In order to reduce the risk for patients by the transplantation of tissues to an acceptable level, it is necessary to operate an effective quality management system. The system may include extensive testing of donor blood and tissue samples, but this alone is not sufficient guarantee of safety

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and efficacy and the system should include other management and control measures. Those involved in procuring, processing and supplying tissues for transplantation shall perform in addition, a risk analysis of procedures prone to error and to disease transmission. The results of this risk analysis should be used to develop safe procedures and implement a quality management system based on clearly identified requirements for tissues. The quality requirements should form the basis of all quality assurance and quality control programmes. It is necessary to define the quality requirements not only for the final product, but also for the starting material collected, reagents and equipment used, staff competencies, testing techniques, packaging materials, labels and process intermediates. These quality requirements are best prescribed and quantified in written specifications. These specifications determine the quality control testing or inspection performed on which the release decisions are based. The quality requirements will be based on characteristics that effect both patient safety and maintaining the clinical effectiveness of the product. It is recognised that quality has to be managed in an organisation and that a systematic approach is the only way to ensure that the quality of products produced and services delivered consistently meets the quality requirements. The high level of quality assurance required for safety, critical therapeutic medical products and clinical services can only be achieved through the implementation of an effective quality management. The international standard for quality management is the ISO 9000 series. Specific principles to be incorporated into the quality systems covering the manufacture and quality control of medicines are known as good manufacturing practice (GMP). The ISO standards, GMP or other applicable standards and other applicable inter-governmental, national, regional and local law or regulation, should be consulted when developing a quality management for tissue banking organisations and other procurement organisations.

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The basic elements of an appropriate quality management system are organisational structure and accountability, documentation, the control of the various processes, and record keeping. Organisational structure and accountability are necessary to achieve the quality requirements and for reviewing the effectiveness of the arrangements for quality assurance. There should be a suitably qualified and experienced member of staff appointed who verifies that the quality requirements are being met, and that there is compliance with the quality management system. The quality manager should be a designated individual who should be independent of production (not directly responsible for or involved in the procurement, processing and testing of tissue) and preferably of other responsibilities within the tissue bank. The quality manager should be generally familiar with the specific work being reviewed and be responsible for each quality assurance review. This individual should report, for his function, specifically to the medical director a n d / o r his/her designee. Where a tissue bank is operated within a large organisation with its own quality department and possibly its own quality manager then strong working links should exist between the tissue bank's quality manager and the relevant quality department staff, as well as to the medical director. Documentation serves the proper flow of information, it should be properly controlled, and properly stored and retrieved. The rationale behind a documentation system is to meet certain objectives. The objectives of thorough documentation are to define the system of information and control, to minimise the risk of misinterpretation and error inherent in oral or casually written communication and to provide unambiguous procedures to be followed. Documents should clearly state the quality requirements, organisational structures and responsibilities, the organisation's policies and standards, the management and technical procedures employed and the records required. All procedures in the processing of tissue should be documented and the documents controlled. Documentation should be legible, readily identifiable and retrievable. Documentation

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should clearly identify the way in which it is to be used and by whom. Documentation should be available to staff to cover all procedures. Any correction should be handwritten clearly and legibly in permanent ink and signed and dated by an authorised person. The system for document control should identify the current revision status of any document and the holder of the document. The system in place should demonstrate that all controlled documents meet the following criteria: • • • •

they are current and authorised; they are reviewed at regular intervals; multiple copies are controlled with a distribution list; obsolete documents are removed and controlled to prevent further use; and • changes to documents should be acted upon promptly. They should be reviewed, dated and signed by the authorised person and formally implemented.

As regards storage and retention of documentation, documented procedures should be established and maintained for identification, collection, filing, storage, retrieval and maintenance of all documents. Master copies of obsolete copies should be archived in a secure and safe environment for 10 years or in accordance with applicable inter-governmental, national, regional and local law or regulation. The control of processes refers to written instructions of standard operating procedures (SOPs) shall be produced where it is essential that tasks must be performed in a consistent way. Equipment, processes and procedures shall be validated as effective before being implemented or changed. Equipment essential to the quality of the product shall be routinely serviced and calibrated, if appropriate. The processing environment and staff performing processes shall meet minimum, prescribed standards of cleanliness and hygiene. The tissue bank shall maintain a SOPs manual which details in writing all aspects of these standards. The SOPs shall be utilised to ensure that all material released for transplantation

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meet at least minimum requirements defined by professional standards and applicable inter-governmental, national, regional and local law or regulation. The SOPs manuals should include, where relevant, but should not be limited to the following: • standard procedures for donor screening, consent, retrieval, processing, preservation, testing, storage and distribution; • quality assurance and quality control policies; • laboratory procedures for tests performed in-house and in contracted laboratories; • specifications for materials used including supply, reagents, storage media and packaging materials; • personnel and facility safety procedures; • standard procedures for facility maintenance, cleaning and waste disposal procedures; • methods for verification of the effectiveness of sterilisation procedures; • equipment maintenance, calibration and validation procedures; • environmental and microbiological conditions and the methods used for controlling, testing and verification; • physiological and physical test specifications for materials; • methods for determination of shelf life, storage temperature and assigning expiry dates of tissues; • determination of insert and or label text; • policies and procedures for exceptional release of material; • procedures for adverse events reporting and corrective actions; and • donor/recipient tracking and product recall policies and procedures. All SOPs, their modification and associated process-validation studies shall be reviewed and approved by either the medical or administrative director as dictated by content. All medically related SOPs shall be reviewed and approved by the medical director. Copies of the SOPs manual shall be available to all staff, and to authorised individuals for inspections upon request. Upon

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implementation, all SOPs shall be followed as written. SOPs shall be updated at regular intervals to reflect modifications or changes. The authorised person, depending on the content shall approve each modification or change. Appropriate training shall be provided to pertinent staff. Obsolete SOPs manuals shall be archived for a minimum of 10 years taking into account the shelf life of the material. Record keeping refers to contract records, donor tracking, inventory, adverse events, and electronic records. Records shall be confidential, accurate, complete, legible and indelible. All donor, processing, storage, and distribution records should be maintained for 30 years or in accordance with applicable inter-governmental, national, regional and local law or regulation. Records shall hold all information that identifies the origins of the product and to demonstrate that the product meets all the quality requirements. Records shall show that all the required processing steps and all quality control tests have been performed correctly by trained staff and that the product has only been released for use after the correct authorisation. Records shall also demonstrate correct handling and storage of materials and track the final status of products, whether transplanted, discarded or used for research. The use and storage of records shall be controlled. When two or more tissue banks participate in tissue procurement, processing, storage or distribution functions, the relationships and responsibilities of each shall be documented and ensure compliance with relevant scientific and quality professional standards by all parties. Tissue banks should perform on-site audits of contract laboratories to ensure their compliance with relevant scientific and professional standards, technical manuals and the tissue bank's own requirements. Each component shall be assigned one unique identifier that shall serve as a lot number to identify the material during all steps from collection to distribution and utilisation. This unique number shall link the final packaged material to the

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donor. This number shall be used to link the donor to all tests, records, organs and other material, and for tracking purposes to the recipient. Records shall include identification and evaluation of the donor, blood testing and micro-biological evaluation of the donor, conditions under which the material is procured, processed, tested and stored and its final destination. Records shall indicate the dates and identity of staff involved in each significant step of the operation. A record of unprocessed, processed, quarantined and distributed tissues shall be maintained. A file of recipient adverse events shall be maintained including any non-compliance. If a computer record-keeping system is used, there shall be a system to ensure the authenticity, integrity and confidentiality of all records but retain the ability to generate true paper copies. A description of the system, its function and specified requirements must be documented. The system shall record the identity of persons entering or confirming critical data. Alteration to the system or programme shall only be made in accordance with defined procedures. When the release of finished batches is conducted by computerised systems it must identify and record the person(s) releasing the batches. Alternative management systems should be available to cope with failures in computerised systems. Methods should be inplemented for detecting, correcting and preventing quality failures from recurring. Quality failures include in-use product deficiencies (complaints, adverse events, etc.), failures to meet quality control specifications and non-compliance with procedures. Methods for detecting failures include quality control tests, inspections, quality audits, staff and end-user feedback. The ability to trace, locate, quarantine and recall materials, consumables and products at any stage, is essential to patient safety. Serious failures shall be thoroughly registered, investigated and appropriate changes to specifications, systems and procedures implemented to prevent further failures of a similar nature.

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The tissue bank shall participate in an audit programme. Quality assurance staff shall perform internal audits. Focused audits shall be conducted to monitor critical areas and when problems with quality have been identified. Regular audits shall be performed by qualified staff who do not have direct responsibility for the processes being audited. The educational and training requirements for each member of staff shall be determined and specified. There shall be regular and formal appraisal of staff competency. Training and education shall include the requirements for quality, standards of practice and good hygiene as well as appropriate continuing professional development. Records of training shall be maintained up to date. 1.1.3.4. Facilities and equipment Facilities and equipment should meet standards of security, environmental standards and sanitation. The facilities of the tissue bank shall be of suitable size and location and shall be designed and equipped for the specialised purposes for which they are to be used. The design of the facilities shall prevent errors and cross-contamination. Critical procedures shall be performed in designated areas of adequate size. Access to the tissue bank shall be limited to authorised persons. Environmental monitoring procedures shall be established, when appropriate, as part of the quality assurance programme. The procedures shall include acceptable test parameters. The monitoring may include particulate air samplings and work surface cultures. Each monitoring activity shall be documented. Facilities used for retrieval, processing or preservation, where there is potential for cross-contamination of material or exposure to blood-borne pathogens, shall be subjected to routine, scheduled and documented cleaning procedures. Equipment and instruments shall be of appropriate quality for their intended function. Equipment and non-disposable

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supplies that come into contact with tissue shall be constructed so surfaces do not alter the safety or quality of the material. Equipment shall be designed, manufactured and qualified for appropriate cleaning and shall be sterilised or decontaminated after each use. Multiple uses of disposable instruments for several donors shall be excluded. There shall be SOPs for monitoring, inspection, maintenance, calibration, and cleaning procedures for each piece of equipment. Storage equipment shall be inspected on a regularly scheduled basis. Appropriate certification and maintenance records shall be maintained for equipment and instruments. Each tissue bank shall provide and promote a safe work environment by developing, implementing and enforcing safety procedures. Safety precautions and procedures for maintaining a safe work environment shall be included in the SOPs manual and shall conform to applicable inter-governmental, national, regional and local law or regulation. Safety procedures shall include, but are not limited to the following: • instructions for fire prevention and evacuation routes in case of fire or natural disaster; • procedures for prevention of worker injury including possible exposure to biohazards material; • procedures for proper storage, handling and utilisation of hazardous materials, reagents and supplies; • procedures outlining the steps to be followed in cleaning biohazard spills; • hazardous material training including chemical, biological and radioactive hazards; • immunisation: appropriate vaccinations should be offered to all non-immune personnel whose job-related responsibilities involve potential exposure to blood-born pathogens. Personnel files should include documentation of receipt of vaccination or refusal of vaccination; and • personnel: personnel engaged in the retrieval, processing, preservation and packaging of tissues shall be suitably attired to

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minimise the spread of transmissible pathogens among and between donors, tissue and staff. Any staff member with a serious infectious condition shall be excluded from the tissue banking activities until the condition is resolved. Human tissue and other hazardous waste items shall be disposed of in such a manner so as to prevent hazards to tissue bank personnel or the environment and shall conform to applicable inter-governmental, national, regional and local law or regulation. Dignified and proper disposal procedures shall be applied to human remains. 1.2.

Implementation

1.2.1. Donor selection The suitability of a specific donor for tissue allograft donation is based upon medical and behavioural history, medical records review, physical examination, cadaveric donor autopsy findings (if an autopsy is performed) and laboratory tests. 1.2.1.1. Donor's history The medical and behavioural history includes donor history review and exclusion and selection criteria. Donor evaluation includes an interview of the potential living donor or the cadaveric donor's next of kin, performed by suitably trained personnel, using a questionnaire. A qualified physician shall approve donor evaluation. 1.2.1.2. Exclusion and selection criteria for the donor The following conditions contraindicate the use of tissues for therapeutic purposes: • history of chronic viral hepatitis; • presence of active viral hepatitis or jaundice of unknown etiology;

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• history of, or clinical evidence, or suspicion, or laboratory evidence of HIV infection; • risk factors for HIV, HBV and HCV have to be assessed by the medical director according to existing national regulations taking into account national epidemiology. Annex 1.2 includes a generally agreed list of risk factors; • presence or suspicion of central degenerative neurological dseases of possible infectious origin, including dementia (e.g. Alzheimer's Disease, Creutzfeldt-Jakob disease or familial history of Creutzfeldt-Jakob disease and multiple sclerosis); • use of all native human pituitary derived hormones (e.g. growth hormone), possible history of dura-mater allograft, including unspecified intracranial surgery; • septicemia and systemic viral disease or mycosis or active tuberculosis at the time of procurement preclude procurement of tissues. In case of other active bacterial infection, tissue may be used only if processed using a validated method for bacterial inactivation and after approval by the medical director; • presence or history of malignant disease. Exceptions may include primary basal cell carcinoma of the skin, histologically proven and unmetastatic primary brain tumour (see Annex 1.3); • significant history of connective tissue disease (e.g. systemic lupus erythematosus and rheumatoid arthritis) or any immunosuppressive treatment; • significant exposure to a toxic substance that may be transferred in toxic doses or damage the tissue (e.g. cyanide, lead, mercury and gold); • presence or evidence of infection or prior irradiation at the site of donation; and • unknown cause of death. If at the time of death the cause of death is unknown, autopsy shall be performed to establish this cause. As regards specific tissue selection criteria, cornea donors with solid extra-ocular malignancies are generally accepted.

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1.2.1.3. Examination and autopsy Prior to procurement of tissue, the donor body shall be examined for general exclusion signs and for signs of infection, trauma or medical intervention over donor sites that can affect the quality of the donated tissue. If an autopsy is performed, the results of the cadaveric donor autopsy report shall be reviewed by the medical director or designee before tissue is released for distribution. 1.2.1.4. Transmissible diseases blood tests Tissues shall be tested for transmissible diseases in compliance with law and practice in the country concerned. In the case of living donors, applicable consent procedure for blood testing shall be followed. Tests shall be performed and found acceptable on properly identified blood samples from the donor using recognized, and if applicable, licensed tests and according to manufacturer's instructions. Tests shall be performed by a qualified, and if applicable, licensed laboratory and according to good laboratory practice (GLP). Blood for donor testing should be drawn at or within seven days of the donation and preferably within 24 hours after death. For potential tissue donors who have received blood, blood components, or plasma volume expanders within 48 hours prior to death, if there is an expected hemodilution of more than 50%, based on calculation algorithm (see example of algorithm in Annex 1.4), a pre-transfusion blood sample shall be tested. The living donor or cadaver donor's next of kin or physician shall be notified in accordance with state laws of confirmed positive test results having clinical significance. Confirmed positive donor infectious disease tests shall be reported to local/national health authorities, when required. A sample of donor serum shall be securely sealed and stored frozen in a serum archive in a proper manner until 5 years after

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the expiration date of the tissue or according to applicable intergovernmental, national, regional and local law or regulation. Minimum blood tests shall include: • • • •

human immunodeficiency virus antibodies (HIV-l/2-Ab); hepatitis B virus surface antigen (HBs-Ag); hepatitis C virus antibodies (HCV-Ab); and syphilis: nonspecific (e.g. VDRL) or preferably specific (e.g. TPHA).

Optional blood tests could be necessary for compliance with applicable inter-governmental, national, regional and local law or regulation a n d / o r to screen for the following endemic diseases: • hepatitis B core antibodies (HBc-Ab): HBc-Ab should be negative for tissue validation. Though, if the HBc-Ab test is positive and the HBs-Ag is negative, confirmation cascade should be entered. If the antibodies against the surface antigen are found (HBs-Ab), the donor can then be considered to have been recovered from an infection and the tissue can then be used for transplantation; • antigen test for HIV (p24 antigen) or HCV, or validated molecular biology test for HIV and HCV (e.g. PCR), if performed by an experienced laboratory; • antibody to HTLV 1: depending on the prevalence in some regions; • cytomegalovirus (CMV), Ebstein-Barr virus (EBV) and toxoplasmosis antibodies: for immunosuppressed patients; and • alanine aminotransferase (ALT) for living donors. In addition to the general testing requirements, testing living donors of tissue for alanine aminotransferase (ALT) is recommended. Retesting of living donors for HIV and HCV at 180 days is recommended. If another method of increasing safety, rather than retesting (antigen testing, molecular biology or viral inactivation method) is used (and allowed by applicable regulation), it shall be documented and validated. In general, positive results for HIV, hepatitis and HTLV-1 are reasons for exclusion.

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Specifically, in life threatening situations for the recipient (e.g. related HPC donation), positive results for hepatitis are no reason for exclusion, in accordance to applicable regulations. In these situations, tissues with a higher risk for recipient may be offered as long as full information is given to the recipient or, if it is not possible, to his relatives. 1.2.1.5. Bacteriological studies of donor and tissues In applying bacteriological testing methods, representative samples of each retrieved tissue have to be cultured, if the tissues are to be aseptically processed without terminal sterilisation. Samples shall be taken prior to exposure of the tissue to antibiotic containing solution. The culture technique shall allow for the growth of both aerobic and anaerobic bacteria as well as fungi. Results shall be documented in the donor record. Blood culture, if procurement is performed on a cadaver donor, may be useful in evaluating the state of the cadaver and interpreting the cultures performed on the grafts themselves. They shall be reviewed by the medical director or designee. Bacteriological bioburden limits refer to low and high virulence microorganisms. If bacteriological testing of tissue samples obtained at the time of donation reveals growth of low virulence microorganisms, which are commonly considered nonpathogenic, the tissue may not be distributed without being further processed in a way that effectively decontaminates the tissue. Tissue from which high virulence microorganisms have been isolated are not acceptable for transplantation, unless the procedure has been validated to effectively inactivate the organisms without harmful potential effects, taking into account possible endotoxins. 1.2.1.6. Non-microbiological tests Non-microbiological tests depend upon the tissues and cells to be transplanted. Haematopoietic progenitor cell donor selection requires as a minimum:

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• ABO blood and rhesus group; • human leucocyte antigen typing (HLA); and • whole blood cell count. 1.2.1.7. Age criteria Donor age criteria for each type of tissue shall be established and recorded by the tissue bank. 1.2.1.8. Cadaver donor retrieval time limits Tissues shall be retrieved as soon after death as is practically possible. Specific time limits vary with each tissue obtained, which shall be determined by the medical director. Usually, procurement of tissues should be completed within 12 hours after death (or circulatory arrest if also an organ donor). If the body has been refrigerated within 4 to 6 hours of death, procurement should preferably start within 24 hours and no later than 48 hours. 1.2.2. Tissue retrieval There shall be documented procedures, which detail all requirements for retrieval to ensure that these processes are carried out under controlled conditions. Retrieval shall be performed using techniques appropriate to the specific tissue recovered, taking into consideration the eventual utilisation of the tissue. 1.2.2.1. Non-living donor tissue retrieval Issues relating to non-living donor tissue retrieval are the determination of death, identification of the donor, retrieval conditions, and body reconstruction. Tissue bank physicians or physicians involved in removal or transplantation shall not pronounce death nor sign the

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death certificate of any individual from whom tissue will be collected. Inter-governmental, national, regional and local law or regulation concerning determination of death shall be respected. Precise identification of the cadaver donor shall be performed before procurement begins. Procurement shall be accomplished in an operating room or adequate mortuary facility. All instruments and equipment used for procurement shall be sterilised between procurements. Tissues may be removed using either aseptic or clean/nonsterile procurement techniques, as follows: • Aseptic technique: aseptic technique shall be observed throughout the procurement procedure. Procurement sites shall be prepared using a standard surgical technique; all methods shall be consistent with standard operating room practice. • Clean/non-sterile technique: allografts procured using clean/ non-sterile techniques are suitable for transplantation, if efficient validated sterilising methods are used to eliminate pathogens after retrieval. Samples for microbiological testing shall be taken, where applicable. Following tissue procurement, the donor's body is to be reconstructed to closely approximate its original anatomical configuration and to make usual funeral proceedings possible. 1.2.2.2. Surgical residues collection Surgical residues shall be collected under aseptic conditions during a surgical procedure in the operating theatre. 1.2.2.3. Living donor tissue retrieval Tissues must be removed under conditions representing the least possible risk to the donor, in properly equipped and staffed institutions.

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1.2.2.4. Packaging and transportation to the tissue bank Each tissue segment shall be packaged individually as soon as possible after retrieval, using sterile procurement containers, in a manner which will prevent contamination. Containers shall conform to inter-governmental, national, regional and local law or regulation, as appropriate. Proper reagents or preservation solution shall be used, as specified in SOPs. Procedures shall be used for ensuring and documenting proper temperature storage during transit. Procurement container integrity requires that after filling and closing the container, it shall not be re-opened nor the tissue removed until further processing by the tissue bank. Procurement container labelling requires that at all times, the container shall be labelled with the donor and tissue identification, in such manner that traceability of tissues will be achieved. The container shall be labelled as containing human tissue, the name and address of the shipping facility and the name and address of the intended receiving facility. Containers shall comply with additional labelling requirements established by common carriers or by inter-governmental, national, regional and local law or regulation. 1.2.2.5. Retrieval documentation Appropriate records of each donation procedure and all tissues retrieved shall be available and kept by the tissue bank. All retrieved tissue shall be provided with an accompanying retrieval form including, at a minimum: • • • • •

the donor identity; the date, time and place of the procedure; the identity of the person(s) performing the retrieval; the tissue(s) retrieved; and donor and tissue selection information.

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1.2.3. Tissue banking general procedures 1.2.3.1. General specifications The specific methods employed for processing may vary with each type of tissue and with the manner in which it has been retrieved. Each type of tissue shall be prepared according to a written procedure, which shall conform with these standards and other applicable standards, resulting in processed tissues appropriate for safe and efficient clinical use. All steps involved during the processing of tissues shall be validated, when appropriate, to demonstrate the effectiveness of procedures. When computers are used as part of a processing or quality management system, the computer software shall be validated. When validation cannot be adequately evidenced through testing, validation shall be evidenced through documentation demonstrating adequate design, development, verification and maintenance procedures. As regards quality controls, tests and procedures shall be performed to measure, assay or monitor processing, preservation and storage methods, equipments and reagents to ensure compliance with established tolerance limits. Results of all such tests or procedures shall be recorded. Appropriate records of each tissue processed shall be kept by the tissue bank. Records shall allow traceability of tissues, including the different steps in the preparation, the date and time of the procedure, the identity of the person performing the procedure and the record of the materials used. Laboratory results (e.g. microbiology/processing cultures) and other test results used to determine final release shall be archived by the tissue bank distributing the tissue. 1.2.3.2. Unique tissue identification number Each individual tissue shall be marked with a unique identification number to relate each specimen to the individual donor.

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1.2.3.3. Reagents, container and packaging The reagents used in preservation and processing shall be of appropriate grade for the intended use, be sterile, if applicable, and conform to existing regulation. The origin, characteristics and expiration date of reagents shall be monitored and recorded. The type of tissue container may vary with the type of tissue and processing. The containers shall maintain the tissue sterility and integrity, withstand the sterilisation and storage methods utilised and avoid the production of toxic residues. They shall conform to applicable inter-governmental, national, regional and local law or regulation. Each tissue container shall be examined visually for damage or evidence of contamination before and after processing and prior to its dispatch. Packaging shall ensure integrity and effectively prevent contamination of the contents of the final container. It shall conform to applicable transportation regulation. 1.2.3.4. Pooling Tissue from each donor shall be processed and packaged in such a way as to prevent contact and cross-contamination with tissues from other donors. If tissues are subsequently treated in batches (e.g. sterilisation), a unique batch number shall be assigned and added to the records of the tissues. Pooling of donors is not recommended and should only be accepted for specific tissues. The size of the pool should be limited to the minimum number of donors and traceability to each donor has to be ensured. If pooling is used for specific tissues, a fully documented rationale and risk assessment shall be undertaken to document safety. 1.2.3.5. Environmental control Processing steps shall take place in an appropriately controlled environment. Tissue processing in an open system shall

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have the environmental conditions and monitoring of the area clearly defined (such as for a "clean room" or laminar flow cabinet). Records shall be maintained to demonstrate that the area is monitored for microbiological contamination and air control. 1.2.3.6. Storage conditions Acceptable temperature ranges for storage shall be established. Low temperature (refrigerated or frozen) storage devices and incubators shall be connected to a central alarm system or each shall be equipped with an audible alarm system, that will sound when the temperature deviates from the acceptable storage range. The alarm system shall be connected to an emergency power source. Continuous recording and daily review of data are recommended. There shall be a system of quarantine for all tissues to ensure that they cannot be released for clinical use until they have met the defined acceptability criteria for release. Storage areas of quarantined or unprocessed tissue shall be separate from storage areas of tissue approved for processing or ready for distribution. The storage areas shall be clearly labelled as containing quarantined, released for processing or processed finished tissue. 1.2.3.7. Documentation reviewing and tissue inspection Staff shall inspect the tissue container upon arrival from the procurement facility in order to ensure the integrity of the container(s), the presence of proper identification and documentation. The donor's medical history, the physical examination, the results of tissue procurement microbiologic tests and donor blood testing, and if performed, the results of an autopsy, shall be reviewed by the medical director or designee. Quarantined tissues

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shall be reviewed prior to distribution after all testing has been satisfactorily completed. Specimen sizing may be made by actual measurements or by imaging sizing techniques. Prior to the release of tissue into the finished inventory, a final review shall be made of donor suitability, procurement, production, processing records, quality control tests. The finished tissue, containers, closures and labels shall be inspected and approved by the medical director or designee. Prior to distribution, final inspection of the container, label and documentation shall be performed to ensure accuracy and integrity. 1.2.3.8. Non-conforming tissues and expiry dates Tissues failing any portion of the review process shall be maintained in quarantine pending disposal and shall not be released for clinical use. There shall be a documented policy for discard of tissue unsuitable for clinical use. Expiry dates shall be established for all tissue released from a tissue bank. If the dating period is 72 hours or less, the hour of expiration shall be indicated on the label. Otherwise, the dating period ends at midnight of the expiration date. 1.2.4. Specific processing procedures Section 1.1 relating to written procedures, process validation, quality control and record management always applies. None of the tissues rejected due to the ineligibility of the donor can be used for transplantation, even after processing including sterilisation or disinfection. Even if terminal sterilisation or disinfection using physical or chemical agents is used, the procurement and processing shall be adequate to minimise the microbial content of tissues in order to enable the subsequent sterilisation-disinfection process to

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be effective. Appropriate indicators for sterilisation must be included in each sterilisation batch. 1.2.4.1. Disinfectant or antibiotic immersion If disinfectants or antibiotics are used after retrieval, the tissues shall be immersed in a disinfectant or in an antibiotic solution following sterility testing and before final packaging. The type of solution used shall be specified on documentation. 1.2.4.2. Fresh tissue Fresh allografts (e.g. small fragments of articular cartilage and skin) are aseptically procured in an operating room. Fresh tissue is usually stored refrigerated at 4°C or in accordance with written procedures. Fresh tissue shall not be used in a patient until donor blood testing is completed according to these standards, available bacteriologic results are acceptable and donor suitability has been approved by the medical director or designee. 1.2.4.3. Frozen tissue After aseptic procurement in the operating room, frozen tissue is placed in a -40°C or colder controlled environment within 24 hours of procurement. Subsequent manipulation of tissues (e.g. cleaning and cutting) shall be undertaken aseptically. 1.2.4.4. Cryopreserved tissue A cryopreservative solution (e.g. DMSO or glycerol) is usually added to treat the tissue prior to freezing. Documentation of the concentration of cryoprotectants and nutrients or isotonic solutions in the cryopreservative solution shall be maintained. Properly packaged specimens are frozen by placing the specimens below -40°C, or may be subjected to control rate

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freezing using a computer assisted liquid nitrogen freezing device. If a programmed control-rate freezing method is employed, a record of the freezing profile shall be evaluated, approved and recorded. 1.2.4.5. Freeze-dried tissue Various protocols for freeze-drying tissues exist. Freezedrying is a method for preservation, but is not a sterilisation method; sterility shall be assumed by aseptic protocol or additional sterilisation. After a standardised procedure for freeze-drying has been developed, a quality control programme for monitoring the performance of the freeze-dryer shall be documented. Freezedried tissues shall be stored at room temperature or colder. Each freeze-drying cycle must be clearly documented, including length, temperature and vacuum pressure at each step of the cycle. Representative samples shall be tested for residual water content. 1.2.4.6. Simply dehydrated tissue The use of simple dehydration (evaporation) of tissues as a means of preservation shall be controlled in a manner similar to freeze-drying. Temperatures of simple dehydration shall be below 60°C. Each dehydration cycle shall be monitored during operation for temperature. Following dehydration, representative samples shall be tested for residual moisture. 1.2.4.7. Irradiated tissue Commercial or hospital radiation facilities are available for ionising irradiation. The minimum recommended dose for bacterial decontamination is 15 kGy (kiloGray). The minimum

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recommended dose for bacterial sterilisation is 25 kGy (kiloGray). Viral inactivation would require higher doses and depends on numerous factors. For this reason no specific dose can be recommended, but shall be validated, when applicable. The used protocol shall be validated taking in account the initial bioburden, and shall be performed by facilities following good irradiation practices (see IAEA Code of Practice for the Radiation Sterilisation of Biological Tissues). Sterilisation by ionising radiation shall be documented. The processing records include the name of the facility and the resultant dosimetry for each batch. 1.2.4.8. Ethylene oxide sterilised tissue Care should be taken when applying the ethylene oxide sterilisation method since the residues may have toxic effects already demonstrated for musculo-skeletal allografts in the literature. Following appropriate processing procedures, the tissues are placed in ethylene oxide permeable containers and exposed to the ethylene oxide gas mixture following the manufacturer's suggested guidelines. The conditions of exposure may need to be individualised depending upon the nature of the specimens to be sterilised. A quality control programme shall demonstrate that equipment meets requirements in temperature, humidity and gas concentration for the selected period. Following ethylene oxide sterilisation, an appropriate aeration procedure shall be followed, to allow removal of residual ethylene oxide a n d / o r its breakdown products (ethylene chlorhydrin and ethylene glycol). Ethylene oxide sterilisation controls are required. Chemical indicator strips shall be included in each batch. A validated procedure shall be run with each lot of tissue to document that sterilisation has been achieved. Monitoring for residual levels of

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chemicals or their breakdown products shall be conducted from representative samples of the finished tissues of each batch. 1.2.4.9. Other processing methods As regards other inactivation methods, some chemical agents only have a decontamination role. Other agents may have an inactivation effect on specific pathogens. The efficiency of these agents towards the treated type of tissue shall be validated. The use of chemical and possible presence of trace residuals shall be included in the information accompanying the tissue. Under specific conditions, heat may be used to decontaminate or sterilise some type of tissues. The used protocol shall be validated taking in account the initial bioburden and shall be performed by a recognised facility. Several methods and procedures for the formation of demineralised bone are available and acceptable. Controlled quality reagents shall be used. Residual calcium obtained by the method shall be determined. 1.2.5. Labelling 1.2.5.1. General requirements There shall be written procedures designed and followed to ensure that correct labels and labelling are used for tissue identification. Standard measurement nomenclature shall be used to describe tissues and the processing they have undergone. The tissue label applied by the tissue bank facility shall not be removed, altered or obscured. When visual inspection through the container is possible, a sufficient area of the container shall remain uncovered to permit inspection of the contents. 1.2.5.2. Labelling of tissue containers Tissue containers shall be labelled so as to identify, as a minimum:

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• • • • •

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the human nature of the contents; product description; the name and address of tissue bank; tissue identification number; and expiration date.

The following information shall be included on the label, if possible, otherwise on the accompanying documentation: • amount of tissue in the container expressed as volume, weight or dimensions or such combination of the foregoing as needed, for an accurate description of the contents; • sterilisation or inactivation procedure used, if applicable; • batch number, if applicable; • potential residuals of added preserving and processing agents/ solution (e.g. antibiotics, ETOH, ETO, DMSO); and • recommended storage conditions. 1.2.5.3. Package insert All tissues shall be accompanied by a document describing the nature of tissue and processing methods and instructions for proper storage and reconstitution, when appropriate. Specific instructions shall be enclosed with tissue, which require special handling. Accompanying documentation shall contain all the information described for container labelling and the following additional information: • origin of tissue (country of procurement); • the nature and results of biological tests performed on the donor using appropriate and licensed tests; • processing methods used and results of sterility tests or inactivation controls; • special instructions indicated by the particular tissue for storage or implantation. Tissue that is to be reconstituted at or prior to the time of use shall include information on the conditions, under which such tissue shall be stored and reconstituted prior to implantation;

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• indications and contraindications for use of tissue, when necessary; and • statement that each tissue shall be used for a single patient only. 1.2.5.4. Tissue outer package labelling Labelling of the tissue outer package shall conform to transportation regulations, when applicable. 1.2.6. Distribution 1.2.6.1. General Tissues can be distributed for a specific patient to a physician, dentist and other qualified medical professional or to a storage facility located in another institution for local use or distributed to another tissue bank. Distribution for therapeutic use shall be based on medical criteria on equitable bases, in accordance with inter-governmental, national, regional and local law or regulation and practice. There shall be written procedures and documentation for all tissues distributed. The clinical team using the tissue shall have instructions for contacting the tissue bank for any question they have and shall be made aware of the following: • action to be taken in the event of loss of integrity of the package; • action for reporting of adverse event; and • action for the return or the disposal of unsuitable or unused tissue. 1.2.6.2. Traceability There shall be an effective system that enables the traceability of tissues between the donor, the processed tissue and the recipient. It is the responsibility of the hospital tissue storage and

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distribution facility or clinician to implement recipient records and to inform the tissue bank of the destination of tissues (implantation date, surgeon and recipient identification). Tissue banks shall maintain records which document the destination of distributed tissue: implantation (date, surgeon and recipient identification), destruction (date and place) and of any adverse event reports. 1.2.6.3. Transportation Maintenance of (upper a n d / o r lower parameters) environmental conditions during transit, as defined in the written procedure of the tissue bank, shall be ensured. Use of hazardous elements such as dry ice or liquid nitrogen shall comply with relevant regulations. 1.2.6.4. Accompanying documentation The release of tissue from storage shall include all documentation originating from the tissue bank. Surgeons shall be aware that copies of this documentation shall be maintained in the recipient's medical records. 1.2.6.5. Return into inventory Issued tissues shall not be returned to the tissue bank without prior consultations with the medical director or designee. Tissue must be in its original unopened container and the storage conditions must have been maintained as required. 1.2.6.6. Adverse events Reports of adverse events shall be evaluated by the institution where the tissue was used and reported immediately to the tissue bank.

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All adverse events shall be reviewed by the medical director and appropriate action documented, in accordance with inter-governmental, national, regional and local law or regulation. Identified transmission of disease shall be reported to the public health authorities, processing institutions, to the donor's personal physician, if clinically relevant and to physicians involved in implantation of the tissue, in accordance with intergovernmental, national, regional and local law or regulation on confidentiality. When donor to recipient disease transmission through tissue use is discovered, all facilities involved in the procurement and distribution of organs or tissues from the infected donor shall be notified without delay. Written reports of the investigation of adverse events, including conclusions, follow up and corrective actions, shall be prepared and maintained by the tissue bank in an adverse event file. 1.2.6.7. Recall A written procedure shall exist for recall of tissues. 1.2.6.8. Distribution to storage facilities outside the tissue bank (depot) When a storage facility is located outside the tissue bank, the institution where this facility is located is responsible for establishing acceptable storage and record keeping procedures to ensure the maintenance of the safety and efficacy of tissue from receipt to use and the traceability of tissue and recipients. The relevant part of these standards shall be made available to these institutions. These storage facilities (depot) shall be subjected to quality audit and control from the tissue bank. Labels on tissue containers shall not be altered, made invisible or removed.

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Tissue storage shall conform with guidelines established by the distributing tissue bank. Records shall document, as a minimum, the receival date of tissue and the destination (transplant date, the recipient's identity and transplant surgeon). These destination records shall be transmitted to the tissue bank. 1.2.6.9. Distribution to another tissue bank The associated tissue bank should adhere to these standards. 1.2.6.10. Acquisition of tissue from another tissue bank Prior to acquiring tissue from another tissue bank, the medical director shall approve the acquisition, and ensure that the tissue bank works according to these standards or according to comparable recognised standards. Labels on processed tissue acquired from another tissue bank shall not be altered, made invisible or removed. Accompanying documentation from the original tissue bank shall be forwarded with the tissue to the clinical team. After implantation, the destination record (transplant date, the recipient's identity and transplant surgeon) shall be forwarded to the original tissue bank. Annex 1.1. Glossary ADVERSE EVENTS [Synonym ADVERSE OUTCOME/REACTION]: An undesirable effect or untoward complication in a recipient consequent to or reasonably related to tissue transplantation. ALLOGRAFT: A graft transplanted between two different individuals of the same species. ASEPTIC RETRIEVAL: The retrieval of tissue using methods that restrict or minimise contamination with microorganisms from

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the donor, environment, retrieval personnel a n d / o r equipment. BRAIN DEATH/ BRAIN STEM DEATH: Complete and irreversible cessation of brain stem and brain encephalic functions and certified according to national laws. Synonym: death. CLEAN ROOM: A room in which the concentration of airborne particles is monitored and controlled to defined specification limits. COMPLIANCE: Conforming to established standards or regulations. CONTAINER: An enclosure for one unit of transplantable tissue. CONTROLLED ENVIRONMENT: An environment, which is controlled with respect to particulate contamination, both viable and non-viable particles. May also include temperature and humidity controls. CORONER: (see medical examiner). CORRECTIVE ACTION: Steps taken to ameliorate non-compliance. COST: The actual costs for retrieval, processing, preservation, storage, distribution, education, research and development. CROSS-CONTAMINATION: The transfer of infectious agents from tissues to other tissue or from one donor's tissue to another donor's tissue. DEATH: (see brain death). DISINFECTION: A process that reduces the number of viable cellular microorganisms, but does not necessarily destroy all microbial forms, such as spores and viruses. DISTRIBUTION: Transportation and delivery of tissues for storage or use in recipients.

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DONOR MEDICAL HISTORY INTERVIEW: A documented dialogue with an individual or individuals who would be knowledgeable of the donor's relevant medical history and social behaviour; such as the donor, if living, the next of kin, the nearest available relative, a member of the donor's household, other individual with an affinity relationship a n d / o r the primary treating physician. The relevant social history includes questions to elicit whether or not the donor met certain descriptions or engaged in certain activities or patterns of behaviour considered to place such an individual at increased risk for HIV and hepatitis or other diseases. DONOR REGISTRY: A formal compilation of an individual's intent relating to donation that may be maintained by a governmental agency or private establishment. DONOR SELECTION/DONOR SCREENING: The evaluation of information about a potential donor to determine whether the donor meets qualifications specified in the SOPs and standards. This includes but is not limited to, medical social and sexual histories, physical examination and laboratory test results (and autopsy findings, if performed). DONOR: A living or deceased individual who is the source of tissue for transplantation in accordance with established medical criteria and procedures. END-USER: A healthcare practitioner who performs transplantation procedures. FACILITY: Any area used in the procurement, processing, sterilisation, testing, storage or distribution of tissue and tissue components. FINISHED INVENTORY: Storage of finished tissue. FINISHED TISSUE: Tissue that has undergone all of the stages of processing, packaging and is approved for distribution.

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GIFT DOCUMENT: A legally recognised document in which an individual indicates his/her wishes as they relate to donation of organs and tissues. GOOD TISSUE BANKING PRACTICES: Practices that meet accepted standards as defined by relevant government or professional organisations. HPC: Haematopoietic progenitor cells. INSPECTION: An examination of a tissue bank to ascertain good tissue banking practices. INSPECTION: An examination of a tissue bank to ascertain good tissue banking practices LABELING MATERIAL: Any printed or written material including labels, advertising, a n d / o r containing information (for example package insert, brochures, pamphlets) related to the tissues. LABELING: Includes steps taken to identify the material and to attach the appropriate labels on the container or package so that it is clearly visible. Includes the package insert which is the written material accompanying a tissue graft bearing information about the tissue, directions for use and any applicable warnings. MEDICAL EXAMINER [synonym coroner]: Governmental official (usually a pathologist) charged with investigating deaths and determining cause of death. NATIONAL REGULATORY AUTHORITY [NRA]: A body appointed by the government with the goal of controlling tissue banking practices. NEXT OF KIN: The person(s) most closely related to a deceased individual as designated by applicable law. NON-COMPLIANCE: Non-conformance to established standards or regulations.

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OPEN SYSTEM: A system which has been breached but where every effort is made to maintain sterility by the use of sterile material and aseptic handling techniques in a clean environment. ORGAN (see vascular organ). PACKAGING (see container). PROCESSING: Any activity performed on tissue, other than tissue recovery, including preparation, preservation for storage a n d / or removal from storage, to ensure the quality a n d / o r sterility of human tissues. QUALITY: Totality of characteristics of a product, process or system that bear on its ability to satisfy customers or other interested parties. QUALITY ASSURANCE (part of quality management): Planned and systematic actions necessary to provide confidence in fulfilling quality requirements (see quality requirements). QUALITY AUDIT: A documented review of procedures, records, personnel functions, equipment, materials, facilities, a n d / o r vendors in order to evaluate adherence to the written SOPs, standards, or government laws and regulations. QUALITY CONTROL (part of quality management): Operational techniques and activities that are used to fulfil requirements for quality. QUALITY MANAGEMENT: All activities of the overall management function that determine the quality policy, objectives and responsibilities, and their implementation by means of quality planning, quality control, quality assurance and quality improvement, within the quality system. QUALITY REQUIREMENTS: Requirements for the characteristics of a product, a process or a system.

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QUALITY MANGEMENT SYSTEM (see quality management). QUARANTINE: The status of retrieved tissue or packaging material, or tissue isolated physically or by other effective means, whilst awaiting a decision on their release or rejection. RECALL: The requested return of finished tissue known or suspected to be non-compliant to the tissue bank, in accordance with the instructions contained in an advisory notice. RECIPIENT: An individual into whom organs, tissue is transplanted. RETRIEVAL [synonyms: recovery, procurement, removal, harvest]: The removal of tissues from a donor for the purpose of transplantation. SAFETY: A quality of tissue indicating handling according to standards and substantial from the potential for harmful effects from recipients. STANDARD OPERATING PROCEDURES [SOPs]: A group of standard operating procedures detailing the specific policies of a tissue bank and the procedures used by the staff/personnel. This includes, but is not limited to procedures to: assess donor suitability and retrieve, process, sterilise, quarantine, release to inventory, label, store, distribute and recall tissue. STERILISATION: A validated process to destroy, inactivates, or reduces micro-organisms to a sterility assurance level of 10" 6 . STERILITY ASSURANCE LEVEL: The probability of detecting an unsterile product, tissue. STORAGE: Maintenance of tissues in a state ready for distribution. TERMINAL STERILISATION: Sterilisation that takes place at the end of processing the tissue, in the final packaging.

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TISSUE: Human tissue includes all constitutive parts of a human body, including surgical residues and amnion, but excluding organs, blood and blood products, as well as reproductive tissues such as sperm, eggs and embryos. New products engineered from human tissue are included. The word "tissue" in this text applies to all types of tissues, including corneas and cells. TISSUE BANK: An entity that provides or engages in one or more services involving tissue from living or cadaveric individuals for transplantation purposes. These services include assessing donor suitability, tissue recovery, tissue processing, sterilisation, storage, labelling and distribution. TRACEABILITY: The ability to locate tissue during any step of its donation, collection, processing, testing, storage and distribution. It implies the capacity to identify the donor and the medical facility receiving the cells a n d / o r tissue or the recipient. TRANSPLANTATION: The removal of tissues a n d / o r cells and grafting of these tissues whether immediately or after a period of preservation a n d / o r storage. Transplantation may be from one person to another (allogeneic) or from a person to him/ herself (autologous). VALIDATION: Refers to establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. A process is validated to evaluate the performance of a system with regard to its effectiveness based on intended use. VASCULAR ORGANS: Any part of a human body consisting of vascularised, structured arrangement of cells, which once removed, cannot be replicated by the body. Examples: heart, liver, lung, kidney, pancreas and intestine.

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Annex 1.2. Guidelines of Factors to be considered for determining risk for h u m a n immunodeficiency virus or B or C hepatitis The following categories involve an increased degree of risk: (a) Men who have had sex with another man in the preceding 12 months. (b) Persons who report non-medical intravenous, intramuscular or subcutaneous injection of drugs in the preceding 12 months. (c) Men and women who have engaged in sex in exchange for money or drugs in the preceding 12 months. (d) Persons with a history of chronic hemodialysis. (e) Persons with a history of haemophilia or related clotting disorders who have received human-derived clotting factor concentrates. (f) Persons who were sexual partners of persons having a history of HIV or B or C hepatitis, manifestations, or risk factors previously described, in the past 12 months. (g) Percutaneous exposure or contact with an open wound, non-intact skin or mucous membrane to blood thought to be at high risk for carrying HIV or hepatitis in the preceding 12 months. (h) Inmates of correctional systems in past 12 months. (i) Diagnosed or treated for syphilis or gonorrhea in past 12 months. (j) A potential tissue donor who has received a blood transfusion within 12 months prior to death may only be accepted as a tissue donor after individual approval from the medical director. (k) The donor is not eligible if in a deferral status of any blood services donor deferral register. The local blood centre (s) shall be checked each time where possible (blood donor card available). (1) Tattoo, ear piercing, body piercing, a n d / o r acupuncture, unless by sterile, non-reused needle or equipment, in the preceding 12 months.

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A n n e x 1.3. Primary tumours of the central nervous system: evaluation of a suitable donor. A reference list No contraindication: pituitary adenoma; pinelcytoma; hemangioblastoma; schwannoma; choroid plexus papilloma; ependimoma; oligodendroglioma differentiated; craniopharyngioma; benign meningioma; pilocytic astrocytoma; and epidermoid tumours. Contraindication: medulloblastoma; chordoma; glioblastoma multiforme; highly anaplastic oligodendroglioma; anaplastic epidimoma; anaplastic meningioma; primary CNS lymphoma; pineoblastoma; CNS sarcomas; astrocytoma grade II; and astrocytoma grade III. Annex 1.4. Example of algorithm for calculating the hemodilution of a donor having received blood, blood components, or plasma v o l u m e expanders w i t h i n 48 hours prior to death The following equation allows calculation of a potential donor 50% plasma volume: 50% plasma volume (ml) = 21 x donor's body weight (kg). The equation as been calculated as follows: total blood volume per kg = 1kg x 70 ml = 70 ml; total plasma volume per kg = 70 ml (total blood volume per kg) x 1.0 - 0.40 (normal adult hematocrit) = 70 ml x 0.60 = 42 ml 50% plasma volume per kg = 42 ml (total plasma volume per kg) x 0.50 = 21 ml per kg.

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Annex 1.5. References and contact addresses REFERENCES: American Association of Tissue Banks (AATB). Standards for Tissue Banking (1984, 1985, 1987, 1989, 1993, 1996, 1998, 2001). Australian Code of Good Manufacturing Practice — Human Blood and Tissues. Therapeutic Goods Administration, 2000. Council of Europe Guide on Safety and Quality Assurance for Organs, Tissues and Cells (Version 11. CDSP, Released for Consultation 1/2001). European Association of Tissue Banks (EATB). EATB General Standards for Tissue Banking (1995). EATB and EAMST Standards for Musculoskeletal Tissue Banking (1997, revised 1999). EATB Standards for Skin Banking and Banking of Skin Substitutes (1997). IAEA Code of Practice for the Radiation Sterilisation of Biological Tissues (IAEA, Vienna). Radiation and Tissue Banking. G.O. Phillips eds. World Scientific. Singapore, New Jersey, London, Hong Kong (2000). UK Code of Practice for Tissue Banks. Department of Health. United Kingdom (2001). CONTACT ADDRESSES: American Association of Tissue Banks (AATB) 1350 Beverly Road, Suite 220A, McLean, VA 22101, USA. www.aatb.org. Asia-Pacific Surgical Tissue Banking Association Dr Norimah Yusof Malaysian Institute of Nuclear Technology Research, Bangi, 43000 Kayang, Malaysia. Email: [email protected].

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Council of Europe Karl Friedrich Bopp, Health Department, Council of Europe, 67075 Strasbourg Cedex, France. www.coe.int. European Association of Tissue Banks (EATB) C / - Dr Heinz Winkler, Vienna, Austria. www.eatb.de. Latin American Association of Tissue Banking. II. Guide for legal and regulatory control 11.1. Introduction This section is intended to assist governmental control authorities (GCA) and tissue banks in their joint task of improving the quality of human tissues for transplantation through regulation and legislation that interface with standards. Each member of the IAEA and their regulatory/ legislative bodies must necessarily determine the appropriate path for such regulation/law to follow, based on the technical capabilities of their region, religious beliefs and practices and healthcare systems. Within these key topics, many options are available for consideration. 11.2. Historical progression The first tissue banks were started in the 1950's, primarily in response to needs for bone, corneas and skin. Through the 1960's and 1970's, tissue banks began to proliferate, although they were usually small programmes that primarily served the hospital at which the tissue bank was located. Laws relating to organ and tissue donation, declaration of death and donor consent were passed in many countries in the 1970's through the 1980's. Starting in the mid-1980's, standards for tissue banking were developed, and often were accompanied by accreditation

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programmes organised by tissue banking associations. Even in countries with well-established tissue banks, the development and enforcement of regulations and laws did not occur until the 1990's, when concerns regarding safety of donated tissues increased. In the new century, tissue banking regulations and laws have been passed in developing countries, as have expanded laws in other countries with well-developed tissue donation systems. II.3. Laws and regulations Laws and Regulations concerning a wide range of topics are necessary, including: (1) (2) (3) (4) (5)

donation/transplantation/recovery/waiting lists; consent; organisation of the tissue bank; interrelationships with organ donation programmes; registration/licensing/accreditation/authorisation of the tissue bank; (6) import/export of tissue; (7) financial aspects of tissue banking; and (8) enforcement and compliance. II.3.1. Donation/transplantation/recovery/waiting lists (a) A law defining death (including brain stem death or brain death) is mandatory for the purpose of cadaveric donation of vascular organs and tissues. Because tissues may also be retrieved from a brain dead organ donor, the standards should reference brain death laws, if the tissue bank is prepared to accept tissues from donors meeting brain death criteria. Ideally, this law will also address how death must be declared, and by whom (e.g., brain death may be determined by a registered medical practitioner not involved with the recovery or transplantation of organs/tissues and using clinical criteria).

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(b) A law or regulation covering the mechanisms for organ and tissue donation is mandatory. These laws must outline how an individual may become or refuse to be a donor, the definition of organs and tissues that may be donated, the existence of a referral system, regulating to whom and how organs and tissues may be donated (allocation rules) and allow compensation for donation-related expenses. (c) Regulations that address at a minimum the donation, recovery, processing, storage and distribution of tissues are key to insuring the safety of the recipient. These regulations must include a list of tissue that are applicable to the regulations, guidelines or rules for donor screening criteria, donor approval systems, documentation, systems to guard against cross-contamination of tissue, labelling, quality systems, processing, validation of systems, storage, distribution and traceability of tissues. These regulations should also be based upon standards established for/ by the tissue bank. (d) Regulations regarding the donation of tissue from living donors (including amnion and surgical residues) are necessary and should be based upon the tissue banking standards. II.3.2. Consent (a) Laws addressing consent for donation are generally in place throughout the world, and vary widely, not only in content but also in practice. At a minimum, a consent law must include who may donate (e.g., the individual prior to his/her death, the individual's next of kin following death, or a patient prior to the donation of living tissues, etc.), whether the consent is presumed or informed ("opting out" or "opting in") and whether the individual's wishes may be countermanded by his/her next of kin. In addition, a mechanism for an individual to change his/her mind about donation prior to death must be included as part of the law/regulation.

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Finally, laws covering donor registries may also be considered as a way of insuring that an individual's choice is carried out, and as a way to increase donation rates in the region. (b) Presumed consent. Many countries have adopted presumed consent laws, in which an individual is assumed to be a donor unless he/she has specifically indicated his/her wish not to be a donor. This decision may be made officially through a non-donor registry (e.g., Belgium, France, Portugal) or informally (e.g., family discussion). This system is also known as "opting out". The presumed consent laws in several countries imply the family confirmation of presumed consent. Despite the fact that presumed consent laws are in place in many countries, few tissue, eye or organ recovery agencies will proceed with the retrieval process without first discussing donation with the patient's family. They either obtain the next of kin's informed consent for donation or verify the patient's desire to be a donor. In other countries, however, consent confirmation for tissue donation may not be routinely obtained from family members. In some countries, the medical examiner/coroner may allow the recovery of corneas and other tissues without family consent. However, this practice is under increasing scrutiny, due to the need for a family interview in order to determine medical suitability of the donor, and due to the perception that it may violate a donor family's rights. (c) Informed consent. Informed consent generally involves a discussion with the family of a recently deceased person regarding his/her desire or intent to be a donor, or in the absence of such knowledge or executed gift document, the family's desire to donate organs, eyes or other tissues for transplantation or research. In general, the consent conversation provides the potential donor family information about the recovery process and the uses of tissue for transplantation or research, what a "reasonable person" would want to know in order to make an informed decision.

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(d) Living donor consent. Regulations for the donation of tissues from living donors should require, as a minimum, that informed consent be obtained from the donor or his/her legal guardian if he/she is not of majority age. Surgical residue collection (e.g., femoral head, skin and amnion) implies information and consent from the patient before collection. 11.3.3. Organisation of the tissue banks Regulations addressing the organisation of the tissue bank should reference international standards for tissue banks and may include: personnel; training; building design and facilities; quality management; and equipment requirements. 11.3.4. Interrelationships with organ donation programmes (a) Collaboration between tissue banks and organ donation/ transplantation programmes is necessary to minimise confusion among the general public and donor hospitals. It may be advisable to include language in laws or regulations that encourages such collaboration. Collaboration between recovery agencies can benefit all. It eliminates duplication of efforts (personnel, organisation, donor promotion/enlightenment programmes), minimises unnecessary expenditures and maximises recovery of organs and tissues when consent for all types are obtained at once. In addition, it reduces the possibility that a bereaved family will be approached with multiple requests to donate. (b) Because laws exist regarding organ donation in many areas, tissue banking laws and regulations should be written so as to coincide with them wherever possible. 11.3.5.

Registration/licensing/accreditation/authorisation

(a) At a minimum, regulations should provide some mechanism for tissue banks to be identified through registration with the

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National Regulatory Authority (NRA) in order for the NRA to review the tissue banks' practices and to ensure compliance with established regulations. (b) Licensing or official authorisation to operate may be preferred. Regulations requiring licensing must take into account the resources required (financial, personnel, technical) to perform in-depth inspections or evaluations of tissue banks. If the NRA does not have the requisite resources, registration can be a reasonable alternative. (c) In some cases, the NRA is unable to adequately inspect or license tissue banks. It may, however, choose to contract such activities to another agency or private accrediting body, such as one that accredits laboratories, hospitals or tissue banks. 11.3.6. Import/export of tissue (a) With the global economy now extending into tissue donation and transplantation, it is critical for laws and regulations to address the import and export of donated human tissues. For instance, export of donated human tissues might be allowed only if all needs in the country have been met. Or, export of tissues might be allowed outright, depending on the laws and regulations of the other country. (b) Import of tissue requires specific rules in order to protect tissue recipients and compliance with these standards or equivalent standards, including ethical aspects, donor consent and safety issues. 11.3.7. Financial aspects of tissue banking (a) Tissue banks may be funded in a variety of fashions, including: governmental agency funding, private funding, funding through investors or through public or private hospitals or universities. Laws and regulations outlining how tissue banks

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receive compensation or reimbursement for their costs, whether they may charge patients or hospitals for tissue are all necessary. (b) The required financial structure of a tissue bank should also be established (non-profit or for-profit or public). (c) Monetary payment or advantages for the donation may not be made to living donors, cadavers donor's next of kin or any donor-related party, excluding compensation for donationrelated expenses. However, there are some locations that are considering pilot programmes that would allow for some moderate financial compensation or reimbursement for travel or funeral expenses to donor families. Commercial sale of tissues is of ethical and safety concern. However, many laws allow for the cost recovering of all tissue transplantation operations, including research/development and educational costs. Several tissue processing technologies are covered by patent rights that should be respected. Sale of tissues is a very vague statement and regulatory and legislative bodies would be well advised to clearly define and regulate what is allowable and what is not acceptable. II.3.8.

Enforcement/compliance

(a) Laws and regulations must include enforcement and compliance of the regulations, for without such enforcement the regulations will be far less effective. (b) Enforcement and compliance should include inspections of tissue banks and systems for addressing non-compliance or violation of laws and regulations. These could include requirements that the tissue bank destroy tissue, quarantine or retain tissue until corrective action is completed, notify hospitals, surgeons or patients of non-compliance, or issue a recall for all non-compliant tissue distributed. Penalties for non-compliance (e.g., closure of the tissue bank, financial penalties, civil a n d / or criminal prosecution) should also be considered and fully outlined.

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(c) Adverse events/self-reporting of non-compliance: the regulations should include requirements that the tissue bank have a system for receiving reports of adverse events, and for addressing those reports. In addition, the regulations should require that the tissue bank notify the NRA in the event of serious instances of non-compliance with standards, SOPs a n d / or regulations. (d) Internal audits: the regulations should include the requirement that the tissue bank perform periodic internal audits in order to assure compliance with standards, SOPs a n d / or regulations. If multiple organisations or tissue banks are involved in the same tissue banking process, the regulations should address which organisation is ultimately responsible for the tissue. However, a tissue bank that engages another organisation or tissue bank under a contract, agreement or other arrangement, to perform any step in the process, should be responsible for ensuring that the work is performed in compliance with the requirements established in the laws and regulations. II.4.

Conclusion

The development and implementation of appropriate laws and regulations is a complex, time-consuming and difficult undertaking. In order for such a system to become a functional reality, it is vital for tissue banks to enlist the support of key stakeholders such as end-users (surgeons, dentists, physicians, etc.), tissue recipients and tissue donor families. It may also be possible to enlist the support of the general public and charitable organisations that support programmes intended to better the well being of their fellow citizens. The importance of donor families should be emphasised, as they can be a powerful advocate for donation, if they are respected and included in the process of improving donation and transplantation. If they are ignored, disrespected or

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marginalised, they can become an even more powerful group, raising ethical questions about the donation and transplantation system that can result in an overall decrease in donation rates. The IAEA will encourage all tissue banks participating in the IAEA radiation and tissue banking programme to apply these standards, in accordance with their national conditions, with the purpose of ensuring the safe clinical use of the tissues produced.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

10 IN VIVO ASSESSMENT OF GAMMA IRRADIATED BONE: OSTEOCONDUCnVITY AND OSTEOINDUCTIVITY

Y. YU, J.B. CHEN, J.-L. YANG and W.R. WALSH Orthopaedic Research Laboratories Prince of Wales Hospital University of New South Wales Sydney 2031 NSW, Australia R. VERHEUL, N. JOHNSON AND DA.F. MORGAN Queensland Bone Bank Princess Alexandra Hospital Health Service District Wooloongabba 4102 QLD, Australia

1. Introduction The presence of bone defects either through osteolysis, tumour or trauma in revision arthroplasty (hip, knee and shoulder), can present a significant surgical challenge. Impacted morsellised allograft has been used to fill bony defects in revision arthroplasty with success (Gie et ah, 1993; Slooff et al, 1996; Bradley, 2000; Fetzer et al, 2001; Schreurs et al, 2001). Clinical use of allogenic bone grafts has increased significantly during the last decade, reaching over 150,000 annually in the United States (Eastlund, 1995; Tomford, Mankin, 1999). However, infection 321

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rates of 5 to 18% have been reported with the use of massive bone allograft in revision and bone tumor surgery (Lord et al, 1988; Hernigou et al, 1993). Careful procurement and processing are critical to avoid contamination and disease transmission. Gamma irradiation is a widely accepted secondary sterilisation procedure (Jonk, Ashby and Heimer, 1981; Pellicci et al, 1982; Ivory and Thomas, 1993; Justice, 1993). A dose of 30 kGy or more is required for the sterilisation of frozen allograft (Brandley et al, 1994; Pruss et al, 2002). However, a dose of 20 kGy or higher has been reported to alter the mechanical properties of bone (Loty et al, 1990; Bradley et al, 1995; Cornu et al, 2000). The effects of gamma irradiation on the osteoinductive and osteoconductive properties of allogenic bone grafts have yet to be reported. An optimal dose of gamma irradiation in combination with other factors such as temperature, tissue processing, donor selection and screening testing requires further investigation (Campbell and Oakeshott, 1995; Khan, Ibbotson and Stockley, 1998; Tomford and Mankin, 1999). In this study we tested new bone formation induced by human morsellised bone graft treated with 0, 15 or 25 kGy gamma irradiation using an athymic rodent "tibial window" defect model. We aimed to determine the effect of gamma irradiation on osteoconductivity and osteoinductivity of human morsellised bone graft. 2. Materials and Methods 2.1. Morsellised h u m a n femoral head processing Seven human femoral heads, harvested from primary total hip replacements with ethical approval, were processed under aseptic conditions by the Queensland Bone Bank. All soft tissue, cartilage and any extraneous materials were removed from the femoral head using a reciprocating saw (Stryker Instruments, Kalamazoo, MI, USA). The femoral head was placed into a Tracer Bone Mill (Tracer Designs Inc, Santa Paula, USA) and a

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small barrel was used to mill the bone into 2-6 mm particles. The morsellised bone was placed into an autoclaved stainless steel strainer with pore size 0.8 mm, rinsed with 0.9% sodium chloride using a high-pressure pulsatile lavage (Surgilav plus, Stryker Instruments, USA). After further rinsing with Ringers lactated solution (Baxter Healthcare, Australia) the morsellised bone was defatted in 6% hydrogen peroxide and disinfected by rinsing/ soaking in 70% ethanol with 2% methanol (v/v). The morsellised bone was drained and divided into three equal aliquots and stored at -70°C. Gamma irradiation (Australian Nuclear Science & Technology Organisation) of 0, 15 or 25 kGy in doses by Cobalt 60 was applied to the samples in dry ice in cylindrical stainless steel containers. The samples were stored at -70°C prior to use. 2.2. Sample randomization The seven femoral heads were numbered 1-7, and the three aliquots of each femoral head were treated with different doses (0, 15 and 25 kGy) of gamma irradiation and randomly assigned a number between 01-21. The samples were allocated in a blinded fashion for surgical implantation. 2.3. N u d e rat bilateral tibial w i n d o w defect Twenty-six 13-week old male CBH/rnu rats, weighing 300 g, were used in this study following approval of the local animal ethical committee. The animal was anaesthetised with fluothane inhalation together with oxygen. A bilateral tibial window (5 x 8 mm) was created from the medial aspect of the tibia 3 mm from the knee joint line using a Hall Burr (S6-122, Midas Rex®, Medtronic). The posterior cortex was left intact (Fig. 1). The surgical site was rinsed with 15 ml of 0.9% sodium chloride and packed with the randomly numbered morsellised bone graft. Autograft was harvested from the tibia cortex from four animals and used as positive controls (allograft) in two animals (four

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Fig. 1. Nude rat tibial window defect (5x8 mm) created at surgery (a, left tibia) and by Faxitron X-ray photography taken immediately after surgery (b, right tibia).

defects). Four empty defects served as negative controls. The surrounding muscle and the overlying fascia were closed in layers with 3-0 absorbable suture followed by the skin closure. The animals were housed in isolation cages allowing free movement, and sacrificed at three or six weeks following surgery. 2.4. Assessment of bone formation and bone remodeling Samples of morsellised bone graft were processed at time zero of implantation to provide histology of the bone chips prior to any new bone formation. The tibias of the sacrificed rats at three weeks or six weeks were harvested and immediately fixed in 10% buffered formalin. Anteroposterior and medial lateral radiographs were taken using a Faxitron X-ray Machine (MA20, Faxitron X»ray Corporation, Wheeling, IL, USA) using highresolution film photography. The tibias were decalcified in 10% formic acid in 10% buffered formalin, embedded in paraffin,

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sectioned (5 /im) from the medial aspect onto slides and stained with Harris haematoxylin & eosin (H&E) for the assessments of bone formation under a light microscope. Care was taken to ensure that new bone formation was assessed rather than allograft in the defects. Bone formation at three weeks was assessed by measuring the thickness of newly formed bone from the intact host cortex. The tissue morphology of each section, such as the presence of fibrous tissues, blood vessels, osteoblastlike cells, osteoclast-like cells as well as newly formed bone in the defect areas (especially in distance from the intact host cortex), was viewed and recorded. Bone remodelling at six weeks was assessed by estimating the percentage of the defect area where the newly formed cortex and bone marrow had formed in place. Two investigators performed the assessments in a blinded fashion. 2.5. Statistical analysis A one-way analysis of variance (ANOVA) and the non-parametric Kruskal-Wallis tests were used for the comparisons of the thickness of newly formed bone from the intact cortex at three weeks; the remodeling rate of tibial cortex at six weeks in the defects packed with morsellised bone grafts treated with 0, 15 or 25 kGy gamma irradiation after assessing homogeneity of variances, with the Levene test. A post-hoc Least-Significant Difference test was applied for measuring differences between individual groups. Statistical analysis was performed using the SPSS/Windows 11.0 statistical package (SPSS, Inc, Chicago, IL). 3. Results Faxitron X-rays of the tibial windows at three weeks showed a similar packing density in all groups (Fig. 2). H&E staining of the sections showed that at three weeks, the empty defects were filled with fibrous tissue without new bone formation [Fig. 3(a)]. The defects packed with rat allograft showed comprehensive

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Fig. 2. Faxitron X-ray showing a similar density of the packed defects with the morsellised human bone grafts treated with 0 (a), 15 (b) and 25 kGy (c) of gamma irradiation.

new bone formations [Fig. 3(b)]. New bone formation was found in all defects packed with morsellised human bone treated with different doses of gamma irradiation. The newly formed bone was mainly located around the implanted human bone chips

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Fig. 3. H&E staining of the three-week defects which were left empty at the surgery (a) or packed with freshly harvested rat allograft (b). No bone formation was noted in the empty defect, and a comprehensive bone formation was found in the defect packed with rat allograft bone (IC = intact cortex).

adjacent to the intact host cortex. More new bone formation was found in the defects packed with non-treated and 15 kGy gamma irradiation groups, compared with the 25 kGy group (Fig. 4). The average thickness of the newly formed bone from the intact host cortex of each section was recorded [Fig. 5(a)].

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Fig. 4. H&E staining of three-week defects packed with morsellised human bone grafts treated with 0 (a), 15 (b) or 25 kGy (c) of gamma irradiation. New bone formations were noted around the implanted bone chips (solid arrows) adjacent to the intact host cortex (IC). More newly formed bones were found in the 0 (a) and 15 kGy groups (b) compared with the 25 kGy section (e), which showed fibrous tissues in the defect area.

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gamma irradiation (0,15, 25 kGy) (a)

gamma irradiation (0,15, 25 kGy) (b) Fig. 5. The average thickness of newly formed bone from the intact cortex in each section at three weeks was illustrated (a). The individual percentage of remodeling rate of the tibial cortex at six weeks was also recorded (b). Both the bone formation and bone remodeling reduced with the increase in dosage of gamma irradiation in most cases.

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Week 6: Bone remodelling (percentage, mean ± SD)

1.28 + 0.38

58.57 ± 13.45

15 kGy b

1.04 + 0.47

51.42 ± 13.45

c

0.55 ± 0.34

31.14 ± 9.44

p = 0.554

p = 0.361

ANOVA

F = 6.068; p = 0.010

F = 9.427; p = 0.002

Kruskal-Wallis test

H = 7.379; p = 0.025

H = 10.730; p = 0.005

p = 0.009 a/c; p - 0.034 b / c

p = 0.002 a/c; p = 0.019 b / c

Doses 0a

25 kGy

Levene test

LSD

Statistical analysis (ANOVA and the Kruskal-Wallis test) of the individual data showed a significant difference between the 0, 15 kGy and 25 kGy groups (Table 1). The effect of gamma irradiation on bone formation was dose dependent. The 25 kGy group had a significantly lower level of new bone formation compared with the 0 and 15 kGy groups. Histology of the morsellised human bone graft at time zero revealed uniform particle size and morphology as well as the absence of any cellular constituents. Histology at three weeks revealed osteoblast-like cells, osteoclast-like cells, newly formed bone and bone marrows adjacent to the implanted human bone chips distant from the intact host cortex in the 0 and 15 kGy groups (Fig. 6). Soft tissue and blood vessels were found in all defects, and there was no significant difference of the number of blood vessels between the groups. At six weeks, bone marrow was present, and remodeling of the tibial cortex was noted. The defects packed with the bone chips that were not irradiated

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Fig. 6. Osteoinductivity of the 15 kGy irradiated bone grafts at three weeks was demonstrated by the presence of osteoblast-like cells (solid arrows), osteoclastlike cells (open arrow) lying around the implanted bone chips distant from the intact host cortex. Newly formed woven bone (WB) and bone marrow (BM) from the implanted bone chips were noted.

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Fig. 7. H&E staining shows the tibial cortex remodeling at six weeks in the defects packed with the morsellised human bone grafts treated with 0 (a), 15 (b) or 25 kGy of irradiation. Lower grade of tibial remodeling and more fibrous tissue formation were noted in the 25 kGy defects (c) compared with the 0 (a) and 15 kGy (b) groups.

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(0 dose) showed the highest remodeling rate, followed by the 15 kGy group. The remodeling rate was significantly lower in the defects packed with 25 kGy treated bone chips compared to the others [Fig. 5(b), Table 1, Fig. 7]. 4. Discussion The effect of gamma irradiation of morsellised human bone graft on new bone formation remains unclear. The current study found a negative dose-dependent effect of gamma irradiation on bone formation and remodeling of morsellised human bone chips in the nude rat defect model. A statistically significant decrease in new bone formation was found with 25 kGy gamma irradiation when compared with the 0 and 15 kGy groups. With careful processing of the morsellised bone grafts (including high pressure hosing, defatting, and soaking), 15 kGy of gamma irradiation may be sufficient to eliminate contamination and disease transmission, especially to those low-risk donor samples. Recent study support that 15 kGy gamma irradiation does not compromise the natural course of allogenic cortical bone graft incorporation (Jinno et al.f 2000). Bone formation may be augmented in three ways as result of bone grafting; osteogenesis, osteoinduction and osteoconduction (Tomford and Mankin, 1999). Osteogenesis can occur when using the so-called "gold standard" autograft bone. In osteogenesis, bone-forming cells and growth factors derived from the matrix of bone graft directly form bone. Osteoinduction occurs after the use of a bone graft that induces host cells that invade the graft, to form new bone. Demineralised bone (DBM) is believed to be osteoinductive through a demineralisation process in which growth factors in the graft are exposed. Osteoconduction refers to a situation when a bone graft acts as a scaffold on which new host bone can be formed. Most mineralised bone allografts currently provided by bone banks are osteoconductive. The addition of exogenous growth factors to these types of grafts may improve their osteoinductivity. To date, no studies have proven

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that bone grafts substitutes act as other than scaffolds on which new bone is formed by the host. In this study we found new bone and bone marrow formation as well as active osteoblast-like cells, and osteoclast-like cells, lying around the morsellised human bone chips, which were surrounded by soft tissues and were far away from the intact host cortex (Fig. 6) in the 0 and 15 kGy groups. The mechanism of new bone formation in those cases may be due to two factors. Firstly, the allograft was prepared from live donors, which allowed growth factors to remain active. Secondly, the allograft was morsellised into small chips (2 mm), which may have increased the available surface area for new cellular attachment and may have some growth factors exposed. The growth factors in the bone matrix may have stimulated mesenchymal cells in the surrounding soft tissues to differentiate into osteoblasts a n d / or osteoclasts and stimulated new bone formation a n d / o r bone remodeling. These findings suggest at least an osteoconductive mechanism, and possibly an osteoinductive mechanism in these groups, considering the soft tissue envelope surrounding the grafts; while new bone formation was noted in the interior of the defect. Our findings on the significant reduction of new bone formation from the intact cortex in the 25 kGy group compared with the 0 or 15 kGy groups, also suggest that the morsellised bone grafts with a zero or low dose of irradiation have some ability to stimulate host bone formation rather than only act as a scaffold. Our results indicate that the morsellised human bone graft may retain some osteoinductivity when treated with a low dose (up to 15 kGy) of gamma irradiation. The use of low risk donors and low dose gamma irradiation may provide a clinically useful combination for morsellised allograft. 5. Summary Morsellised human bone grafts treated with 0, 15 or 25 kGy gamma irradiation were implanted into tibial window defects of nude rats to determine the effect of gamma irradiation on their

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osteoconductivity and osteoinductivity. At three weeks, postoperation osteoconductivity was assessed by measuring the thickness of newly formed bone around the implanted bone chips from the intact host cortex. Analysis of individual data showed a significant difference of the thickness of newly formed bone between the 0, 15 and 25 kGy groups (ANOVA and LSD tests, p < 0.05). The effect of gamma irradiation on osteoconductivity was dose dependent. The 25 kGy group had a significantly lower level of new bone formation compared with the 0 and 15 kGy groups. Osteoinductivity of the morsellised grafts was evidenced by the notes of active osteoblast-like cells and new bone formation adjacent to the implanted bone chips, which were surrounded by soft tissues distant from the host cortex. Six weeks post operation, bone marrow was present and remodeling of the tibial cortex was noted. The 25 kGy group, again, showed the significantly lower remodelling rate when compared with other two groups. Our data indicate that 25 kGy gamma irradiation reduces the osteoconductive and osteoinductive properties of the morsellised human bone graft. 6. References BURING, K. and URIST, M.R. (1967). Effects of ionising radiation on the bone induction principle in the matrix of bone implants, Clin. Orthop. 55, 225. BRADLEY, G.W. (2000). Revision total knee arthroplasty by impaction bone grafting, Clin. Orthop. 113-118. CAMPBELL, D.G. and OAKESHOTT, R.D. (1995). Bone allograft banking in South Australia, Aust. N.Z. ]. Surg. 65, 865-869. CORNU, O., BANSE, X., DOCQUIER, P.L., LUYCKX, S. and DELLOYE, C. (2000). Effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone, /. Orthop. Res. 18, 426-431.

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EASTLUND, T. (1995). Infectious disease transmission through cell, tissue, and organ transplantation: Reducing the risk through donor selection, Cell Transplant. 4, 455-477. FETZER, G.B., CALLAGHAN, J.J., TEMPLETON, J.E., GOETZ, D.D., SULLIVAN, P.M. and JOHNSTON, R.C. (2001). Impaction allografting with cement for extensive femoral bone loss in revision hip surgery: A 4- to 8-year follow-up study, /. Arthroplasty 16, 195-202. FIDELER, B.M., VANGSNESS, C.T. Jr, LU, B., ORLANDO, C. and MOORE, T. (1995). Gamma irradiation: Effects on biomechanical properties of human bone-patellar tendon-bone allografts, Am. J. Sports Med. 23, 643-646. GIE, G.A., LINDER, L., LING, R.S., SIMON, J.P., SLOOFF, T.J. and TIMPERLEY, A.J. (1993). Impacted cancellous allografts and cement for revision total hip arthroplasty, /. Bone Joint Surg. (Br) 75, 14-21. HERNIGOU, P., DELEPINE, G., GOUTALLIER, D. and JULIERON, A. (1993). Massive allografts sterilised by irradiation. Clinical results, /. Bone Joint Surg. Br. 75, 904-913. IVORY, J.P. and THOMAS, I.H. (1993). Audit of a bone bank, /. Bone Joint Surg. (Br) 75B, 355-357. JINNO, T., MIRIC, A., FEIGHAN, J., KIRK, S.K., DAVY, D.T. and STEVENSON, S. (2000). The effects of processing and low dose irradiation on cortical bone grafts, Clin. Orthop. Rel. Res. 375, 275-285. JONK, L.M., ASHBY, J.A.S. and HEIMER, E.R. (1981). Allogenic bone transplantation, S. Afr. Med. J. 60, 453-457. JUSTICE, K.C. (1993). Recovery and banking: Allograft bone, Setnin Peri-operative Nursing No. 2. KHAN, M.T., STOCKLEY, I. and IBBOTSON, C. (1998). Allograft bone transplantation: A Sheffield experience, Ann. R. Coll. Surg. Engl. 80, 150-153.

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LORD, C.F., GEBHARDT, M.C., TOMFORD, W.W. and MANKIN, H.J. (1988). Infection in bone allografts. Incidence, nature, and treatment, /. Bone Joint Surg. Am. 70, 369-376. LOTY, B., COURPIED, J.P., TOMENO, B., POSTEL, M., FOREST, M. and ABELANET, R. (1990). Bone allografts sterilised by irradiation. Biological properties, procurement and results of 150 massive allografts, Int. Orthop. 14, 237-242. PELLICCI, P.M., WILSON, P.D. Jr, SLEDGE, C.B., SALVATI, E.A., RANAWAT, C.S. and POSS, R. (1982). Revision total hip arthroplasty, Clin. Orthop. 170, 34-41. SCHREURS, B.W., VAN TIENEN, T.G., BUMA, P., VERDONSCHOT, N., GARDENIERS, J.W. and SLOOFF, T.J. (2001). Favourable results of acetabular reconstruction with impacted morsellised bone grafts in patients younger than 50 years: A 10- to 18-year follow-up study of 34 cemented total hip arthroplasties, Acta. Orthop. Scand. 72, 120-126. SLOOFF, T.J., BUMA, P., SCHREURS, B.W., SCHIMMEL, J.W., HUISKES, R. and GARDENIERS, J. (1996). Acetabular and femoral reconstruction with impacted graft and cement, Clin. Orthop. 108-115. TOMFORD, W.W. and MANKIN, H.J. (1999). Bone banking. Update on methods and materials, Orthop. Clin. North Am. 30, 565-570.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

11 DISINFECTION OF FEMORAL HEADS FOR BONE GRAFTING USING THE MARBURG BONE BANK SYSTEM (LOBATOR SD 1) A RETROSPECTIVE EVALUATION OF QUALITY CONTROL IN THE ENDO-KLINIK BONE BANK

LARS FROMMELT Institut fur Infektiologie klinische Mikrobiologie und Krankenhaushygiene ENDO-KLINIK Holstenstr. 2, D-22607 Hamburg LUTZ GURTLER Friedrich Loeffler Institut fur Medizinische Mikrobiologie Ernst Moritz Arndt Universitat, Matin-Luther-Strasse D-17487 Greifswald THOMAS VON GARREL Klinik fur Unfall-, Wiederherstellungs- und Handchirurgie Philipps Universitat, Baldingerstrasse, D-35043 Marburg

1. Introduction According to German federal law, bone grafts are drugs, and as such, are produced under the control of local and federal 339

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authorities. The intention is to protect the bone graft recipient from any harm — especially to infections — whether they are due to bacteria or viruses. Bacteria are often thought to be less harmful, but particular aspects in the pathogenesis of foreign body infection show an other scenario (Frommelt, 2001): In the presence of a foreign body, the inocculum of bacteria to induce infection is reduced by 2 to 3 logio steps as shown by Elek and Conen (1954). This may be of importance because the graft itself may also act as a foreign body. 1.1. The E N D O - K L I N I K b o n e bank In the ENDO-KLINIK bone bank, femoral heads from living surgical donors are used as allogenic structural bone grafts. These femoral heads are obtained from patients undergoing total hip replacement, and the donors are selected after consideration of the results of the medical examination; the patients history; laboratory tests (for syphilis antibodies, hepatitis B virus (HBsAg, Anti-HBc); hepatitis C virus (Anti-HCV); and human immunodeficiency virus (HIV) (Anti-HIV 1/2) by enzyme immuno assay and nucleinic acid test for HVC und HIV), by microbiological quality assessment in accordance with current German and European regulations and standards. Additionally, the femoral heads undergo a thermal incubation at 82°C for 10 minutes in the Marburg bone bank system (Lobator SD-1) to reduce the number of microbiological pathogens. For quality assurance we introduced the following critical points for preventing hazards which are defined as follows: (a) Bone tissue (1): Cancellous bone harvested in the operating theatre. (b) The processing fluid for thermal disinfection, which is a rinsing culture of the graft. (c) Bone tissue (2) obtained at the time of graft implantation in the operating theatre. This specimen is not relevant for the release of the femoral heads for grafting, but for infection control in the ENDO-KLINIK.

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The femoral heads are released for grafting when no bacterial or fungal growth from bone tissue (1) and from the processing fluid can be detected. The following study presents the bacteriological results from the tests required in our quality assurance system. The aim of the study is to show that processing according to the Marburg Bone Bank System (Lobator SD 1) is capable of, and effective in destroying vegetative bacterial contamination of the graft material. 2. Materials and Methods For every graft a microbiological evaluation was conducted as part of internal quality control procedures. Cancellous bone samples were taken simultaneously and evaluated bacteriologically. After completion of the thermal disinfection process, the disinfection fluid was evaluated for microorganisms. Only when both tests revealed no bacterial growth, was the femoral head released for transplantation in so far as that bacteria was involved. The aim of the testing was to show that processing with the Lobator SD 1 System is capable of, and effective in destroying vegetative bacterial contamination of the grafts. 2.1. Acquisition and disinfection of the graft material The graft material was obtained during primary hip replacement surgery performed in an operating theatre with conventional ventilation according to DIN 1946 Part 4. A cancellous bone sample, measuring about 5 mm in diameter, was taken from the prospective graft material and tested for bacterial presence. In the operating room the graft was transferred to a disinfection container (Telos Co., Hungen, Germany). The container was filled with sterile Ringer's lactate solution. The disinfection container was transported to the laboratory, and the graft was processed

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in the Lobator SD-1 in a separate area in the laboratory. After completion of thermal disinfection, a sample of the disinfection fluid was removed using a sterile syringe under germ-free conditions. It was then tested for bacteria and fungi in the VITAL blood culture system (bioMerieux Co., Marcie Etoile, France). All steps in the bacteriological processing which were at risk of bacterial contamination, were performed in a "class 100" workbench with laminar airflow. 2.2. Study time period From November 1994 to December 31, 1997 a total of 2,458 grafts were tested. A sample of the bone tissue of each graft was tested after harvesting, including the disinfection solution after processing. No grafts were excluded from the study. 2.3. Microbiological studies 2.3.1. Primary cultures The cancellous bone samples obtained from the grafts during the operation were incubated for 14 days at 35°C in an incubation chamber in a high layer culture as described by Lodenkamper (1956). They were observed and controlled daily. The cultures were prepared under sterile conditions in a "class 100" workbench. At the end of the incubation period, microscopic preparations were evaluated using Gram's staining, and placed in superficial cultures on Columbia blood agar (Difco Co., Detroit, USA). If no bacteria or fungi were found during the observation period, in the subsequent microscopic evaluation, or in the following superficial culture, the culture was determined to have "no growth". From the thermal disinfection fluid 10 ml were extracted, an aerobic and anaerobic blood culture bottle (Hemoline VITAL duo, bioMerieux, Marcy Etoile) was inoculated, and incubated

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for 14 days in the VITAL blood culture system. At the end of the observation period or if indicated by the apparatus, an aliquot of 5 ml was centrifuged and subjected to a microbiological evaluation with Gram's staining. Additionally a superficial culture was placed on Columbia blood agar aerobically, and on Brucella blood agar (Difco Co., Detroit, USA). If no microorganisms were found during the observation period and in the subsequent testing, the culture was determined to have "no growth". 2.3.2. Diagnostics of microbial growth When microbial growth was found, an orienting diagnostic schedule was performed, which excludes Staphylococcus aureus, phaemolysing streptococci, enterococci, faecal germs and Pseudomonas aeruginosa. To test for yeast, a diagnostic to exclude Candida albicans was prescribed. Anaerobes were divided on the basis of microscopic and culture morphology into Propionibacterium spp. and other anaerobes. For Clostridia and Bacillus spp. a further differentiation was prescribed. The following categories were established: for Staphylococcus aureus, /3-haemolysing streptococci, enterococci, faecal germs, non-fermenting gram-negative rods, C. albicans, Clostridia, and bacilli, the taxonomy on species level. The remaining findings were divided in the following categories: Staphylococcus spp. (CNS), Streptococcus spp., aerobic diphtheroid rods, non-fermenting rods, Propionibacterium spp. and other anaerobes. 2.3.3. Documentation The test orders and the observations were continuously registered in a log book and the results for every femoral head investigated during the study were transferred to a Microsoft Excel data sheet on a Microsoft Windows-based personal computer. The data were processed for the number of events and the percentage per year in the study period.

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3. Results 3.1. Bacterial contamination A total of 2,458 grafts were included in the study. Of the bone tissue samples taken before thermal disinfection 233 were found to have growth (9.07%). Three cases of bacterial growth were found (0.12%) among the samples from the disinfection fluid. These three cases appeared during the first month after thermal disinfection using the Marburg Bone Bank System (Lobator SD-1) was commenced. The details of the results are presented in Tables 1 and 2.

Table 1. Germs found in bone tissue samples upon removal of the grafts (prior to thermal disinfection).

Year 1994 (Nov & Dec) 1995 1996 1997 Total (1994-97)

Femoral heads number (all) No growth Growth

% Growth

127 849 770 712

120 776 697 642

7 73 73 70

5.51 8.60 9.48 9.83

2,458

2,235

223

9.07

Table 2. Germs found in the disinfection fluid after thermal disinfection.

Year 1994 (Nov & Dec) 1995 1996 1997 Total (1994-97)

Femoral heads number (all) No growth Growth

% Growth

127 849 770 712

124 849 770 712

3 0 0 0

2.36 0.00 0.00 0.00

2,458

2,455

3

0.12

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Disinfection of Femoral Heads for Bone Grafting

Table 3. Distribution of the germs (cancellous bone samples) 1994-97 Rank listing, n = 238 for 223 grafts (7 grafts showed the presence of more than one species).

Type of genera 1. 2. 3. 4. 5. 6.

Number absolute

Relative [%]

.31 80 16 8 2 1

53.04 33.62 6.37 3.36 0.84 0.42

238

100.00

Propionibacterium spp. Staphylococcus spp. (CNS) Aerobic diphtheroid rods Other anaerobes (apart from Clostridia) Non fermenters Streptococcus spp.

Total

Notes: CNS = coagulase negative staphylococci; Non fermenters = other than P aeruginosa; Streptococcus spp. — not Lancefield groups A-C, E-F or Enterococcus spp.

Table 4. Distribution of the type of germs (1994-97) by years, n = 238 for 223 transplants (7 transplants showed more than one type of germs). Type of germ Staphylo-

Year

coccus

Diphthe-

Non

Strepto-

> 1

Propioni-

spp.

roid

Other

fer-

coccus

species/

bacteria

(CNS)

rods

anaerobes

menters

spp.

transplant

1994 (Nov & Dec)

3

2

2

0

0

0

0

1995

46

21

3

5

0

0

2

1996

44

22

6

2

1

0

2

1997

38

35

5

1

1

1

3

Total

131

80

16

8

2

1

7

Notes: 994 = November and December; 1995-97 = each 12 month; Staphylococcus spp. (CNS) = coagulase negative staphylococci; diphtheroid rods = aerobic diphtheroid rods; Non fermenters = not P. aeruginosa; Streptococcus spp. = not Lancefield groups A - C , E - F or Enterococcus spp.

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3.2. Types of germs found Of the 223 contaminated bone tissue samples, seven were shown to have several different types of bacteria. The distribution of the germs is presented in a rank listing in Table 3. The details of the distribution of the isolates are given in Table 4. Samples from the disinfection fluid showed bacterial growth of coagulase negative staphylococci in three cases, while the corresponding samples of cancellous bone obtained before thermal disinfection remained sterile. 4. Discussion 4.1. Germs isolated before thermal disinfection During the study 223 (9.07% isolation rate) bone samples out of 2,458 grafts taken in the operating theatre at the time of primary hip replacement, were found to have bacterial growth. The number of cultures showing growth increased slightly from 1995 to 1997. So far no explanation has been found for this increase. No changes in the processing and preparation of the primary cultures were made. The rate of bacterial growth in bone grafts as reported in the literature, shows variability from less than 1% up to 92% (Hustedt and Kramhoft, 1996; Knapler et at, 1992; Deijkers 1997; Veen et ah, 1994; Garrison and Morse, 1993). However, these data are difficult to compare because a variety of methods with different sensitivities was used, and in some studies, cadaverous material and material from living donors were investigated together. In this study, the intention was to exclude living donors with low-grade infections at the graft site. Donors who were suspected of being infected after intraoperative inspection of the site were excluded before the femoral head was harvested. We used a sample of cancellous bone from the femoral head to prepare the microbiological cultures for investigation of bone

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infection. Bone biopsy turned out to be an appropriate method to recover bacteria from bone tissue, as Veen and co-workers (1994) showed impressively. The sensitivity of bone biopsies with respect to bacterial growth is superior to the use of swabs with and without enrichment cultures. Unfortunately the presence of bacteria does not automatically mean the presence of an infection. 4.1.1. Significance of germs isolated Due to the fact that in an operating room people are active and working, germs deriving from the bacterial flora of the skin are found in the air even though the air leaving the filters of the air conditioning units is sterile. Another principal source of bacteria is the patient's own flora. Most of the bacterial findings in the sample have to be suspected as contaminants from human bacterial flora which came into contact with the surface of the specimen during harvesting. An additional source of contaminants is the processing of the samples in the laboratory. Here we were able to control the rate of bacterial contamination by using a clean air workbench. Sorted by rank, the distribution of the germs in this study differs from the distribution of bacteria in the air of clean rooms as reported by Schulz (as quoted by Wallhauser, 1995) and the distribution found in human skin as found by Maibach and Aly (1981). Schulz reported about 65% coagulase negative staphylococci, about 20% micrococci, about 10% bacilli and less than 5% of yeasts in the air of clean rooms. Other bacteria are small in number and are not enumerated individually. These data correlate well with our observations of air samples taken during surgical procedures, but in the distribution of the germs found in this study, there was a high incidence of diphtheroid gram-positive rods (Propionibacteria or aerobic germs). These bacteria are residents of the normal flora of the human skin (Maibach and Aly, 1981) and are able to contaminate the site of surgery during the procedure, and can thus enter the surgical

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wound. These germs are thought to come directly from the skin rather than from contaminated air. Otherwise the high incidence of Propionibacteria was unexplainable. The rate of contamination was due to the aseptic surgical wound. Fitzgerald and co-workers (1973) demonstrated a rate of contamination in orthopaedic surgery of 30%. These bacteria are airborne and are residents of the patient's skin flora surrounding the surgical wound itself. Other authors dealing with bone grafts report a rate of contamination of less then 1% up to 92% recovered from bone obtained under "sterile" conditions (Hustedt and Kramhoft, 1996; Knapler et al, 1992, Deijkers, 1997; Veen et al., 1994; Garrison and Morse, 1993). This wide range can be explained by the different methods of processing. Another concurring explanation is that in some studies cadaveric donors are included. As Deijkers and co-workers (1997) report, more than one graft is obtained from these donors, and they showed that the rate of bacterial contamination increases in relation to the time before the graft is harvested. Our study included only living donors who were undergoing primary artificial joint replacement of the hip. From these donors only the femoral head was obtained for grafting. The rate of contamination detected depends on two crucial conditions: the sensitivity of the method used for detection of germs, and the size of surface area, which can be contaminated. The bio burden level of these contaminations appears to be low in our experience. With few exceptions we found two or three colony-forming units in the high layer agar, so that the bio burden of the samples was low in number. Lodenkamper's method using high layer cultures — in which the sample is embedded in the nutrient agar — is able to show colony-forming units of facultative anaerobic and anaerobic bacteria, and with slight restrictions for strict aerobics. Following data from the literature and especially a study of von Garrel, Mutters and co-workers (2002) showed that there is a ranking in sensitivity: direct enrichment broth, e.g., blood

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culture systems, are superior to filtration methods, and swabbing is less sensitive than the methods mentioned before. 4.2. Germs isolated after thermal disinfection After thermal disinfection, direct enrichment methods were used to detect remaining bacteria. This method appears to be suitable with respect to sensitivity. We found Staphylococcus spec. (CNS) three times after Lobator SD 1 processing. The isolates were obtained during the first month following introduction of the Marburg Bone Bank System. After the first month no germs were recovered for more than three years. We concluded that these isolates were due to handling problems when starting a new system (short learning curve), and have their origin in the technician's skin flora during handling of the disinfection fluid samples. 5. Conclusions The Marburg Bone Bank System using the Lobator SD 1 is effective in eradicating germs contaminating bone grafts (femoral heads) from living donors. Furthermore, this thermal disinfection system is able to inactivate vegetative forms of bacteria but not spores, and appears to be especially suited to processing bone allograft from living donors where bio burden levels are expected to be low. Our data strongly suggests that when the Marburg bone bank system is properly used, aerobic and anaerobic cultures of the graft material are no longer required for screening transplants when bacterial contamination with lowgrade pathogens occurs. However, these bacterial cultures are of value in internal process controls and for screening of grafts which are taken from infected donors or which are contaminated with high-grade pathogens, like Escherichia coli or Salmonella spec. In order to minimise the risk of bone infection for the graft recipient, we recommend discarding these grafts from infected donors if contaminated with high-grade pathogens.

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The methods used for recovering germs should be very sensitive, and an international standard for the microbial processing of bone grafts should be established. 6. References DEIJKERS, R.L.M., BLOEM, R.M., PETIT, P.L.M., BRAND, R., VEHMEYER, S.B.W. and VEEN, M.R. (1997). Contamination of bone allograft, /. Bone Joint Surg. [BrJ 79-B, 161-166. ELEK, S.D. and CONEN, P.E. (1957) The virulence for Staphyloccus pyogenes for men: A study on the problems of wound infection, Br. J. Exp. Pathol. 38, 573-586. FITZGERALD, R.H., PETERSON, L.S.A., WASHINGTON II, J.A., VON SCOIJ, R.E. and COVENTRY, M.E. (1973). Bacterial colonisation of wounds and sepsis in total hip arthroplasty, /. Bone Joint Surg. A 55-A, 1242-1250. FROMMELT, L. (2000) Periprosthetic infection — Bacteria and the interface between prosthesis and bone. In: Interfaces in Total Hip Replacement, I.D. Learmonth, ed., Springer, London. GARRELL, V.T., MUTTERS, R., GARBAS, J., JUNGE, A. and GOTZEN, L. (2002). Optimisation of microbiological screening for bacterial contamination in allogenic bone transplants — Comparative study of various microbiological investigative techniques. Submitted for publication — personal communication. GARRISON, C.P. and MORSE, B. (1993). Comparison of bacterial contamination of cadaveric bone allograft collected under operating room and morgue conditions with and without the use of a decontamination process. AATB 17th Annual Meeting 1993, Oct. 22nd-25th, Boston, USA. HUSTED, H. and KRAMHOFT, M.U. (1996). Microbiology of femoral head grafts in bone banks, Ugeskr Laeger 28, 62606262.

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KNAEPLER, H., GARRELL, V.T., SEIPP, H.M. and ASCHERL, R. (1992). Experimentelle Untersuchungen zur thermischen Desinfektion und Sterilisation allogener Knochentransplantate und deren Auswirkungen auf die biologische Wertigkeit, Der Unfall-chirurg 95, 477-485. LODENKAMPER, H. and STIENEN, G. (1956). Zur Therapie anaerober Infektionen, Dtsch med Wschr 81, 1226-1231. MAIBACH, H.J. and ALY, R. (1981). Skin Microbiology. Springer, New York, p. 234. VEEN, M.R., BLOEM, R.M. and PETIT, P.L.C. (1994). Sensitivity and negative predictive value of swab cultures in musculoskeletal allograft procurement, Clin. Orthop. Relat. Res. Na. 300, 259-263. SCHULZ, H. (1990). Bedeutung des Menschen als Kontaminationsquelle, Pharma. Techn. J. 11, 31-32.

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SECTION III: ETHICAL AND SOCIAL ASPECTS OF TISSUE BANKING

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

12 TISSUE BANKING AND TRANSPLANTATION: THE ETHICAL CHALLENGES

PAUL L. R O M A I N , M.D. D e p a r t m e n t of Rheumatology C a m b r i d g e H e a l t h Alliance a n d H a r v a r d Medical School

1. Introduction A number of difficult ethical dilemmas and conflicts pervade the field of human tissue banking and tissue transplantation, which have come under increased public scrutiny in recent years. Some aspects of these issues, especially the legal and commercial concerns, have been the focus of thoughtful recent reviews and commentaries (Indech, 2000; Mahoney, 2000; Charo, 2002). The present discussion will briefly outline why this is an issue of increasing importance for workers in the field to address; identify selected issues of particular current concern; review applicable ethical considerations; and discuss strategies for addressing some of these dilemmas. Less than two weeks before the 2002 Annual Meeting of the American Association of Tissue Banks (AATB), a page one story in The New York Times reported that the US Food and Drug Administration (FDA) had cited and partially shut down the nation's largest processor of donated human tissue because of

355

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concerns that "the company could not adequately assure patients that its soft-tissue products were freed of deadly bacteria and fungi" (Blakeslee, 2002). Another article, published in Business Week earlier in the same year, focused more on tissue used for research and issues of confidentiality (Raeburn, 2002). It suggested that "somewhere in a laboratory there may be a scientist who knows far more about you than you do yourself", and cited a 1999 study by President Clinton's National Bioethics Advisory Commission, noting that "current federal regulations ... are inadequate to ensure the ethical use of human biological materials in research." These articles highlight the potential public interest in tissue banking. They also serve as a reminder that one test of the ethical appropriateness (or need for further ethical analysis) of a policy, procedure or practice may be the simple question: "Would I want this widely known?" This raises practical, as well as philosophical concerns. For example, the present-day reluctance of African-Americans to donate organs or to participate in research studies is considered by many to be an unintended legacy of the infamous Tuskegee study of the natural history of untreated syphilis in a group of African-American men, continued even when treatment became available, and which became publicly notorious in the 1970s. While we assume that any similar abuse of patient and research subject rights is unlikely to be repeated, two of the several important lessons that can be drawn from this history are of particular relevance to the present discussion. First, behaviours or practices collectively accepted under particular social, cultural or economic forces as proper or good, can on later reflection reveal themselves as deeply morally flawed. The other lesson is that the collective memory of such events by those who have been part of a victimised group, can be a powerful force in shaping attitudes. If the public views the privilege of using donated tissues for clinical care and research as an act of abuse, a substantial change in the regulation of such activities and tissue supply may be the consequence.

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2. Principles of Biomedical Ethics The relevant principles that help to frame the discussion of ethical concerns in biomedicine have evolved slowly over many centuries, but the confluence of a number of relatively recent and contemporary social forces has had substantial impact on our current understanding (Evans, 2000). These principles were reframed and articulated in the 1970s in their current broadly accepted form in the landmark publications of the US National Commission for the Protection of Human Subjects in Biomedical and Behavioral Research; The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research (National Commission, 1978) and the first edition of Principles of Biomedical Ethics (Beauchamp and Childress, 1979). The four principles are: respect for autonomy, nonmaleficence, beneficence and justice. Application of these principles is not a simple calculus — indeed, conflicts between the principles often help to define the nature of an ethical conflict, and they are particularly helpful as organising precepts and as the foundation of a common vocabulary. They are however, not the only important factors in analysing ethical problems in biomedicine. A number of important values and contextual issues help to shape the context and weight of the four principle factors in such analyses. Among these are: religious and cultural values; legal constraints; social factors; economic constrains and rewards; institutional practices; respect for human dignity; and professional values and moral virtues such as compassion, discernment, trustworthiness, integrity and conscientiousness (Beauchamp and Childress, 1994). Debates — for example, regarding the allocation of organ transplants and tissues that are in limited supply, or of other medical resources — often revolve around questions of justice. Although this helps to shape the discussion of these issues it does not "solve" them. Justice has been defined as the ethics of fair and equitable distribution of burdens and benefits within a community, or more concisely as: "to each their due." Yet the

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meaning of "to each their due" can have at least four quite different meanings: to each equally, to each according to their contribution, to each according to merit, or to each according to need. Clearly, each meaning can result in quite different solutions to problems of organ scarcity, or to the broader areas of health care policy where justice and beneficence are often the focus of discussion. 3. Ethical Challenges in Tissue Banking and Transplantation The ethical challenges of tissue banking and transplantation are numerous, but can be broadly divided into issues related to the collection and distribution of tissues for clinical use on the one hand, and research use of tissues on the other. Major concerns derive from the mix of non-profit and for-profit sectors, with differing goals and values. Issues related to tissue collection include balancing altruism and incentives, and maintaining practices that would be generally considered as affording appropriate respect for the gift of donated tissue. While in organ transplantation the granting of a "valuable consideration" is not acceptable, defining "reasonable payment" which may be acceptable for tissue (particularly processed tissue) is not always easily negotiated. Who is a vendor and who is a donor? How should each be treated or compensated? Should tissue obtained with an understanding that it is for clinical use, be used for research, and under what circumstances? What are the standards for obtaining consent in acquiring and using tissue? Do the standards dictate the voluntary informed consent expected in clinical medicine and clinical research, or (as in some countries) is simple assent or even lack of specific refusal from a member of the family, adequate? Does the community or a nation have a right to the tissues of the deceased as a vital societal resource? If not a public resource, do we consider the body of the deceased a commodity or a gift, the use of which may be governed by laws applicable to property, or is there a

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better framework derived from legal and cultural understanding of the body as "quasi-property"? Some of these dilemmas are reflected in the various terms used to refer to the harvest, salvage, retrieval or procurement of human tissues, each encompassing its own shade of meaning for the same act, or acts which appear quite similar. Who should have access to such tissues? How is "fair" distribution of resources assured where scarcity exists? What does fairness mean in these contexts, and how do we account for multicultural and legal differences in multinational markets? Substantial public debate surrounds questions such as the limits or appropriateness of foetal tissue use, and the definitions of the boundaries of life and death. How can concerns of confidentiality be reconciled effectively with issues of healthand donor-partner risk? How can potential conflicts of interest be minimised and actual conflict of interest be prevented from occurring? In research, additional issues are highlighted. The nature and content of appropriate consent for both surgical and cadaveric materials is a matter of debate, informed by many of the concerns noted above. Issues of anonymity and confidentiality, particularly with respect to genetic studies, are of real concern. The difficult question of how to address commercial use of research tissues or their derivatives, and who should fairly profit from commercial uses of such tissues and their related products, has not been satisfactorily or consistently resolved. Finally, how can the trust of the public be maintained and justified so that organ procurement organisations, tissue processors, suppliers, clinicians and researchers can meet their goals as well as their obligations? 4. Guidelines for Commercial Use of H u m a n Tissues The American Medical Association (AMA) has provided guidelines for commercial use of human tissue in its Code of Ethics (AMA, 2002), and the AATB is revising its own Code of

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Ethics; but adherence to these codes is neither mandatory practice nor enforced by regulations sufficient to address the issues raised above. For example, the AMA policy applies only to physicians, who are expected to abide by the following guidelines: (1) Informed consent must be obtained from patients for the use of organs or tissues in clinical research. (2) Potential commercial applications must be disclosed to the patient before a profit is realised on products developed from biological materials. (3) Human tissue and its products may not be used for commercial purposes without the informed consent of the patient who provided the original cellular material. (4) Profits from the commercial use of human tissue and its products may be shared with patients, in accordance with lawful contractual agreements. (5) The diagnostic and therapeutic alternatives offered to patients by their physicians should conform to standards of good medical practice and should not be influenced in any way by the commercial potential of the patient's tissue. The cornerstone, as well the potential Achilles' heel, of these policies is informed consent, as will be discussed below. Complicating the consent processes are the multiple custodians of tissues, many of whom do not know and cannot control many of the subsequent uses of these materials. Another factor complicating the commercial use of tissues has been the often unpredictable potential for tissue obtained by donation (sometimes for free and often for no more than a nominal sum), to have unexpected and sometimes very substantial financial value for an investigator, university or biomedical business concern. The debate over the proper ethical and legal framework from which to address this issue remains unresolved, with conflicting arguments between a market solution and a donation paradigm — the former, arguing for fair compensation only through commodification of human tissues

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as private property of the donor, and the latter (the "gift" or donation paradigm) arguing for such tissues to be viewed as a public resource instead of as a market commodity (Charo, 2002). The difficulties in resolving this conflict have been emphasised by the controversial decision of the California Supreme Court in Moore v. Regents of the University of California. The verdict supported disclosure of financial or research interests that might affect professional judgement regarding recommendations to patients, but upheld a position that the interests of society in new medical products were dominant over the possible individual interest in their tissue as private property (California Supreme Court, 1990; Harrison, 2002; Jordan and Price, 2002). While institutional review boards (IRB) will have an important role in reviewing and approving (with modifications as necessary) the human-subject research, which may also involve collaboration with commercial tissue banks or "biobanks", these new and important relationships raise important ethical questions that the IRB's will need to consider carefully (Rothstein, 2002). Even with careful IRB review, however, the "gift/community resource versus private property" debate will continue, and future legal decisions on this issue are likely to affect the decisions made by prospective research subjects with regard to their willingness to participate in human research protocols involving the taking or donation of their tissues. A proposal for an intermediate approach to compensation has been suggested to address the various ethical and legal problems inherent in each of the gift and market-driven strategies (Harrison, 2002). This proposal suggests compensation based on a liability rule that "would allow for donation of most human materials, but provide remuneration for unusually valuable tissue through a transparent, collectively-guided process conducted at a distance from the original contribution, and at a time when use of the tissue had established its commercial importance." Harrison suggests that removal of tissue should be both consensual and consistent with the contributor's dignity and bodily integrity; that the use of such tissue for research and commercial

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purposes should be consensual as well; and that private sales from the original tissue source would be banned. The proceeds from use for research or commercial purp-oses, however, should be shared with tissue sources with respect to ethical and practical considerations by use of a statutorilyestablished compensation tribunal. Approaches to implementing such a proposal, its impact and relationship to international legal and ethical standards, are also addressed. These issues will likely remain a matter of intense debate. What incentives are ethically permissible for organ and tissues donation for clinical use? This is also an area of increasing controversy, with pressure mounting to find ways to increase the availability of solid organs for transplant. Note that musculoskeletal tissues and skin, for example, are often stored for months after being "altruistically donated by grieving families" — yet these tissues are subsequently turned into sources of income by for-profit processors and distributors. Several editorialists have recently commented that this aspect of transplantation practice in the US "has circumvented the intention of the National Organ Transplant Act and makes the future of altruistic organ donation uncertain." (Delmonico et al., 2002). Given the limits of the consent process for donation as currently practised, the potential risks of this mix of altruism and profittaking may also lead to uncertainties for tissue transplantation, as discussed below. 5. Voluntary Informed Consent Several criteria have been widely agreed upon for the definition of voluntary informed consent in medical care and research. Clinical ethicists stress the view that "informed consent is a process, not a piece of paper," to emphasise the content and discussion that facilitates meaningful voluntary and informed consent in medical and research practice. The written "consent form" is meant to document such consent, but it is not a substitute. Documentation components include the following:

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description of the nature and purpose of the decision; discussion of alternatives; discussion of risks and benefits; discussion of related uncertainties; dssessment of the patient or subject's understanding; and elicitation of the patient or subject's preferences.

Discussion of the medical consequences and benefits alone is insufficient when confidentiality, commercial and other concerns are also at issue. Moreover, the consent requirements for organ and tissue procurement vary for different tissues, and between different countries (Indech, 2000; Childress, 1995). Express donation by the next-of-kin, or before death, by the individual donor, is common in a many countries around the world, including the United States and the United Kingdom, for procuring solid organs and sometimes for tissues. Presumed donation laws are often used for obtaining corneas, and sometimes for solid organs, but corneas are sometimes obtained in some states by routine removal or salvage. In certain situations tissues can be viewed as conscripted due to legal or forensic requirements. In some cases, tissues for research are obtained under conditions considered as abandonment, while sale remains common for sperm used for artificial insemination. 6. Government Analysis and Overview of Informed Consent in Tissue Banking Public interest in tissue banking, as represented by the interest of the federal government in what it views as the "tissue banking industry", is evident in several reports and new regulations issued in 2001 by the US Department of Health and Human Services (DHSS) and the Food and Drug Administration (FDA) concerning the need for greater oversight, safe practices, and informed consent, including in January, 2001, the Office of the Inspector General (OIG) of the US Department of Health and Human Services (DHSS) report on informed consent in tissue

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donation. (US DHSS OIG, 2001a; 2001b; US DHSS FDA, 2001). This report included recommendations to the DHSS, and to the tissue banking industry that merit particular attention. For the report on informed consent, information was obtained through extensive interviews with senior staff from organisations involved in obtaining, processing and distributing human tissues; and through questionnaires administered to donor families. Officials of leading organisations, including the AATB and others, were interviewed, and their documents and procedures reviewed in detail. State and Federal laws and regulations related to tissue banking were reviewed as well. The investigators found that the expectations and altruistic motives of donor families formed the foundations of tissue banking. The goals include a desire to enhance the lives of others; respect for the donor and the family; and trust in the tissue banking community. It was noted, however, that the reality of tissue banking raises some underlying tensions with the families' assumptions, given the commercialisation of tissue banking, the view of tissue as a commodity, and the uses of tissue for cosmetic purposes. In addition, fundamental difficulties limit the amount of information given to families. Consent is requested at a time of extreme vulnerability (as already alluded to above); families may not desire detailed information regarding tissue banking; and obtaining consent and medical information requires timeconsuming and invasive questioning about a recently deceased loved one. The investigators also found that current practices in requesting consent raise concerns about how and what information is provided to families. Such practices include obtaining consent by telephone, and reliance on staff from other organisations, some with little training and accountability. Finally, little written material is provided at the time of donation. The investigators noted that until recently, standards governing how families are approached, and what they are told about tissue donation, have been nonexistent. Although there are no federal laws, regulations or direction from the States' Uniform Anatomic Gift Acts on the manner of obtaining consent, or what

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information should be provided by tissue banks, several organisations have proposed elements of informed consent for tissue donation, including the AATB, AOPO and the EBAA, as well as the National Donor Family Council of the National Kidney Foundation. The AATB, moreover, is incorporating these statements into accreditation standards. Recommendations made to DHSS include that the Health Resources and Services Administration (HRSA) should work with groups representing donor families and the tissue banking industry, to develop guidelines for conveying information to families about tissue donation; and that the Health Care Financing Administration (HCFA) should address informed consent for tissue donation through the Medicare conditions of participation. With regard to their recommendations to the tissue banking industry, they advise the following: at the time of obtaining consent, tissue banks should provide families with written materials that provide thorough and fuller disclosure about the uses of tissue and the nature of the gift, including a copy of the consent form; information for follow-up contact if concerns or questions arise; a "full description of the uses to which donated tissue may be put"; and "a list and description of other companies and entities with which the bank has relationships for processing and distributing tissue." The recommendations to the industry also include working with groups representing donor families, to explore a process for periodic public disclosure about tissue bank financing, and fostering greater accountability for the performance of those who request consent for donation. 7. Preventive Ethics It has long been said that "an ounce of prevention is worth a pound of cure." This is true in medical ethics as well. Preventive ethics will include taking actions that foster communication and disclosure regarding methods, uses, goals and incentives; actions that derive from and demonstrate respect for persons, including

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their values and goals, as well as fostering fairness and respect for the gift; actions that limit potential conflicts of interest; and actions that establish guidelines through open deliberation. Such actions are the keys to maintaining trust, and such trust is one key to the future of maintaining and enhancing the opportunities to derive the medical and scientific benefits afforded by tissue banking and transplantation. 8.

Acknowledgements

This manuscript was presented in part as a plenary address on August 26, 2002 at the 26th Annual Meeting of the American Association of Tissue Banks and the Third World Congress on Tissue Banking. The author wishes to thank Dr. Sam Doppelt for his role in stimulating and helpful discussions. 9. References AMERICAN MEDICAL ASSOCIATION (2002). AMA Code of Ethics Policy E-2.08. Commercial use of human tissue. (www.ama-assn.org) BEAUCHAMP, T.L. and CHILDRESS, J.E (1979). In: Principles of Biomedical Ethics, Oxford U. Press, New York. BEAUCHAMP, T.L. and CHILDRESS, J.F. (2001). In: Principles of Biomedical Ethics, Oxford U. Press, New York. BLAKESLEE, S. (2002). Recall is ordered at large supplier of implant tissue. In: The New York Times, New York, August 15, pp. A l , A16. CALIFORNIA SUPREME COURT (1990). Moore v. Regents of the University of California. 793 P.2d 479 (Cal. 1990). CHARO, R.A. (2002). Skin and bones: Post-mortem markets in human tissue, Nova Law Rev. 26, 421-450.

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CHILDRESS, J.F. (1995). Organ and tissue procurement. II. Ethical and legal issues regarding cadavers. In: Encyclopedia of Bioethics, W.T. Reich, ed., Macmillan, New York, pp. 18571865. DELMONICO, F.L., ARNOLD, R., SCHEPER-HUGHES, N., SIMINOFF, L.A., KAHN, J. and YOUNGER, S.J. (2002). Ethical incentives — Not payment — For organ donation, New Engl. J. Med. 346, 2002-2005. HARRISON, C.H. (2002). Neither Moore nor the market: Alternative models for compensating contributors of human tissue, Am. J. Law Med. 28, 77-105. INDECH, B. (2000). The international harmonization of human tissue regulation: Regulatory control over human tissue use and tissue banking in select countries and the current state of international harmonization efforts, Food Drug Law J. 55, 343372. JORDAN, C M . Jr, and PRICE, C.J. (2002). First Moore, then Hecht: Isn't it time we recognise a property interest in tissues, cells, and gametes? Real Prop. Prob. Tr. J. 37, 151-190. MAHONEY, J.D. (2000). The market for human tissue, Va. Law Rev. 86, 163-223. NATIONAL COMMISSION FOR THE PROTECTION OF HUMAN SUBJECTS IN BIOMEDICAL AND BEHAVIORAL RESEARCH. (1978). The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research. GPO: Washington D.C. RAEBURN, P. (2002). Human tissue: Handle with care. It's getting more important in medical research, but ethicists have grave privacy concerns. In: Business Week, New York, April 15, pp. 75-77. ROTHSTEIN, M.A. (2002). The role of IRBs in research involving commercial biobanks, /. Law, Med. Ethics. 30, 105-108.

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US DEPARTMENT OF HEALTH AND HUMAN SERVICES. OFFICE OF THE INSPECTOR GENERAL. (2001). Informed Consent in Tissue Donation. Expectations and Realities. OEI-01-0000440, January. US DEPARTMENT OF HEALTH AND HUMAN SERVICES. OFFICE OF THE INSPECTOR GENERAL. (2001). Oversight of Tissue Banking. OEI-01-00-00441, January. US DEPARTMENT OF HEALTH AND HUMAN SERVICES. FOOD AND DRUG ADMINISTRATION. (2001). Current Good Tissue Practice for Manufacturers of Human Cellular and Tissue-Based Products; Inspection and Enforcement; Proposed Rule. Federal Register. Part VIII. 21 CFR Part 1271, January 8.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

13 THE VOLENDAM BURN DISASTER AND THE IMPORTANCE OF INTERNATIONAL COLLABORATION IN TISSUE BANKING

JEROEN V A N BAARE N e t h e r l a n d s Bone b a n k F o u n d a t i o n Leiden, The N e t h e r l a n d s GER K R O P M A N Euro Skin bank, Beverwijk, The N e t h e r l a n d s

1. Introduction In the treatment of burns — either superficial or deep, partial or full thickness — where sufficient autograft is lacking, wound coverage by fresh or stored human allograft skin is still the gold standard for many physicians involved in burn care. To provide allograft skin on a continuous basis, and when allograft skin is readily available, long-term storage of allograft skin is necessary, preferably in an accredited skin or tissue bank. Euro Skin Bank has been delivering allograft skin for more than 25 years. The skin bank started as a local bank in 1976 in the vicinity where the Beverwijk steel factories were located. Factory employees with burns were transported to the Red Cross hospital in Beverwijk. To avoid the use of skin from living donors, which left unacceptable scars on the living donors, the

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skin of cadaveric donors was used. As two other burn centres were set up in The Netherlands, the skin bank started to operate on a national level. Operating as a national skin bank resulted in a concentration of know-how, reduction of costs and competition in skin banking. The long-term existence of the skin bank was also a result of the support by the Dutch Burns Foundation. The Dutch Burns Foundation is a fund-raising organisation, and is successfully involved in programmes for burn patients, public education for preventing burns, and in efforts to support research projects and the national skin bank. In 1992, the name of the national skin bank was changed to Euro Skin Bank because of increasing international demand for human allograft skin, and to express international collaboration. Euro Skin Bank has been preserving donor skin in glycerol for many years now, and interest in the clinical use of this kind of allograft is still increasing. Currently, over 300 skin donors are procured each year, and more than 1.6 million square centimetres are distributed throughout the world each year. Euro Skin Bank has been working in close collaboration with several tissue banks, especially to support the establishment of skin banks in several countries. An example of a successful result of international colla-boration is that with the tissue bank in Brno, which is located at the University Hospital, Czech Republic, and is supervised by Dr. Jiri Adler. 2. Operational Readiness Under normal circumstances, allograft skin is distributed to burn centres and hospitals for the treatment of one of very few patients at one time. Large disasters in which a great number of burn patients are involved, are very uncommon, and always in unusual circumstances. For example, in August 1988, an airplane crashed during an air show in Ramstein, Germany. An airplane crashed in the suburbs in Amsterdam in October 1992, and another Dutch airplane crashed in December 1992 in Faro, Portugal.

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Euro Skin Bank has always been prepared to provide large amounts of allograft skin in case of disasters. To be prepared for a disaster, there is frequently the question of how much skin should be stored in a skin bank to handle a situation with many burn victims. As The Netherlands is very well organised, no terrorist attacks were expected, and since the burn prevention programmes by the Dutch Burns Foundation were very successful, the definition for a disaster in The Netherlands was calculated in 2002 to involve approximately 15-20 patients. The unexpected happened on New Year's Eve, 2000-2001, in a night club in Volendam, a small village north-east of Amsterdam (Fig. 1). That night, 300 (young) people were in the club, where only 85 were allowed. The decoration on the ceiling was not protected against fire, and fire-exits were blocked or not present, or emergency stairs were missing. People were wearing

Amstetf T.

NETHERLANDS The Hague

BELGIUM

Fig. 1. The deadly cafe fire in Volendam, The Netherlands.

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highly flammable clothes, mainly consisting of acrylic a n d / o r polyester material (Fig. 2). The fire was caused by some small fireworks which came into contact with the decoration on the ceiling. The fire lasted only 10 seconds. Directly after the fire, 203 patients were admitted to 27 hospitals, mainly injured with burns and inhalation trauma. During the fire three people died. The first week after the fire, another seven people died due to burns and inhalation trauma, and 90 burn patients were in the hospital. In the following months, four patients subsequently died. The day after the disaster it was unclear how many burn patients were admitted to hospitals. As all Intensive Care units were occupied, patients were also transported to hospitals outside The Netherlands. Euro Skin Bank had 75,000 cm 2 of glycerol-preserved allograft skin in storage. It was obvious that more allograft skin was needed than the skin bank had in stock. Also, the "regular" burn patients were being admitted to hospitals in Europe and these burn centres also needed allograft skin to treat them.

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Therefore, a number of skin banks were contacted in order to procure a big supply of allograft skin. Sam Whyatt of the Stephen Kirby skin bank in London, UK, was contacted, and shipped 10,000 cm2. Daniel Lismont of the tissue bank in Leuven, Belgium, shipped 5,000 cm2. Dr. Jiri Adler from the tissue bank in Brno, Czech Republic, shipped 20,000 cm2. Also, Glenn Greenleaf from LifeCell in New Jersey, USA, was contacted. Glenn Greenleaf is one of the most dedicated persons in skin banking and burn wound treatment over the last decade, with many national and international contacts. He called a number of skin banks in the USA and found LifeNet prepared to ship another 20,000 cm2 of allograft skin to Euro Skin Bank. In total, 200,000 cm2 was needed to treat all burn patients of the Volendam burn disaster. As already mentioned previously, disasters are seldom expected. This was also the case with the terrorist attack on the World Trade Centre in New York on September 11, 2001 (Fig. 3). Euro Skin Bank contacted Glenn Greenleaf immediately to offer

Fig. 3. World Trade Centre on September 11, 2001 (from CNN exclusive).

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any help and assistance where possible. As with many disasters there was no good overview of the extent of casualties, and of how much skin would be needed. Eventually, no extra skin was needed as, unfor-tunately, burn-victim mortality was high. The American Association of Tissue Banks has now already set up an Emergency Preparedness Plan to have a quick overview of where and how much allograft tissue is available. International collaboration is therefore very important, but is only possible through long-term relationships. All aspects of tissue banking should be considered when retrieving allograft tissue from other tissue banks, to ensure the tissue is safe and of high quality, including prior consent, screening procedures, disease testing, good lab storage and reliability. These cases demonstrate the importance of international collaboration well before disasters and terrorist attacks ever occur.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

14 THE NEED FOR A TISSUE BANK IN A DISASTER: EXPERIENCE IN ARZOBISPO LOAYZA NATIONAL HOSPITAL AFTER THE TRAGEDY IN "MESA REDONDA", LIMA, PERU

MARCO ANTONIO GARCES MORALES and CESER ALEJANDRO REYNAGA LUNA Plastic and Burns Department of Loayza Hospital Lima, Peru

"When we know what do we have to do in order to save a life, we don't argue ... we do it" ... Peter Safar, 1987

1. Introduction This experience is based on the participation of a human group, which was responsible for the plastic surgery and burns treatment service of Loayza Hospital during the disaster in Mesa Redonda on December 29, 2001. At 7:10 p.m. on a Saturday, a great tragedy occurred in central Lima. It started in the cradle of a flea market, where there was a minimum of safety regulations, and the incident lasted for five hours, leaving a macabre scene of death and desolation. 375

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It all began with the explosion of 90 tons of gunpowder, generating temperatures of 1,200°C that claimed more than 660 victims: 300 dead, 352 who disappeared, and caused a loss of 100 million dollars. 2. General Analysis To be able to understand our task, we needed to define the basic concepts. A disaster is a big catastrophe, and an unfortunate and mournful event. To be able to confront it, it is necessary to have a plan in order to respond to the great number of deaths and injuries, and be prepared to attend to them with efficiency, and to adopt the necessary security measures, which will contribute to its future prevention. The tragedy in Mesa Redonda was very complicated. Its root cause was human and technical failure. Based on the number of victims, this tragedy was defined as very serious by Mabrouk, 1981 and Gomez, 1988. The victims' injuries were burns, inhalation and other bodily damage produced in different parts of the body by fire, gunpowder explosions, building collapses, physical crushing, toxic gases and electric discharges. 3. On-Site Management of the Mesa Redonda Tragedy Priority in the tragedy area was given to the evacuation of victims who could receive immediate treatment in an adequate location, with the possibility of saving their lives. This first intervention was handled by the emergency systems of the Peruvian civil defense: firemen, policemen, health ministry and social insurance department, and civilians. There were more than 5,000 people involved, with no escape paths, trapped in traffic jams, dodging fireworks all over the streets. The lack of a fireproof system, and constructions that lacked security measures, formed a fatal urban trap, as found in most cosmopolitan cities of Third World countries.

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During the first 15 minutes, a fireball formed, with the temperature reaching between 700-1,200 degrees Celsius. This is an important point, since it explains the evaporation of the bodies. For every three bodies present, one evaporated. There was chaos, panic and confusion surrounding the area. Hundreds of phone calls were received and needed to be responded to immediately. The first evaluation suggested that 300 people were trapped. Evacuation of the wounded was started but the situation became uncontrollable. Later, defensive actions were taken against the fire; security perimeters were established, and the electricity supply was cut. There was an increase of water pressure directed to the area, and water trucks started to arrive. An incident command center was installed, along with the opening of sectors for medical assistance, search and rescue, fireproofing, logistics, security and public relations. The coordination of assistance to patients within Loayza Hospital started towards the daily changing of shift, and because of the holiday period, there were many beds available. There was great solidarity perceived among members of the health team; personnel on vacation and those on leave returned immediately to their working positions. 4. Management of the Tragedy in the Emergency R o o m The first check-up took place in the tragedy area. It was fast and simple, based on the tragedy classification 919970, which is a simplified variant of the start method. Respiration, circulation and consciousness were evaluated, with control of the unconscious victims and haemorrhage control. The second check-up was undertaken in the emergency room, taking into account the age, sex, extension and depth of the burns and inhalation harm suffered by the patient. The victims were later taken to the medical area where the third and most important check-up was made. Of the 147 patients, 55 were hospitalised in the medical area, which had been adapted for the burns, plastic surgery and general treatment.

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5. The Plastic Surgery and Burns Service An operational command centre was installed to manage the circumstances of the tragedy, and to appraise the physical resources, equipment, logistics (skin substitutes), communication, and transportation. The Peruvian Government ordered, by supreme resolution, free treatment in the hospitals for those people with burns and related injuries. Of the 147 patients in the emergency room, 92 received outpatient treatment (analgesics, oral hydro reposition, sedatives) and 55 were hospitalised. The treatment started with the participation of a multidisciplinary team to achieve an effective union between the medical and paramedical teams for the treatment of the burn patients according to the following scheme: LOCALISATION

EVOLUTIVE STATE

\ / ATMOSPHERE CARE

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BURNED PATIENT ,

HEALTH SYSTEM

CAUSE-BURN

/ \ BURNS EXTENT

6. Materials and Methods There were more female patients (60.71%) than males (39.29%). The proportion by age was greater among those 21-40 year old patients (75%) and among 0-20 year old patients (10.71%) and those from the 41-60 age group (14.29%). The burns extent (ruler of 9 and with the palm of the hand) of less than 10% was 48.15% in patients, and greater than 10% in 51.95% of patients. In terms of depth of burn (using the Benaim and American classification) the patients had second to third degree burn degrees in relation to seriousness (Garces abbreviated index of the burninjury). Other diagnoses were: inhalation damage, thermal keratoconjunctivitis, diabetes, cervical concussion, bronchial asthma, and fractures.

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The treatment started with the removal of burnt cloth; cooling of the burn in patients with less than 10% burns; cleaning of the burned areas with iodine; administration of 1% silver diazine, and the application of the occlusive method with gauze. This was necessary because of the lack of sufficient supplies from an available tissue bank, which would have allowed sufficient temporary biological dressings. This procedure was followed every day in patients with greater than 10% of burns. Also, resuscitation isotonic solutions were applied. The patients with inhalation damage were treated with oxygen, hyperbolic oxygen, artificial respirators and treatment for respiratory rehabilitation. Nevertheless, there was a need for a bronco-fibrescope to diagnose and treat the lower and upper respiratory paths. The surgical treatment proceeded with the elimination of crust (dead skin). From the second to fifth day, the surgical teams worked double shifts from Monday to Sunday. They made eight interventions per day with the help of doctors from Israel and China. They used manual and electric dermatoms, compressed air and skin meshes, and also the escarectomy technique according to the depth and place of the burn, including tangential escarectomy, tangential debridement, aponeurotic escarectomy and the following surgeries: surgery heels + escarectomy; escarectomy + autograft; escarectomy + heterograft; escarectomy + autograft + heterograft; and local flaps. The skin substitutes used were autografts (microcrafts, stamps, laminates, and meshes). For the transitory patients cadaveric skin (from the USA), pig skin in laminates of 10 x 10 cm, rings in laminates of 30 x 30 cm were used. In the post operative stage, sulfamylon solution in 5% and silver nitrate in 0.5% were used. 7. Discussion Accidents in the home, during transportation, at work, at entertainment centres, and during civil violence or wars, produce great numbers of victims. The cost is more than 550 million dollars and in the United States these accidents cause 33% of the

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hospitalisations, with each hospitalisation costing US$34,000 and up to US$317,000 for severe cases. For this reason, it is important to take all possible preventive measures. The medical management of the Mesa Redonda tragedy was carried out under the unified temporary command system, due to the lack of resources. This included an incident action plan. The transportation of the patients was carried out as quickly as possible because the tragedy was out of control. In the hospital the treatment needed was determined in three check ups, according to the patient's condition. For these reasons, it was necessary to have human resources which can work in motivated and compact multidisciplinary teams to provide adequate diagnoses, and to start the respective treatments promptly. The treatment was undertaken, using a simple protocol starting with a resuscitation period (parlank formula). For local treatment, only silver diazine at 1% was used. Surgical treatment started between the second and fifth day according to the depth, extension and localisation of the burn. In patients with extended burn areas, skin substitutes were used, which had been supplied by the tissue bank located at the Children Health Institute (Insititut de Nino), Lima. Donations of pig skin came from China, allografts from the United States and fresh and refrigerated tissue rings prepared by our service. The treatments utilised 661,500 cm 2 of skin substitutes. The actual needs were greater due to the escarectomy and need for autografts after the escarectomy. It is known that the preparation of the bed is very important for the grafts, especially for the patients with large burn areas and where only small areas of donated skin are available. 8. Conclusions (1) To deal with a tragedy involving a high number of casualties, the common areas at the hospital should be converted into restricted areas.

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(2) The initial patient treatment must be undertaken using a simple protocol. (3) Escarectomy must be carried out between the second and fifth days. (4) There is a need for a multi-disciplinary and trans-disciplinary team. (5) Use of skin substitutes available (pig skin, anionic membrane and the products of tissue banks) is urgently required and logistically planned for, before contingencies. (6) The creation of tissue banks in all the countries is necessary so that they can together contribute when there is such an emergency. The value of a man should be judged according to what he gives and not according to what he is able to receive. Albert Einstein

9. References AMERICAN BURN ASSOCIATION (1984). Guidelines for service standards and severity classification in the treatment of burn injuries, Am. Col. Surg. Bull. 69, 24-28. ARMSTRONG, C.E., Jr, SCHAEFFER Y C.P. Y C. P. ARTZ (1984). Treatment of. ARTIGAS, N. Y COL. (1984). Normas Medico Quirurgicas Para El Tratamiento De Las Quemaduras. Editorial Andres Bello, Santiago De Chile. ARTURSON, G. (1981). The Los Alfaques Disaster: A boiling liquid expanding — Vapour Explosion, Burns 7, 233-251. ARTURSON, G. (1987). The Tragedy of San Juanica: "The Most Severe LPG Disaster in History", Burns 13, 87-102.

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ASISTENCIA SANITARIA INTERNACIONAL (1989). Medicina De Catastrofe. Boletin Del Dpto De Med De Catast. Dir. Nac. Defensa Civil Min. De Defensa. Buenos Aires 2(9), 10. BABCOCK. C.L (1974). High Rise. Building Fires and Fire Safety. NEPA, pp. 3. BACKES, A.T. (1973). Great Fire of America. Wankesha Country Beautiful Corp., pp. 124-130. BECK, A.T. Y COL (1961). An inventory for measuring depression (B. D. I), Arch. Gen. Psychiatry 4, 561. BENAIM, F. (1956). "Diagnostico De Gravedad De Las Quemaduras", Bol. Soc. Arg. Cir. Plast. 1. BENAIM, F. (1959). Diagnostico De Gravedad De Las Quemaduras, Bol. Soc. Arg. Plast. 1. BENAIM, F. (1962). Tratamiento De Urgencia De Las Quemaduras Graves. Vol. 1, pp. 13-34. Buenos Aires: Edic. Fanetti. BENAIM, F. (1968). Quemaduras. En Michans, J.R. (Ed.) Patologia Quirurgica, 2a. Edic, Vol. 1, Cap. 6. Buenos Aires. El Ateneo, pp. 134. BENAIM, F. (1971a). Burn Centers And Prevention Campaigns in Latin America Countries. En Matter, P., T.L. Barclay Y Z. Koniciova (Eds- Transaction of the thrid international congress on research in burns, Bern: Hans Huber Publishers, pp. 39. BENAIM, F. (1971b). Personal opinion on a uniform classification of the depth of burns. Transaction of the third international congress on research in burns. Bern: Hans Huber Publishers, p. 715. BENAIM, F. (1978). Cuidado Progresivo Y Equipamiento Especializado En Las Unidades De Quemados, Cir. Plast. Arg. 2, 28-35.

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BENAIM, F. (1982). Evolution De La Asistencia Al Quemado En La Argentina, Rev. Arg. De. Cir. 43, 32-40. BENAIM, F. (1984a). El Especialista En Quemados, Rev. Arg. Quern. 2(3). BENAIM, F. (1984b). Qeumaduras. En Torres, R. Tratado De Cirugia, Cap. 10. Interamericana, Mexico, pp. 259-304. BENAIM, F. (1984C). Plan De Regionalizacion Para La Atencion Del Paciente Quemado En La Republica Argentina, Rev. Ar. De. Cir. 46, 170-175. BENAIM, F. (1984d). Asistencia Interdisciplinaria Para El Paciente Quemado, Rev. Arg. Quern. 2(2). BENAIM, F. (1986). Quemaduras. En Coiffman, F. Y Col, ed., Texto De Cirugia Plastca, Reconstructiva Y Estetica, Tomo 1, Cap. 3. Salvat, Barcelona, pp. 243-282. BENAIM, F. (1989a) Catastrophe En Niteroi, Brazil 1961. Fire in a Circus. Argentinian Help to the Burn Victims. Ludwingshafen International Symposium on Disaster Management of Burns. September 1-2, Ludwingshafen, W. Germani. BENAIM, F. (1989b). Quemaduras. En Boretti, J.J. Y C. Lovesio, eds., Cirugia, Cap. 15. El Ateneo, Buenos Aires, pp. 202-216. BENAIM, F. Y D. SCHNEERSON (1964). Conducta Medica En Caso De Catastrofe De Quemaduras. Symposium Conducta Medica En Caso De Catastrofe. Academia Nacional De Medicina, Buenos Aires, pp. 313-324. BENAIM, F. Y COL (1964). Incendios Y Exlposiones. Symposium Conducta Medica En Caso De Catastrofe. Academic Nacional De Medicina, Buenos Aires, pp. 481-487. BEN-HUR, N. Y H. SOROFF (1975). Quemados Y Sindrome De Quemaduras Por Cohetes Antitanque, Cir. Plas. lb. Lat. 1, 77-84.

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BENNETT, C. Y H. BRISTOL (1970). Controlled Survery of Effects on Health of Local Community Disaster, Br. Med. }. 3, 454. BENZAQUIN, P. (1976). Fire in Boston's Coconut Grove. Braden Press, Boston, pp. 204-224. BEST, R. (1978). Reconstruction of a tragedy: The Beverly Hills Supper Club Fire. NFPA, Boston, p. 314. BONTE, F. (1988). Chernobyl Retrospective, Semin. Nucl. Med. 18, 16-24. BOSWICK, J. (1987). Initial care of burn wounds. En Aspen Publishers Inc. (Ed.) The art and science of burn care. Rockville Maryland, Royal Tunbridge Wells. Botha, C. (1986). The dental identification of fire victims, /. Forensic Odontostomatol. 4, 67-75. BUCEK, S. Y V. PINMCOVA (1987). Micridisastres in the Prague Burn Centre. II Congress On Burn Treatment. Kosice-Saca, Czeschoslovakia, p. 69. BURTON, H. (1973). The Morro Castle. Viking Press, New York, pp. 92-103. BUTMAN, A. (1982). Responding to the mass casuality incident. A guide for EMS personnel. Emergency training, Akron, Ohio. CARSWELL, J. Y A. HATHAWAY (1976). A fire at Nakivubo, Kampala: A case Report. II — Infection in a group of burned patients, Burns 2, 184-190. CHAMPION, H. Y COL (1981). The Trauma Score, Ann. Med. 9, 672. CHURCHILL, B. (1953). Panic in Disaster, Ann. Surg. 138, 395. CLASS, A. (1959). Physiological aspects of disaster, JAMA 171, 222. COBB, S. Y E. LINDEMANN (1943). Neuropsychiatric observation during the Coconut Grove fire, Ann. Surg. 117, 814.

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COLE, M. Y COL (1986). Gasoline explosions, gasoline snifting: An epidemic in young adolescent? JBCR 7, 532-534. COMMITTEE ON TRAUMA OF THE AMERICAN COLLEGE OF SURGEONS (1986). Qualification of trauma-care personnel, Am. Col. Surg. Bull. 71, 13-38. COMMITTEE ON TRAUMA OF THE AMERICAN COLLEGE OF SURGEONS (1983). Hospital and pre-hospital resources of optimal care of injured patients, Am. Col. Surg. Bull. 68,11-21. CORNELL, J. (1979). The great International Disaster Book. New York Gulf and Western Corp., pp. 290-292. DAS, R.A. (1984). Circus Fire Disaster In India (Feb. 7,1981), Bull. Clin. Rev. 1, 26-31. DAS, R.A. (1983). 1981 Circus Fire Disaster in Bangalope, India: Causes, management of burn patients and possible presentation, Burns 10, 17-29. DE LEONE, H. (1988). Categorizacion De Pacientes En Caso De Catastrofe. Circulacion. Medic. De Catastrofe. Boletin Del Dpto Med De Catast, Dir. Nac. Defensa Civil. Min. De Defensa. Buenos Aires 1(2), 1. DICKINSON, W., D. SHARPE Y A. ROBERTS (1988). Tangencial excision of scalp burns: Experience from the Brodford fire, Burns 14, 151-155. EDLICH, R. Y COL. (1983). Firefighter's guide to emergency rescue and care of victim burned in structural fire, JBCR 4, 367-373. FREY, R. Y P. (1980). Types and Events of Disaster. Springer, Verlag, New York, pp. 282-285. GOMEZ, M. (1988). Categorizacion De Pacientes En Caso De Catastrofe. Respiracion. Medic. De Catastrofe. Boletin Del Dpto Med De Catastr. Dir. Nac. Defensa Civil. Min. De Defensa. Buenos Aires 1(2), 4.

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GORMICAN, S. (1982). CRAMS Scale: Field triage of trauma victims, Emerg. Med. 11, 132. GUNN, S. (1989). Disaster Medicine and International Relief Multilingual Dictionary. English, French , Spanish, Arabic. Kluwer Academic Publishers, Dordrecht, Holland. GRUPTA, R. Y A. SRIVASTAVA (1988). Study fatal burns cases in Kanpur (India), Forensic Sci. Int. 37, 81-89. HADDEN, W., W. RUTJERFORD Y J. MERRET (1978). The injuries of terrorist bombing in a study of 1532 consecutive patients, Br. J. Surg. 62, 525. HAMILTON, M. (1960). Depression Rating Scale (HDRS), /. Neural Neurosurg. Psychiatry 23, 56.

SECTION IV: TISSUE GRAFTS IN ORTHOPAEDICS

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

15 MAJOR LIMB RECONSTRUCTION USING MASSIVE CADAVERIC ALLOGRAFTS

HENRY J. M A N K I N Orthopaedic Oncology Service Massachusetts General Hospital H a r v a r d Medical School, Boston, M A 02114

1. Introduction Over the past 31 years, limb-sparing bone tumour treatment has depended heavily on the replacement of the resected segment of bone with a cadaveric allograft. Since the pioneer efforts of Frank Parrish in Houston (Parrish et al, 1966; 1973) and Carlos Ottolenghi in Buenos Aires (Ottolenghi et al, 1996), a number of orthopaedic oncologists have utilised the system and reported their findings (Alho et al, 1998; Delloye et aZ.,1988; Dick et al, 1985; Friedlaender et al, 1999; Gouin et al, 1996; Hejna et al, 1997; Hornicek et al, 1998; 1999; Jofe et al, 1988; Makley et al, 1985; Mankin et al, 1982; 1987; 1996; Mnaymneh et al, 1986; 1989; Ortiz-Cruz et al, 1997). Bone banks have appeared, and through the efforts of the American Association of Tissue Banks and their leaders, have developed a set of guidelines to maintain the safety and competence of the parts (Buck et al, 1989; Conway et al, 1990;

389

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Schachar et al, 1991; Strong et al, 1996; Tomford et al, 1983; 1989; 1999). Special attention has been paid to cryopreservation of cartilage (Schachar et al, 1991; 1994; Tomford et al, 1983); prevention of transfer of infection (Buck et al, 1989; Hernigou et al, 1991; Lord et al, 1988; Strong et al, 1991; Tan et al, 1997; Tomford et al, 1990); banking methodology (Tomford et al, 1989; 1999); use of radiation sterilisation techniques (Conway et al, 1990; Loty et al, 1990); and most recently genetic structure of the graft in correlation with the host recipient (Alho et al, 1998; Friedlaender et al, 1976; 1999; Muscolo et al, 1996; Strong et al, 1996). Since 1971, the author and a group of colleagues in the Othopaedic Oncology Service at the Massachusetts General Hospital have performed almost 1,200 operative procedures in which a segment of frozen allograft bone has been introduced into a patient, most frequently after wide or marginal resection of a malignant or aggressive bone tumour. Since 1976, the bone segments have come from a local bone bank, which is now headed by William W. Tomford, a respected member of the AATB and a leader in the field of bone banking (Tomford et al, 1989; 1999). A careful review of the results of the allograft procedures was performed, and the purpose of this presentation is to analyse the outcomes and determine the factors which seem to play a role in the success or failure of the procedure. It should be noted that of the almost 1,200 procedures 75 were hemipelvis grafts, which are considerably different from those for more peripheral parts. The indications, clinical management, complications and outcomes for this group differ from the other procedures, and are therefore not included in this review. The study is limited to a description of the results for 1,098 patients who received their peripheral part alloimplants between November of 1971 and June of 2002. The outcome studies require that only patients followed for two or more years are included, and this number is 1,052.

Major Limb Reconstruction Using Massive Cadaveric Allografts

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2. Materials and Methods Data for the entire allograft series were collected in a Microsoft Foxpro system (Microsoft, Redmond, WA) (Mankin et al, 2002) which houses information for almost 15,000 patients treated by the Orthopaedic Oncology Group since 1972. A special file in this system is used to maintain all of the data regarding the 1,098 allografts performed by the group since 1971. Information included consists of the patient's demographics, diagnosis, MSTS stage (Enneking et al., 1980), anatomical site, resection margin, use of adjunctive treatment modalities, tumour complications, allograft complications, additional operative procedures and outcome. The statistics are in part performed by the Foxpro system, but in addition, a statistical program was used for study of data using Cox regressions (Cox et al, 1972) and KaplanMeier outcome studies (Kaplan et al, 1958) (BMDP System, Los Angeles, CA). The 1,098-patient group consists of 573 males and 525 females with a followup ranging from two to 367 months, with a mean figure of 86 ± 72 months. The average age for the patients was 32 ± 18 years, with a range from two to 86 years. An analysis of the diagnoses for which the procedure was performed, shows that the majority were done for malignant tumours including osteosarcoma (285 patients), chondrosarcoma (148 patients), parosteal osteosarcoma (62 patients), fibrosarcoma or MFH (51 patients), Ewing's sarcoma (50 patients), metastatic carcinoma (47 patients), soft tissue sarcomas (34 patients), and adamantinoma (32 patients). In addition, in the early days of the system, 139 patients with giant cell tumours were also treated with resection and allograft implantation, as were 56 patients with other neoplasms. There were in addition, a total of 182 patients treated for non-tumourous conditions including failed allografts or total joint replacements (90 patients), massive osteonecrosis or traumatic loss (58 patients), fibrous dysplasia (14 patients) and 20 others.

392

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Table 1. Anatomical sites for 1,098 allograft procedures performed between 11/1971 and 6/2002. Osteoarticular (583) Distal femur Proximal tibia Proximal humerus Proximal femur Distal radius Distal humerus Scapula Distal tibia Proximal ulna Talus Elbow Phalanx

268 116 84 41 27 15 10 8 6 3 3 2

Intercalary (295) Femur Tibia Humerus Fibula Ulna Radius Scapula Hand bone Foot bone Sternum Vertebra

122 102 49 6 5 5 2 1 1 1 1

Allograft-prosthesis (138) Proximal femur Distal femur Proximal tibia Entire femur Proximal humerus Scapula Elbow

72 35 16 10 2 1 1

Allograft arthrodesis (82) Distal femur Proximal humerus Proximal Femur Distal Tibia Proximal tibia Distal radius

36 28 8 6 3 1

Major Limb Reconstruction Using Massive Cadaveric Allografts

393

Five hundred and eighty-three of the procedures were osteoartieular (involving a joint); 295 intercalary (introduced to fill a gap in the host bone); 138 were allograft plus prosthesis and 82 were allograft-arthrodesis. The anatomical locations for these segments are shown in Table 1. The outcome for these patients was assessed according to a scheme originally proposed in the 1980,s (Mankin et ah, 1982). The results, or more appropriately, the "outcome" of the procedure, was determined by scoring the result as excellent, good, fair or failure. An excellent outcome is defined as a functionally normal result with basically no limitations or disability, no local recurrence or pain. A good outcome is defined as a pain-free functionally useful limb but limited in terms of agility and sports participation. Patients described as fair are limited in employability, have discomfort and limitations, which require support

(A)

(B)

Fig. 1. A 19 year old woman presented with a painful right humerus in June, 1974. A fibrosarcoma was discovered [Fig. 1(A)] and the proximal humerus resected and replaced with an allograft segment [Fig. 1(B)]. At the age of 47, she remains partially limited in abduction and forward flexion, but otherwise enjoys normal movement.

H.J. Mankin

394

for ambulation. The term failure was reserved for patients who required removal of the graft or amputation. For the statistical studies reported in this presentation, an "excellent" or "good" result is considered to be a successful outcome while a "fair" or "failure" is designated as a failure. Of note is the fact that the failure category includes patients who have had a local recurrence, which led to removal of the graft or amputation. If one is determining the outcome of the grafting procedure rather than the tumour treatment, it is appropriate to remove these cases from the series. Examples of allograft procedures are shown in Fig. 1 through 3. The results of all three of these cases were considered to be "good", and hence their designation as successful outcomes. All three of these patients remain free of disease, have no pain and although two are moderately restricted, they remain quite functional. The patient described as Case 1 has remained a successful outcome for 31 years.

(A)

(B)

(C)

Fig. 2. A 16 year old female was found to have an adamantinoma of the right tibial shaft in September 1988 [Fig. 2(A)]. A resection was performed and an allograft implanted [Fig. 2(B)]. A recent X-ray [Fig. 2(C)] shows excellent healing of the graft. The patient is now 31 years of age and is fully functional.

Major Limb Reconstruction Using Massive Cadaveric Allografts

(A)

(B)

(D)

395

(C)

(E)

Fig. 3. A 16 year old female presented in September 1980 with a painful proximal left tibia. She had two previous operative procedures for a giant cell tumour and each time had a prompt and aggressive recurrence. [Fig. 3(A)]. After complete resection of the proximal end of the tibia an allograft was implanted [Fig. 3(B)] and fixed with a plate and screws. The recipient's patellar tendon was sutured to the donor's tibial tubercle. A recent X-ray [Fig. 3(C)] shows good healing of the graft and preservation of the joint. At the age 38, her function remains good with full extension [Fig. 3(D)] and 90 degrees of flexion [Fig. 3(E)].

396

H.].

Mankin

LIFE TABLE FOR ALLOGRAFT TRANSPLANTATION 77% _j

1

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12 16 20 GRAFT SURVIVAL IN YEARS

Fig. 4. A Kaplan-Meier plot for the entire series allografts followed at least two years. The excessive fall-off in the first three years is noticeable but the overall figure for the entire series is 77% good or excellent.

3.

Results

The overall level of success for patients treated between November 1971 and September 2000 is shown in the Kaplan-Meier graphic in Fig. 4. The success rate for this series is a mean value of 77% according to the described definition of "excellent" and "good" results defined as successful, and "fair" and "failure" defined as failure. The numerical values for the total number of cases in this group is 73% (769/1,052), but if as described above, the 49 local recurrences which led to removal of the graft or amputation are deleted, the value becomes 769/1,003 or 77%. 3.1.

Date of performance

Analysis of the effect of date of performance showed no statistical difference for 131 patients whose surgery was performed

Major Limb Reconstruction Using Massive Cadaveric Allografts

397

prior to 1982, from the 520 performed between 1982 and 1991, and the 352 procedures performed from 1992 to 2000. 3.2. Gender The statistical study comparing the results for 485 female patients (75% success rate) and 518 males (79% success rate) failed to show a significant difference between the two groups. 3.3. Age Four age groups were studied. These included 326 patients whose ages were less than 20, and for whom the result was a 73% success rate; 381 patients between the ages of 20 and 39, for whom the success rate was 78%; 195 patients whose ages fell between 40 and 59, whose success rate was 79%; and 101 patients whose ages were greater than 60 years and whose success rate for the allograft segments was also 79%. Although a slight difference was noted for the younger patients, these data are not statistically significant. 3.4. Anatomical site The variation in anatomical site was statistically significant. Of 353 implanted distal femoral segments, 242 remained successful (69%). Proximal tibial segments were somewhat better in terms of outcome with 112 of the 150 grafts remaining successful (75%). Proximal femoral grafts had a success rate of 79% (108 of 136) and the best of all were the 122 proximal humeral grafts with a success rate of 82% for 122 implants (p < 0.01 by chi square). 3.5. Types of grafts Figure 5 displays the results for the type of graft. As can be noted, the best results are with the intercalary grafts which have

398

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CRAPT SURVIVAL BY TYPES OF GRAFT (P<0.00001)

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Fig. 5. Graft survival by types of graft. The poorest performance in the series was for allograft arthrodeses, which had a mean of 61% graft survival with a very rapid fall off in the first three years. The best results were for the intercalary grafts at 86% (p < 0.00001).

a success rate of 86%. The allograft prostheses and osteoarticular grafts are similar in result at approximately 75% excellent or good. Poorest of all are the allograft-arthrodeses, which have a failure rate of almost 40% (Donati et al., 2002). These data are highly significant according to the Kaplan-Meier plotting system at p < 0.00001. 3.6. Effect of diagnosis, stage and adjuvant therapy Diagnosis and stage of disease had a significant effect on graft survival. These data are clearly evident in Fig. 6 which compares the results for the three major high grade tumours, osteosarcoma (66% success), chondrosarcoma and Ewing's sarcoma (both at over 80% successful). Chondrosarcomas are usually not treated

399

Major Limb Reconstruction Using Massive Cadaveric Allografts

EFFECT OF DIAGNOSIS ON ALLOGRAFT SURVIVAL (P< 0.0008) - B - OSA (245) 667. G CHSA(134)80% E - EfflNCS (43) 61%

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Fig. 6. The various major diagnoses had different outcomes. Best was chondrosarcoma (80% excellent or good) while the poorest was osteosarcoma (66% successful).

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400

H.J. Mankin

with chemotherapy or radiation. Osteosarcomas are most often located adjacent to joints, and require more extensive surgery, since the tumour is not as affected by chemotherapy a n d / o r radiation as a Ewing's sarcoma. Fig. 7 shows the effect of MSTS stage (Enneking et ah, 1980) and this could be easily predicted on the basis of the data shown in Fig. 6. Stage 0 not only includes the giant cell tumours but a number of non-tumourous conditions, so that the resective surgery need not be as wide as for high grade tumours. It is evident on the basis of this study that Stages 0 and 1A and IB would have a higher success rate than 2A, 2B or 3, and that the data are highly significant (Enneking et ah, 1980). It could be anticipated that adjuvant chemotherapy or radiation would play some role in the success of the allograft implant, and this is certainly true as is evident in Fig. 8. A EFFECT OF ADJUVANT THERAPY ON ALLOGRAFT SURVIVAL (P<0.00001) 1

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Fig. 8. Adjuvant therapy had a well defined negative effect on allograft survival. Patients who received chemotherapy or radiation or both did far less well than the patients who did not receive either. The range is from 59% good or excellent for radiation, to 83% for patients who received neither (p < 0.00001).

Major Limb Reconstruction Using Massive Cadaveric Allografts

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EFFECT o r COMPLICATIONS ON ALLOGRAFT SURVIVAL ( P < 0.00001)

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Fig. 9. The effect of complications on allograft survival is displayed in the Kaplan-Meier plot. As can be noted, non-union is less of a problem than fracture, for which the success rate is only 47%. When both occurred the course was disasterous. No fracture or non-union provides a 96% success (p < 0.00001).

patient success tion or 70% (p

who did not receive chemotherapy or radiation has a rate for their graft of 83% while chemotherapy, radiaboth cases a significant lowering of that value to below < 0.00001).

3.7. Effect of allograft complications As can be readily seen in Fig. 9, complications in the allograft procedure were the major causes of failure. Fracture of the graft occurred in 155 cases and resulted in a 47% success rate (Berrey et ah, 1990; Mankin et ah, 1983). Patients with non-unions did somewhat better with a 71% success rate for the 125 patients with this complication (Mankin et ah, 1983). If the patients had both non-union and fracture, the graft survival rate was only

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Fig. 10. Infections are a major problem in alloimplants. If the infection occurs de novo without either fracture or non-union, the number is small (7.3%) and the result does not materially differ from no infection. If, on the other hand, infection occurs along with non-union or fracture, the results are very poor (less than 12% surviving).

33%. If no complications occurred, the result for 599 patients was a 96% success rate. Infection was a major issue as well, but as noted in Fig. 10, only when it was associated with operative procedures to correct non-union or fracture (less than 20% of these grafts survived). Infection alone occurred in 80 patients (7.6%) and had a reasonable outcome with over 60% of the grafts surviving. None of the patients in this series died of infection, and only three of the total infected cases could be tracked to cultures of the donor tissue taken prior to implantation (Hernigou et ah, 1991; Lord et ah, 1988). Only one patient who had a distal femoral allograft implanted 10 years previously, developed AIDS — a complication which

Major Limb Reconstruction Using Massive Cadaveric Allografts

403

was tracked to blood tranfusion. Two other patients who received blood from the same donor developed the disease. Our patient remains alive and retains her graft which appears to continue to function. 4, Discussion The history of massive bone allografting is a checkered one. The earliest reported successful result is a canonical one. Saints Cosmas and Damian in the sixth century A.D. transplanted a limb on a patient's a cancerous growth (Rinaldi et ah, 1987). The graft came from a Moor who had died that morning, and hence the procedure was known as the "Miracle of the Black

Fig. 11. Renaissance artist's depiction of the "Miracle of the Black Leg". In the fifth century AD, the twin physicians Cosmas and Damian performed the replacement surgery on a Justinian, a faithful church retainer with a cancerous limb. Not only was the procedure believed to be successful, but as a result, the twins were canonized.

404

H.J. Mankin

Leg"; numerous artists of the Renaissance captured the event on canvas (Fig. 11) (Rinaldi et al, 1987). Only sporadic cases were reported until the efforts of Lexer at the turn of the century and 25 years later (Lexer et al, 1908; 1925); followed subsequently by Volkov in the 1960s (Volkov et al, 1970). The change that occurred in the treatment protocols for bone tumours in the early 1970s allowed patients with osteosarcoma and Ewing's tumours to have a much greater chance at disease-free survival (Cortes et al.,1972; Goorin et al, 1985; Jaffe et al, 1974; 1976), and the introduction of these agents and the use of neo-adjuvant adminstration of the drugs, made it safer to perform marginal surgery (Rosen et al, 1982). The discovery of the bone scan, use of computerised technology, and the magnetic resonance image, helped the surgeon plan the surgery and made obtaining a wide margin around the tumour simpler (Damadian et al, 1971; 1973; Hounsfield Pt 1 et al, 1973; Hounsfield Pt 2 et al, 1973). As indicated above, it was Frank Parrish (Parrish et al, 1966; 1973) and Carlos Ottolenghi (Parrish et al, 1966) who first defined the technological advances required for competent allograft surgery, and the Navy experience with bone banking (Contreras et al, 1998), along with the special efforts of Friedlaender (Friedlaender et al, 1976), Tomford (Tomford et al, 1983; 1989; 1999), Schachar (Schachar et al, 1991; 1994) and their colleagues that allowed the banks to meet the needs of the surgeons. In recent years, the allograft procedure has had a modest decline in popularity. Some features are now clearly a major problem, such as the use of allograft arthrodesis (Donati et al, 2002) or total joint grafts (Mankin et al, 1983), both of which have a high failure rate. Fractures, and to a lesser extent, nonunions, are clearly the major problems for the patients, and despite attempts to add special hardware, bone graft or other bone stimulants, or immobilise the patient for a prolonged period, these two problems still represent the principal causes of failure (Berrey et al, 1990; Mankin et al, 1983).

405

Major Limb Reconstruction Using Massive Cadaveric Allografts

Infection is a serious problem, although primary infection itself is less difficult to deal with than those that occur in relation to non-union or fracture of the graft (Buck et al, 1989; Lord et al, 1988; Mankin et al, 1983; Strong et al, 1991; Tan et al, 1997; Tomford et al, 1990). Secondary infections following reoperations through a previously operated field in a patient with an altered immune state, lead to a high rate of failure. At the same time as the appearance of these problems in the allograft system, the metallic implants and more specifically, the modular devices, have gained broad appeal and acceptance amongst the orthopaedic oncologists (Chao et al, 1985; Eckardt et al, 1991; Johnston et al, 1987; Malawer et al, 1989; Otis et al, 1987; Ramach et al, 1987; Ritschl et al, 1987; Sim et al, 1979; 1987). The devices are simpler to implant than the allografts, have a much quicker period to functional restoration following surgery, and appear (at least in the short period since their introduction)

COMPARISON OF METALLIC IMPLANTS AND ALLOGRAFTS

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.780

1.50

2.25 3 3.75 DEVICE SURVIVAL IN YEARS

4.50

525

6

Fig. 12. As bad as the results for the allografts seem, it is important to note that 130 metallic implants had a success rate slightly less than the initial allograft values over the first six years of followup.

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to have a better outcome. The period of followup, however, is short, and as can be noted in Fig. 12, the success rate for 130 metallic implants over the six years since first introduced has shown that the complications for these devices are not minor, and loosening, metal or plastic failure may limit their long term survival (Clarke et ah, 1998). It is furthermore evident that if allografts fail, there may be more alternatives than if the modular devices develop serious functional problems. If one considers the future of the allograft system, much has been learned from studies such as this, to define the reasons for successful or poor outcomes. With all the problems and especially the issues now arising in relation to infections, it seems clear that the current rate of success has to be improved if the system can regain its status as an important technique for the treatment of bone tumours. Several issues must be addressed: (a) Bone banks must be more carefully supervised and made to adhere to the guidelines and protocols advanced in the 30 years of utilisation of the system. Donor procurement remains a major issue, and all efforts should be directed to guarantee that high quality graft material will be available to the surgeon in the appropriate size without delay. (b) An attempt should be made to improve the quality of graft fixation with hardware, and define whether additional biologic materials at the host-donor junction site may be successful in improving acceptance of the graft. (c) Surgeons must utilise plastic surgical procedures to avoid inadequate skin coverage over the site of the graft, and thus hopefully reduce the local failure rate. (d) An attempt should be made to assess the antigens in the host and compare them to the donor. Such studies in the past have strongly suggested that a match in Class 2 antigens has a higher rate of success (Friedlaender et ah, 1976). (e) Research must continue to define how the immune system can be altered in either the donor or the host to improve the quality and rate of acceptance of the graft. The use of agents such as are used in organ transplant, have very limited use

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in bone allografts implanted for tumours. Furthermore, there is little reason to introduce a life-threatening programme for a limb-threatening problem such as distal femoral sarcoma. It is likely that such basic research and clinical trial programmes will continue to evolve over the next several years, and improve the status of the allograft in the management of patients with bone tumours. Many research centres are invested and committed to this approach, and it is also evident that if necessary, Saints Cosmas and Damian will add their "moral" support to attempts to improve the system (Fig. 13).

Fig. 13. We are counting on continued assistance in the field of allograft surgery from the twin Saints Cosmas and Damian, here depicted performing their magnificent procedure of replacing the cancerous limb of a patient with the limb of a Moor who had died that morning. Please note that nurses such as these are difficult to find today.

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5. Summary This presentation describes 31 years of application and study of the technology, applications, limitations, complications and outcomes of 1,052 massive allograft implantations — mostly for the treatment of malignant bone tumours. The data obtained show that the success rate for the procedure is approximately 77% and that generally, the more severe the disease, the poorer the outcome. Intercalary grafts have better results than others, while allograft-arthrodeses have the poorest results. The most common complication is fracture of the graft, which materially compromises the success of the procedure. Non-union as a complication has fewer problems, and infection as a primary event only occurred in less than 8% of the grafts. None of the patients died of infection, and the graft was implicated as a cause of disease in only 3 of the 1,052 cases. It is clear on the basis of this study that the principal component of the success of the procedure is a competent and concerned bone bank. Future study and research should lead to improvement in outcome and fewer problems for the patients. 6. References ALHO, A.J., ESKOLA, J., EKFORS, T., MANNER, I., KOURI, T. and HOLLMEN, T. (1998). Immune responses and clinical outcome of massive human osteoarticular allografts, Clin. Orthop. 346, 196-206. BERREY, W.H. Jr, LORD, C.F., GEBHARDT, M.C. and MANKIN, H.J. (1990). Fractures of allografts. Frequency, treatment and end-results, /. Bone Joint Surg. 72A, 822-833. BUCK, R.E., MALININ, T.I. and BROWN, M.D. (1989). Bone tranplantation and human immunodeficiency virus. An estimate of risk of acquired immunodeficiency syndrome (AIDS), Clin. Orthop. 240, 129-136.

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CHAO, E.Y. and SIM, F.H. (1985). Modular prosthetic system for segmental bone and joint replacement after tumour resection, Orthopedics 8, 641-651. CLARKE, H.D., BERRY, D.J. and SIM, F.H. (1998). Salvage of failed femoral megaprostheses with allograft prosthesis composite, Clin. Orthop. 356, 222-229. CONTRERAS, T.J., BLAIR, P.J. and HARLAN, D.M. (1998). Brief history of the United Sates Navy Tissue Bank and Transplantation Program. Captain Kenneth Sell's living legacy. In: Advances in Tissue Banking, G.O. Phillips, D.O. Strong, R. von Versen and A. Nather, eds., River Edge, New Jersey, World Scientific, Vol. 2, pp. 21-28. CONWAY, B., TOMFORD, W.W., HIRSCH, M.S., SCHOOLEY, R.T. and MANKIN, H.J. (1990). Effects of gamma irradiation on HIV-1 in a bone allograft model, Trans. Orthop. Res. Soc. 15, 225. CORTES, E.P., HOLLAND, J.F., WANG, J.J. and SINKS, L.F. (1972). Doxorubicin in disseminated osteosarcoma, /. Am. Med. Assoc. 221, 1132-1138. COX, D.R. (1972). Regression models and life tables, /. Royal Statist. Soc. 34, 187-220. DAMADIAN, R. (1971). Tumour detection by nuclear magnetic resonance, Science 171, 1151-1153. DAMADIAN, R., ZANER, K., HOR, D. and DIMAIO, T. (1973). Human tumours by NMR, Physio. Chern. Phys. 5, 381-402. DELLOYE, C , DENAYER, P., ALLINGTON, N., MUNTING, E., COUTELIER, L. and VINCENT, A. (1988). Massive bone allografts in large skeletal defects after tumour surgery: A clinical and microradiographic evaluation, Arch. Orthop. Trauma. Surg. 107, 31-41.

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DICK, H.M., MALININ, T.I. and MNAYMNEH, W.A. (1985). Massive allograft implantation following radical resection of highgrade tumours requiring adjuvant chemotherapy treatment, Clin. Orthop. 197, 88-95. DONATI, D., GIACOMINI, S., GOZZI, E., SALPHALE, Y., MERCURI, M , MANKIN, H.J., SPRINGFIELD, D.S. and GEBHARDT, M.C. (2002). Allograft arthrodesis treatment of bone tumours: A two center study, Clin. Orthop. 400, 217-224. ECKARDT, J.J., MATTHEWS, J.G. and EILBER, F.R. (1991). Endoprosthetic reconstruction after bone tumour resections of the proximal tibia, Orthop. Clin. NA. 22, 149-160. ENNEKING, W.F., SPANIER, S.S. and GOODMAN, M.A. (1980). Current concepts review: The surgical staging of musculoskeletal sarcoma, /. Bone Joint Surg. 62A, 1027-1030. FRIEDLAENDER, G.E., STRONG, D.M., TOMFORD, W.W. and MANKIN, H.J. (1999). Long term followup of patients with osteochondral allografts. A correlation between immunologic responses and clinical outcome, Orthop. Clin. North Am. 30, 583-590. FRIEDLAENDER, G.E., STRONG, D.M. and SELL, K.W. (1976). Studies on the antigenicity of bone. I. Freezed-dried and deep frozen bone allografts in rabbits, /. Bone Joint Surg. 58A, 854858. GOORIN, A.M., ABELSON, H.T. and FREI, E. (1985). Osteosarcoma fifteen years later, N. Engl. J. Med. 313, 1637-1643. GOUIN, F., PASSUTI, N., VERRIELE, V., DELECRIN, J. and BAINVEL, J.V. (1996). Histological features of large bone allografts, /. Bone Joint Surg. 78B, 38-41. HEJNA, M.J. and GITELIS, S. (1997). Allograft prosthetic composite replacements for bone tumours, Sem. Surg. Oncol. 13, 18-24.

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HERNIGOU, P., DELEPINE, G. and GOUTALLIER, D. (1991). Infections after massive bone allografts in surgery of bone tumours of the limbs. Incidence, contributing factors, therapeutic problems, Rev. Chir. Orthop. 77, 6-13. HORNICEK, F.J., GEBHARDT, M.C., SORGER, J.I. and MANKIN, H..J. (1999). Tumour reconstructions, Orthop. Clin. North Am. 30, 673-684. HORNICEK, F.J., MNYMNEH, W., LACKMAN, R.D., EXNER, G.U. and MALININ, T.I. (1998). Limb salvage with osteoarticular allografts after resection of proximal tibia bone tumours, Clin. Orthop. 352, 179-186. HOUNSFIELD, G.N. (1973). Computerised transverse axial scanning (tomography): Part 1: Description of system, Br. J. Radiol. 46, 148-149. HOUNSFIELD, G.N. (1973). Computerised transverse axial scanning (tomography): Part 2: Description of system, Br. J. Radiol. 46, 1016-1022. JAFFE, N., FREI, E., TRAGGIS, D. and BISHOP, Y. (1974). Adjuvant methotrexate and citrovorum factor treatment of osteogenic sarcoma, N. Engl. J. Med. 291, 994-997. JAFFE, N. and WATTS, H. (1976). Multidrug chemotherapy in the treatment of osteosarcoma, /. Bone Joint Surg. 58A, 634-635. JOFE, M.H., GEBHARDT, M.C., TOMFORD, W.W. and MANKIN, H.J. (1988). Osteoarticular allografts and allografts plus prosthesis in the management of malignant tumours of the proximal femur, /. Bone Joint Surg. 70A, 507-516. JOHNSTON, J. (1987). A modular prosthetic knee systrem for tumour surgeons. In: Linb Salvage in Musculoskeletal Oncology, W.F. Enneking, ed., New York, Churchill Livingstone, pp. 234-237.

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KAPLAN, E. and MEIER, P. (1958). Non parametric estimation for incomplete observations, /. Am. Statist. Assoc. 53, 457-481. LEXER, E. (1908). Die Verwendung der freien Knochenplastik nebst Versuchen uber Gelenkversteifung und Gelenktransplantation, Arch. f. Klin. Chir. 86, 939-954. LEXER, E. (1925). Joint transplantation and arthroplasty, Surg. Gynec. Obstet. 40, 782-809. LORD, C.F., GEBHARDT, M.C., TOMFORD, W.W. and MANKIN, H.J. (1988). The incidence, nature and treatment of allograft infections, /. Bone Joint Surg. 70A, 369-376. LOTY, B., COURPIED, J.P., TOMENO, B., POSTEL, M., FOREST, M. and ABELANET, R. (1990). Bone allografts sterilised by irradiation. Biological properties, procurement and results of 150 massive allografts, Int. Orthop. 14, 237-242. MAKLEY, J.T. (1985). The use of allografts to reconstruct intercalary defects of long bones, Clin. Orthop. 197, 58-75. MALAWER, M.M. and MCHALE, K.A. (1989). Limb sparing surgery for high grade malignant tumours of the proximal tibia. Surgical technique and a method of extensor mechanism reconstruction, Clin. Orthop. 239, 231-248. MANKIN, H.J. (2002). A computerised system for orthopaedic oncology, Clin. Orthop. 398, 252-261. MANKIN, H.J. (1983). Complications of allograft surgery. In: Osteochondral Allografts, G.E. Friedlaender, H.J. Mankin and K.W. Sell, eds., Little Brown and Co. Boston, pp. 259-274. MANKIN, H.J., DOPPELT, S.H., SULLIVAN, T.R. and TOMFORD, W.W. (1982). Osteoarticular and intercalary allograft transplantation in the management of malignant tumours of bone, Cancer 50, 613-630. MANKIN, H.J., GEBHARDT, M.C., JENNINGS, L.C., SPRINGFIELD, D.S. and TOMFORD, W.W. (1996). Long-term results

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of allograft replacement in the management of bone tumours, Clin. Orthop. 324, 86-87. MANKIN, H.J., GEBHARDT, M.C. and TOMFORD, W.W. (1987). The use of frozen cadaveric allografts in the management of patients with bone tumours of the extremities, Orthop. Clin. North Am. 18, 275-289. MNAYMNEH, W. and MALININ, T. (1989). Massive allografts in surgery of bone tumours, Orthop. Clin. North Am. 20, 455467. MNAYMNEH, W., MALININ, T.I., MAKLEY, J.T. and DICK, H.M. (1986). Massive osteoarticular allografts in the reconstruction of extremities following resection of tumours not requiring chemotherapy and radiation, Clin. Orthop. 227, 666677. MUSCOLO, D.L., AYERZA, M.A., CALABRESE, M.E., REDAL, M.A. and ARAUJO, E.S. (1996). Human leukocye antigen matching, radiographic score and histologic findings in massive frozen bone allografts, Clin. Orthop. 326, 115-126. ORTIZ-CRUZ, E., GEBHARDT, M.C, JENNINGS, L.C., SPRINGFIELD, D.S. and MANKIN, H.J. (1997). The result of transplantation of intercalary allografts after resection of tumours. A long term follow-up study, /. Bone Joint Surg. 79A, 97-106. OTIS, J.C. and LANE, J.M. (1987). Non-modular segmental knee replacements: Design and performance. In: Limb Salvage in Musculoskeletal Oncology, W.F. Enneking, ed., New York, Churchill Livingstone, pp. 22-24. OTTOLENGHI, C.E. (1966). Massive osteoarticular bone grafts, /. Bone Joint Surg. 48B, 646-659. PARRISH, F.F. (1973). Allograft replacement of part of the end of a long bone following excision of a tumour: Report of twentyone cases, /. Bone Joint Surg. 55A, 1-22.

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PARRISH, F.F. (1966). Treatment of bone tumours by total excision and replacement with massive autologous and homologous grafts, /. Bone Joint Surg. 48A, 968-990. RAMACH, W., SIGMUND, R., SEKERA, J., SALZER-KUNTSCHIK, M , KOTZ, R., KNAHR, K. and SALZER, M. (1987). Functional results of customised prosthetic devices for the knee region after resection of bone and joints. In: Limb Salvage in Musculoskeletal Oncology, W.F. Enneking, ed., New York, Churchill Livingstone, pp. 215-220. RINALDI, E. (1987). The first homoplastic limb transplant according to the legend of Saint Cosmas and Saint Damian, Hal. J. Orthop. Traumatol. 13, 394-406. RITSCHL, P., BRAUN, O., PONGRACZ, ER, RAMACH, W. and KOTZ, R. (1987). Modular reconstruction system for the lower extremity. In: Limb Salvage in Musculoskeletal Oncology, W.F. Enneking, ed., New York, Churchill Livingstone, pp. 237-243. ROSEN, G., CAPARROS, B. and HUVOS, A.G. (1982). Preoperative chemotherapy for osteogenic sarcoma: Selection of postoperative adjuvant chemotherapy based on the response of the primary tumour to preoperative chemotherapy, Cancer 49, 1221-1230. SCHACHAR, N.S. and MCGANN, L.E. (1991). Cryopreservation of articular cartilage. In: Bone and Cartilage Allografts, G.E. Friedlaender and V.M. Goldberg, eds., Park Ridge IL, American Academy of Orthopaedic Surgeons, pp. 211-230. SCHACHAR, N.S., CUCHERAN, D.J., MCGANN, L.E., NOVAK, K.A. and FRANK, C.B. (1994). Metabolic activity of bovine articular cartilage during refrigerated storage, /. Orthop. Res. 12, 15-20. SIM, F.H. and CHAO, E.Y. (1979). Prosthetic replacement of the knee and a large segment of the femur or tibia, /. Bone Joint Surg. 61A, 887-892.

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SIM, F.H., BEAUCHAMP, C.P. and CHAO, E.Y. (1987). Reconstruction of musculoskeletal defects about the knee for tumour, Clin. Orthop. 221, 188-201. STRONG, D.M., FRIEDLAENDER, G.E., TOMFORD, W.W., SPRINGFIELD, D.S., BURCHARDT, H.C., ENNEKING, W.F. and MANKIN, H.J. (1996). Immunological responses in human recipients of osseous and osteochondral allografts, Clin. Orthop. 326, 107-114. STRONG, D.M., SAYERS, M.H. and CONRAD, E.U. Ill (1991). Screening tissue donors for infectious markers. In: Bone and Cartilage Allografts, G.E. Friedlaender and V.M. Goldberg, eds., Park Ridge IL, American Academy of Orthopaedic Surgeons, pp. 193-209. TAN, M.H. and MANKIN, H.J. (1997). Blood transfusion and bone allografts. Effect on infection and outcome, Clin. Orthop. 340, 207-214. TOMFORD, W.W. and MANKIN, H.J. (1999). Bone banking: Update on methods and materials, Orthop. Clin. North. Am. 30, 553-565. TOMFORD, W.W. (1983). Cryopreservation of articular cartilage. In: Osteochondral Allografts, G.F. Friedlaender, H.J. Mankin and K.W. Sell, eds., Little Brown and Co. Boston, pp. 215-218. TOMFORD, W.W., DOPPELT, S.H. and MANKIN, H.J. (1989). Organisation, legal aspects and problems of bone banking in a large orthopaedic center. In: Bone Transplantation, M. Aebi, and P. Regazzoni, eds., Berlin, Springer Verlag, pp. 145-150. TOMFORD, W.W., THONGPHASUK, J., MANKIN, H.J. and FERRARO, M.J. (1990). Frozen musculoskeletal allografts. A study of the clinical incidence and causes of infection associated with their use, /. Bone Joint Surg. 72A, 1137-1143. VOLKOV, M. (1970). Allotransplantation of joints, /. Bone Joint Surg. 52B, 49-53.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

16 REVISION ARTHROPLASTY USING FRESH FROZEN ALLOGRAFT WITH CEMENTED CUP FOR ACETABULAR BONE DEFICIENCY

W O N G YONG SHON, CHANG YONG HUR a n d S O O N G H Y U N JUNG D e p a r t m e n t of Orthopaedic Surgery G u r o Hospital, Korea University

1. Introduction Acetabular revision with major bone defect is a challenging procedure (Berry and Muller, 1992; Tasty and Harris, 1990; Rosson and Schatzer, 1992; Schatzer et at, 1984). Loss of bone stock is due to osteolysis, which is caused by polyethylene, cement or metal particulate debris (Goldring et at, 1996; Goodman et at, 1985; Howie et at, 1987; Pazzaglia et at, 1985). There are several acetabular revision options, which are: jumbo cups, high hip centre, bipolar hemiarthroplasty, oblong cup or custom-made triflanged acetabular component, and allografts (Gross et at, 1993; Kelly, 1994; McFarland et at, 1991; Surtherland, 1996). Restoration of bone stock is necessary to provide suitable bone support, restore hip mechanic (centre of rotation) and secure prosthesis fixation (Paprosky and Magnus, 1994). In addition, bone grafting will help to restore leg lengths, making future revision possible (Hasegawa et at, 1996). 417

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Massive bone stock may be replenished, usually with allograft bone, because of limited use of the patient's own bone (Gordon et al., 1985; Mulroy and Harris, 1990; Samuelson et al., 1998). There are, of course, certain disadvantages to using allograft bone (Hooten et al, 1994; Pollock and Whiteside, 1992). The problems of disease transmission are well documented (Tomford, 1995). The purpose of this study is to evaluate the use of fresh frozen femoral head allografts which were provided from hospital bone banks, in revision acetabular reconstruction in patients with marked insufficiencies of acetabular bone stock. 2. Materials and Methods From August 1992 to December 1999, we performed 26 acetabular revisions in 25 patients with major acetabular bone deficiency, using frozen femoral head allograft. Two hips in one patient were lost to follow-up, and one hip was excluded because detailed evaluation was impossible due to severe illness. Twenty-three hips were available for clinical and radiographic follow-up at an average of 4.5 years (range 2.1-9.4 years). The average age of the patients at the time of revision was 53 (range 37-71). Fifteen of the patients were male, and the remaining eight were female. Acetabular bone deficiency cases are classified into seventeen hip defects, with Type II (Type IIA in six hips, Type IIB in four hips and Type IIC in seven hips) and six hips with Type III (Yupe MA in three hips and Type IIIB in three hips) according to Paprosky's classification (Paprosky and Magnus, 1994). Each operation was performed by a single surgeon (Dr. Won Yong Shon). A Posterolateral approach to the hip (without trochanteric approach) was used in 12 patients. A Transtochanteric approach was performed in 10 patients, and a Rubash's triradiate extensile approach was performed in one hip. After removal of all the loose components, including metal cups, polyethylene liner or cups, cement, fibrous membrane or

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particulate induced granuloma, the acetabular bony deficiency was evaluated visually again, and the decision as to whether only impacted allografting or use of reinforcing ring with allografting, was made depending on priority for getting primary fixation. All allografts were taken from patients who had had primary total hip arthroplasty, and the fresh frozen femoral head bone banking techniques and donor selection met the criteria of the American Association of Tissue Banks (AATB, 1993). At the time of harvesting in the operating room, material from the femoral head was taken for cultures; then the femoral was placed in a sterile environment and frozen at -70°C. Before use, a frozen femoral head allograft was thawed at room temperature in normal saline solution. The grafts were prepared with a rongeur during surgery, and the morsellous chips were washedout several times with normal saline. The amount of the graft used varied from one to four femoral heads per patient, depending on the severity of the acetabular bone defect, and the graft was mixed with vancomycine (1 gm per hemoral head). The acetabular defect was filled with firmly impacted morsellised cancellous allografts, and the polyethylene cup was fixed using bone cement. The vancomycine and bone cement were mixed intraoperatively. If the cup is not sufficiently supported by these grafts, one of several metal reinforcement rings is used to buttress it, depending on the type of acetabular bone defect. Eight hips were fixed with bone cement only, and 15 hips were fixed with bone cement supported by acetabular reinforcing rings (Kerboull plate in eight hips, Ganz ring in three hips and Muller ring in four hips). Systemic antibiotics (secondgeneration cephalosporine) were administered to all patients for five to seven days postoperatively. All of the patients underwent followed-up both clinically and radiographically. A modified Harris hip score was used for clinical assessment. Radiographic review involved several parameters. Allografts were assessed for union, as evidenced by trabecular bridging of the host-donor

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interface, radiolucent line and resorption of allografts (Gordon et ah, 1985). The allograft was assumed to be incorporated when osseous trabeculae had completely bridged the space between the host acetabulum and the allograft (Lamerigts et ah, 2000). Radiographic evaluation was performed using measurements of vertical (cranial) and horizontal (medial) changes in acetabular position on standard anteroposterior pelvic radiographs. Migration of the acetabular component was considered present if measurements indicated a change in position greater than 2 mm (Yoder et ah, 1988). Radiolucent lines were measured in the three zones specified in the technique of DeLee and Charnley (Delee and Charnley, 1976). 3. Results At the last follow-up examination (mean 4.5 years), three of 21 hips were failures, thus resulting in a success rate of 87%. The procedure was most effective in providing pain relief. The mean Harris hip score of the patients improved from an average of 48 (range 41-67) before surgery, to an average of 84 (range 59-92) at the last follow-up evaluation. Excluding the three unsuccessful patients, the remaining 20 patients required no support when walking, and had no or little pain on the repaired hip. Of the three failures, one was for deep infection at six years after revision operation, and was treated with excision arthroplasty. There was progressive radiographic loosening in the other two hips. One hip showed more than 7 mm in superior migration of cemented polyethylene cup due to resorption of allograft, and re-revision was performed at 3.8 years after revision (Fig. 1). The other hip suffered fixation failure of the reinforcement ring, which also showed migration at 1.2 years after revision. There were three patients who suffered one or two dislocations postoperatively, and all patients were treated with abduction brace successfully. Radiographically, all but two allografts have incorporated to the host-bone. The exact time of

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(a)

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(b)

Fig. 1. Radiograph showing (a) revision with morsellised allograft using cemented fixation in a 67 year old man, (b) acetabular loosening with resorption of allograft at 3.8 years.

Fig. 2. Radiograph showing (a) acetabular loosening in a 58 year old man, (b) revision using morsellised allograft with cemented fixation, (c) restoration of acetabular bone stock with no evidence of loosening at eight years.

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(a)

(b)

(c)

Fig. 3. Radiograph showing (a) acetabular loosening of polyethylene cup in a 44 year old man, (b) revision with morsellised allograft using Kerboull plate and cemented fixation, (c) five years after revision surgery with complete incorporation.

incorporation of allografts was difficult to assess in impacted mosellised allografts, but it seemed to be confirm in serial radiographs (Fig. 2). Remodeling of the acetabular allograft which was defined as trabecular reorientation of the allograft and same density changes as surround host bone was observed at the time of two or three years after revision operation (Fig. 3). There was no definitive resorption of allograft in the remaining 20 hips. 4. Discussion Acetabular reconstruction with bone graft during revision of a total hip replacement was demonstrated as an acceptable method for the treatment of massive deficiency of acetabular bone, because it provides several advantages (Geber and Harris, 1986;

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Gie et al, 1993; Gross et al, 1993; Paprosky and Magnus, 1994). Restoration of hip mechanics (center of rotation of hip), restoration of acetabular integrity and continuity, and acetabular bony stock for the re-revision can be achieved by the use of bone graft in acetabular reconstruction. Autografts are the best material for restoring acetabular bone defects (Goldberg and Stevenson, 1987). However, donor site morbidity and limited availability are the major drawbacks in the use of autograft bone in reconstructive hip surgery (Mulroy and Harris, 1990). The usual alternative to an autograft is the use of allografts. Allografts — biological materials from donors with a different genetic background than the recipients' — are not limited in supply. But allografts are associated with risks of transmission of infectious disease such as tuberculosis, hepatitis, HIV and bacterial infection to recipients, and risks of late graft failure due to weaker biomechanical properties or less incorporation than autografts (Jacobs, 1987; Tomford, 1995). Longer term follow-up results were inconsistent, although numerous authors have reported successful short-term results with the use of structural acetabular allografts in revision surgery (Gordon et al; 1985, Hooten et al., 1994; Pollock and Whiteside, 1992). Kwong et al. reported a failure rate of 47% at an average of 10 years (Kwong et al., 1993). But impaction grafting has been shown to give good results, with predicatable graft incorporation and remodelling in acetabular reconstruction with contained acetabular defects or uncontained defects in combination with a reinforcement ring or cage (Gie et al., 1996; Slooff et al., 1996). Failure rate was three (13%) of 23 cases at an average of 4.5 years' follow-up study in our series; only one hip showed resorption of allograft; the other cases which showed fixation failure of reinforement rig seemed to be failures of primary fixation, excluding one patient with late infection. Of remaining 20 hips, the allograft appeared united to the host bone, incorporated and remodelled in the majority, without resorption on follow-up radiography. Our results suggest that

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the acetabular impaction morsellised cancellous allograft has a biological potential that produces viable new bone under adequate conditions, and biomechanical properties which can endure some load during the process of uniting with the host bone, and during incorporation. Infectious disease transmission is a serious concern for orthopaedic surgeons and patients contemplating bone allograft use (Jacobs, 1987). Good tissue banking practices are required for ensuring recipient safety in bone banking. To minimise the risk of transmission of infectious diseases, good tissue banking practices should be observed by surgeons and bone banks. An important approach is to ensure meticulous donor evaluation, including careful donor screening; screening testing and exclusion criteria; adherence to regulations; aseptic tissue recovery procedures; and a quality assurance programme. The incidence of infection related to the use of large allografts was 4-5% in the cited literature. Of the 23 hips in our series, an infection developed in one (4.5%) hip at six years after acetabular reconstruction with allograft from our bone bank. Staphylococcus epidimis grew on culture. That suggested that the cause of infection seem to be contamination of the allograft during havesting (procurement) or during the revision operation. Therefore, the incidence of infection in patients who had a acetabular reconstruction with allograft taken from our hospital bone bank is not higher than that of other tissue banks. There has been as yet, no report of disease transmission or other infection such as HIV, hepatitis or tuberculosis during follow-up of all patients who received an allograft from our bone bank. 5. Conclusion Our current study shows that acceptable results can achieved from the acetabular revision with fresh frozen morsellised femoral head allografts taken from our hospital bone bank at an average of 4.5 years' follow-up.

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6. Summary Massive deficiency of acetabular bone stock is a challenging problem to patients who need a revision of a failed hip arthroplasty. The goal of reconstruction with an allograft for major acetabular bone defect is to provide support for the cup, to restore bone stock, and to provide approximate fixation. The purpose of this study was to assess the results of using fresh frozen allografts from our surgical bone bank with cemented fixation for revision reconstruction in hips with acetabular bone deficiency, in order to clarify the safety of our hospital bone bank. Twenty-six hips in 25 patients had an acetabular revision with fresh frozen allografts with cemented fixation, between October 1992 and December 1999. Two hips in one patient were lost to follow-up, and one hip was excluded because of severe illness. Twenty-three hips were available for clinical and radiographic follow-up at an average of 4.5 years (range 2.1-9.4 years). The average age of the patients at the time of revision was 53 (range 37-71). Acetabular bone defects were classified into 17 hips with Type II (Type IIA in six hips, Type IIB in four hips and TypellC in seven hips), and six hips with Type III (Type IIIA in three hips and Type IIB in three hips) according to Paprosky's classification. All of the morsellised bone chips were from fresh frozen femoral heads harvested by the authors, and fresh-frozen according to recommended protocols, and then mixed with 1 gm of vancomycine for each femoral head. Fifteen of 23 hips were fixed with reinforced acetabular devices, and the remaining eight hips with cement only. Two acetabular cups (9%) had progressive migration with collapse of allografted bone, and were considered radiographical failures and one cup was revised. One hip (4%) was infected at six years after the operation. All the remaining 20 (87%) of 23 hips were considered a clinical and radiographical success — the allograft incorporated in all cases, with no resorption.

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No other local and systemic complications except dislocation, had occurred in all patients. We believe that the results of the present study support the use of fresh frozen femoral heads from our hospital bone banks, in cases of massive bone loss in a revision hip replacement. 7. References AMERICAN ASSOCIATION OF TISSUE BANKS (1993). Guidelines for the banking of musculoskeletal tissue, Am. Assoc. Tisse Banks 6th ed. BERRY, D.J. and MULLER, ME. (1992). Revision arthroplasty using an anti-protrusion cage for massive acetabular bone deficiency, /. Bone Joint Surg. [Br] 74B, 711-715. DELEE, J.G. and CHARNLEY, J. (1976). Radiographic demarcation of cemented sockets in total hip replacement, Clin. Orthop. 121, 20. GERBER, S.D. and HARRIS W.H. (1986). Femoral head autografting to augment acetabular deficiency in patients requiring total hip replacement: A minimum five-year and an average seven-year follow-up study, /. Bone Joint Surg. [Am] 68A, 12411248. GROSS, A.E. et al. (1993). Bone grafting in hip replacement surgery: The pelvic side, Clin. Orthop. North. Am. 24, 679. GIE, G.A. et al. (1993). Impacted cancellous allografts and cement of revision total hip arthroplasty, /. Bone Joint Surg. 75B, 14-21. GIE, G.A. et al. (1996). Contained morsellised allograft in revision total hip arthroplasty: A minimum of five years follow-up, /. Bone Joint Surg. 78B, 71. GOLDRING, S.R. et al. (1983). The synovial-like membrane at the bone cement interface in loose total hip replacements and its proposed role in bone lysis, /. Bone Joint Surg. 65A, 575.

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GOLDBERG, V.M. and STEVENSON, S. (1987). Natural history of autografts and allografts, Clin. Orthop. 225, 7-16. GORDON, S.L. et al. (1985). Assessment of bone grafts used for acetabular augmentation in total hip arthroplasty: A study using roentgenograms and bone scincitiography, Clin. Orthop. 201, 18-25. GOODMAN, S.B. et al. (1985). The effect of polymethylmethacrylate on bone: An experimental study, Arch. Orthop. Trauma Surg. 104, 150. GOSS, A.E. and CATRE, M.G. (1994). The use of femoral head autograft shelf reconstruction and cemented acetabula components in the dysplastic hip, Clin. Orthop. 298, 60-66. HASEGAWA, Y. et al. (1996). Cementless total hip arthroplasty with utologous bone grafting for hip dysplasia, Clin. Orthop. 324, 179-186. HOOTEN, JP. Jr, et al. (1994). Failure of structural acetabular allografts in cementless revision hip arthroplasty, /. Bone Joint Surg. 76B, 419-422. HOWIE, D. et al. (1987). Bone resorption in the presence of polyethylene wear particles, /. Bone Joint Surg. 69B, 165. JASTY, M.J. and HARRIS, W.H. (1990). Salvaging total hip reconstruction in patients with major acetabular bone deficiency using structural femoral head allografts, /. Bone Joint Surg. 72B, 63. JACOBS, N.J. (1987). Establishing a Surgical Bone Bank. In: Tissue Banking, K.J. Fawcett and H.R. Barr HR, eds., Tissue Banking. American Association of Blood Banks, Arlington, VA, pp. 67-96. KELLT, S.S. (1994). High hip centre in revision arthroplasty, J. Arthroplasty 9, 503-510.

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KWONG, L.M. et al. (1993). High failure rate of bulk femoral head allografts in total hip acetabular reconstructions at 10 years, /. Arthroplasty 8, 341-346. LAMERITS, N. et al. (2000). Incorporation of morsellised bone graft under controlled loading conditions: A mew animal model in the goat, Biomaterials 21, 741-747. MCFARLAND, E.G. et al. (1991). Use of bipolar endoprosthesis and bone grafting for acetabular reconstruction, Clin. Orthop. 268, 128-139. MURLROY, R.D. and HARRIS, W.H. (1990). Failure of acetabular outogenous grafts in total hip arthroplasty: Increasing incidence, /. Bone Joint Surg. 72A, 1536-1540. PAZZAGLIA, U.E. et al. (1985). Involvement of metal particles in loosening of metal-plastic total hip prostheses, Arch. Orthop. Trauma Surg. 104, 164. PAPROSKY, W.G. and Magnus R.E. (1994). Principles of bone grafting in revision total hip arthroplasty: Acetabular technique, Clin. Orthop. 298, 147. POLLOCK, F.H. and WHITESIDE, L.A. (1992). The fate of massive allografts in total hip acetabular revision surgery, /. Arthroplasty 7, 271-276. ROSSON, J. and SCHATZER, J. (1992). The use of reinforcement rings to reconstruct deficient acetabula, /. Bone Joint Surg. 74B, 716-720. SAMUELSON, K.M. et al. (1998). Homograft bone in revision acetabular arthroplasty: A clinical and radiographic study, /. Bone Joint Surg. 70B, 367-372. SCHATZER, J. et al. (1984). A preliminary review of the muller acetabular and Burch-Schneider antiprotrusion support rings, Arch. Orthop. Trauma Surg. 103, 5-12.

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SLOOFF, T.J.J.H. et al. (1996). Acetabular and femoral reconstruction with impacted graft and cement, Clin. Orthop. 324, 108115. SURTHERLAND, C.J. (1996). Early experience with eccentric acetabular components in revision total hip arthroplasty, Am. J. Orthop. 25, 284-289. TOMFORD, W.W. (1995). Transmission of disease through transplantation of musculoskeletal allografts, /. Bone Joint Surg. 77 A, 1742-1754. YODER, S.A. et al. (1988). Total hip acetabular component position affects component loosening rates, Clin. Orthop. 228, 79.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

17 PRESENT WAYS OF TREATING CHONDRAL AND OSTEOCHONDRAL KNEE DEFECTS

PETR VISNA Traumatological Hospital Ponavka 6, 602 00 Brno, Czech Republic JIRI ADLER Tissue Bank, University Hospital Brno Czech Republic L. PASA and R. HART Traumatological Hospital Brno Czech Republic J. FOLVARSKY University Hospital, Hradec Kralove Czech Republic

1. Introduction The aim of today's knee surgery procedures is to minimise the consequences of cartilage injuries, and to renovate the continuity of the articular surface, mainly in the weight-bearing area. The 431

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clinical picture of various lesion types varies according to their localisation and thickness of the defect. The most frequent symptoms of chondral and osteochondral injuries include: • Pain — progressing with the degree of knee flexion, when the mechanical loading is transferred to the defect area. • Haemarthros — the blood effusion develops as a result of uncovered and affected subchondral bone. • Oedema — develops chronically as a result of reactive synovitis following the degradation products released from the injured cartilage. The percentage of findings of osteochondral defects in acute haemarthros varies between 8-11% (Butler, 1988). Osteochondral defects are found mainly on weight bearing areas of the femoral condyle. They occur as a consequence of shearing forces on the knee joint, and are often localised also at the femoropatellar joint, in consequence of patellar luxation. Treatment of chondral and osteochondral defects includes pharmacological and surgical options. 2.

Pharmacological Treatment

The application of chondroprotectives is the leading way of conservative therapy. The most frequently used drugs include hyaluronic acid, chondroitin sulphate and glucosamin sulphate. The positive effect of such therapy can be expected after a longer time period (after two months) due to the low metabolic turn of the cartilage. In cases with symptoms of developed osteoarthritis, best results can be reached with antirheumatic drugs or local applied corticosteroids. Further pharmacological options include the intraarticular application of bioactive growth factors. This type of treatment is used in preclinical studies. Growth factors increase mitogenic

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activity of chondrocytes, as well as DNA synthesis, and enhance intercellular matrix synthesis. Current tested factors include: • • • • • •

epidermal growth factor; basic fibroblast growth factor; insulin-like growth factor; transforming growth factor beta; bone morphogenic protein-2; and cartilage-derived morphogenic protein.

Growth factors are present in subchondral bone and in the synovial membrane under physiological conditions. The stimulating effect on chondrogenesis was documented above all in the transforming growth factor beta (Iwasaki, 1995). The perspectives for clinical practice of these drug types are in primotherapy of partial chondral defects. On the other hand, it is necessary to point out that effectiveness of these substances declines with older patients. 3. Surgical Treatment 3.1. Fragments refixation Separated chondral and osteochondral defects are suitable for refixation only if the fragment is large enough, and has not been crushed into small parts, if the surface cartilage layer is not destroyed (Figs. 1-4). This situation occurs mostly in cases of acute trauma, or in cases of osteochondritis dissecans. The refixation technique varies according to the thickness and size of separated fragments. The best way of osteochondral-fragment fixation is the use of the small screw or Herbert screw, eventually of the absorbable pins (Figs. 5-8). The use of the small screw has some key provisos: (a) Fragment must be reamed very gently to avoid breakage. (b) The screw has to be inserted under compression, and mounted perpendicularly to the joint surface.

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Fig. 1. Free osteochondral fragment on medial femoral condyle.

Fig. 2. Free osteochondral fragment on medial femoral condyle.

Fig. 3. Fragment refixation with two screws.

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Fig. 4. Fragment refixation with two screws.

Fig. 5. Free osteochondral fragment.

Fig. 6. Free osteochondral fragment.

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Fig. 7. Temporary fixation with Kirschner wire.

Fig. 8. Fixation with single screw and antirotation absorbable pin.

(c) When more screws have to be inserted, their divergence increases the stability of osteosynthesis. (d) The screw head has to be inserted under the cartilage surface, to allow free motion and immediate postoperative rehabilitation. A further option for fragment fixation is the usage of tissue glue. Its usage is limited to slim or subtle fragments only.

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Transfixation with Kirschner wires is also possible, but due to the danger of their malmigration, it is not a recommended procedure. When fragment-refixation is not possible or not indicated, their correct extraction is always necessary. The remaining fragments could act as alien-free body, and may induce the knee pseudoblockade and other symptoms — synovial membrane irritation, reactive synovitis, motion limitation and impingement syndrome. 3.2. Abrasive techniques — The subchondral b o n e perforation Abrasive techniques represent one of the most frequently used treatments of chondral cartilage defects yet. These methods follow the presumption that subchondral bone penetration stimulates forming of new reparative tissue. Subchondral bone penetration in full-thickness cartilage defects affects subchondral vessels, and leads to formation of fibrin clots, and to migration of undifferentiated mesenchymal cells. Transformation of those cells forms the base of further reparative tissue with characteristics of fibrocartilage consisting of collagen type I fibrils. Surfaces in the healed defect location remains partly depressed after the healing process, and the quality of new-formed cartilage lacks the quality of the original. The method for uncovering the subchondral bone varies among different authors: • pridie drilling; • microfracture techniques; and • subchondral abrasion. Pridie (Pridie, 1959) provided numerous drillings of subchondral bone with 6 mm bore. Second-look arthroscopy demonstrated healing of defects in the form of numerous islets of reparative tissue. But the islets did not form homogeneous reparative tissue.

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Steadman (Steadman, 2001) perforated the subchondral bone (with a special chisel) 3-4 mm deep, and the distance between single drill holes was 3-4 mm. He suspected formation of socalled superclots, forming the basis for future reparative tissue. The resulting reparative tissue was not of uniform quality. Subchondral abrasion arthroplasty was first described by Johnson (Johnson, 1986), and consists of ablating sclerotic bone layers with a shaver (abrader) to open the haversian canals. Abrasion was provided to the depth of 1-2 mm, and defects were sharply margined. Indications for abrasive techniques are full thickness chronic degenerative chondral lesions (type Ill.a. and Ill.b. according to Noyes-Stabler). According to the majority of studies, abrasive techniques reach good and excellent results in 50-75% of cases (Ewing, 1990). In contrast to those results is the work of Bert (Bert, 1993), who provided the drills in osteoarthrotic knees, and he did not confirm the good results. 3.3. Autologous grafts New developing techniques apply the idea of harvesting the osteochondral graft shaped in cylinders from the non weightbearing area of the knee, and the idea of graft transplantation to the defect in the weight-bearing zone of the cartilage. The recommended place and size of graft differs among authors. Bobic (Bobic, 1996) used cylinders of diameter of 5-10 mm and length of 10-15 mm from the marginal zone of the intercondylic fossa. Hangody (Hangody, 1996) took the grafts from the border zone of medial and lateral condyle. The diameter of grafts was 2.5-6.5 mm and length was 10-15 mm. Both authors used the press-fit technique for the graft application to the defect. Defects were not filled u p with grafts completely: only up to 70% of the area. This method was tested on dogs before its application to human medicine, and the firm connection of the transplanted graft was histologically confirmed. Grafts retained their hyaline-like character after healing; the area in-between grafts was healed by fibrous cartilage. Thus we

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conclude: transplanted grafts do not induce chondrogenesis, but their filling into defects reduces the area of lesser quality fibrous cartilage. The method described is called mosaicplasty. The long-term follow-up published by Hangody is very promising: he reached good and excellent results in 80-94% of patients compared to abrasive techniques (good and excellent results in 50-75%). We started putting Hangody's method into practice at our hospital in 1998 (Figs. 9 and 10), and recommend following.

V ) Fig. 9. Application of big cylinder grafts to the defect.

Fig. 10. Application of small cylinder grafts to the defect.

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3.3.1. Indication criteria (a) Chondral defects of type Ill.a. and Ill.b. according to NoyesStabler classification (Noyes, 1989). (b) Best results can be obtained in isolated cartilage defects, caused by trauma or osteochondritis dissecans. Cartilage defect localised on the weight-bearing area of femoral condyle or at the femoropatellar joint. (c) The indication for patients with combined soft knee injury is problematic. (d) Patients should be under 50 year of age, and the biological status of their knee joint and degree of osteoarthritis development must be considered. (e) Size of defect between 0.5 and 2.5 cm 2 , defects smaller than 0.5 cm 2 are not subject for surgical treatment. Large size defects (above 2.5 cm2) induce problems with harvesting of cylinders. 3.3.2. Contraindications (a) Patients with signs of chronic generalised osteoarthritis. (b) Severe general or metabolic disorders influencing cartilage turnover — (haemophilia, ochronosis). (c) Severe unicompartmental arthrosis of the knee joint. (d) Axial and other biomechanical disorders of the knee joint; instability, varus or valgus deformity, and patellar subluxation. Mosaicplasty is indicated after surgical reconstruction of the above mentioned disorders (after anterior crucial ligament reconstruction, lateral patellar release...). 3.3.3. Results Our study included 30 patients treated by mosaicplasty (19 male, 11 female). Average age was 30.7 (range 17-45 years). The injury was caused by trauma (acute: eight cases, chronic: 10 cases) and 12 cases were indicated for osteochondritis dissecans. Symptom duration before surgery varied from 0-450 days. Size of cartilage

Present Ways of Treating Chondral and Osteochondral Knee Defects Table 1. Type of chondral lesions according Noyes-Stabler. Degree of cartilage defect according to Noyes-Stabler Number of patients, n

Type Ill.a.

Type IILb.

21

9

defect varied from 0.5-2.8 cm 2 , with an average size of 1.5 cm2. The defect was localised on the weight-bearing area of the medial femoral condyle in 20 cases, and on the weight-bearing area of lateral femoral condyle in 10 cases. The degree of cartilage defects was classified according to Noyes-Stabler in Table 1. Concomitant injuries of the soft knee were observed: • • • •

rupture of anterior crucial ligament in 16 patients; lesion of medial meniscus in seven patients; lesion of lateral meniscus in five patients; and patellar chondromalacia up to degree Il.a. in 14 patients.

Cartilage monotrauma was observed on the weight bearing area in nine cases. Defects combined with the above injuries were present in the remaining 21 cases. Open reconstruction technique was used in 12 cases; arthroscopy in 18 cases. Grafts were obtained from the medial margin of the femoral condyle in 18 cases. In 12 cases it was necessary to add grafts from the lateral condyle. We used two to nine cylinders to fill up the defect. Loading was strictly forbidden in the postsurgical period for four weeks. Full loading was allowed individually after 6-16 weeks. Follow-up evaluation was provided at five and 12 months after surgery (Table 2). The results document significant improvements in groups of surgically treated patients. Their Lysholm score before operation was 54.3 points, but five months after surgery it was 77.5 points; and 1 year after surgery the score reached 85.2 points. The surgically treated group of patients was further divided into two parts according to the type of defect (Table 3). Observed

44

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Table 2. Defects treated with mosaicplasty. Lysholm knee score before surgery, five and 12 months after surgery. Number Lysholm Lysholm Lysholm

of patients, n score before surgery score 5 months after surgery score 12 months after surgery

30 54.3 77.5 85.2

Table 3. Lysholm score according to defect type. Degree of cartilage damage according to Noyes-Stabler

Type III.a.

Type IILb.

Number of patients, n

21

9

Lysholm score before surgery

55.5

51.5

Lysholm score 5 months after surgery

79.3

73.2

Lysholm score 12 months after surgery

86.4

82.4

results were significantly better in groups of patients with defect III.a. as opposed to before surgery. Three patients underwent second look arthroscopy 3-5 months after mosaicplasty. During the second look arthroscopy we observed healed cylinders and a soft graft surface. The graft surface was evaluated according to the cartilage repair assessment system (ICRS — cartilage score). The average ICRS cartilage score was 8.33 points (near normal graft surface). 3.4. A l l o g e n o u s grafts The allograft is used to solve large osteochondral defects (area above 3 cm2, depth above 10 mm). The advantage of the osteochondral allograft is the possibility of exact correction according to defect size. The clinical experiences documented

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Fig. 11. Healing of allogenous graft in the area of tibial plato.

Fig. 12. Healing of allogenous graft in the area of tibial plato.

good healing of grafts into defects, including good reparation of the articular surface (Figs. 11 and 12). In our hospital we used fresh frozen allografts. Use of allografts is indicated in cases of deep large defects reaching the subchondral bone that are not indicated for primary fixation, and in cases of secondary replacement following unsuccessful primary fixation. Grafts can be connected to the defect by the press-fit technique or by small screws or Herbert screws. The long time results are satisfactory: 75-94% of good results after two to five years (Flynn, 1994).

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3.5. Autologous chondrocyte transplantation The limited ability of chondrocytes to repair the articular surface defect is generally known. The aim of different investigators was to find a method to produce cartilage in places of chondral and osteochondral defects. It was demonstrated that chondrocytes and undifferentiated mesenchymal stem cells, after placement to the defect, survive and produce the cartilaginous matrix (Britberg, 1994). Various ways of cells fixation to the defect exist. Wakinati (Wakinati, 1994) demonstrated new formation of hyaline cartilage in rabbits after filling the defect with allogenous chondrocytes in collagenic gel. One of the most important studies in this field was published by Brittberg (Britberg, 1994). The cartilage was harvested from the non weight-bearing area of the knee joint in patients diagnosed with deep chondral defects on the femoral condyle or patella. Chondrocytes were cultivated in vitro for 14 to 21 days, and later injected into the defect and covered by a periosteal flap. In 14 patients out of 16 with documented condylar defects and in two patients out of seven with patellar defects good or excellent results were achieved after transplantation. The biopsy from the former defect's location demonstrated formation of new cartilage with a hyaline-like structure in two out of three of patients. This topic is also intensively studied at our hospital. We use the method of chondrocyte retrieval and in vitro cultivation similar to that described by Peterson (Peterson, 2000). The transplantation phase using a periosteal flap is in our opinion not optimal due to the fixation of the periost with stitches to the cartilage, which is not always ideally strong or even waterproof. For this reason modifications of the original technique are tested in many hospitals. The goal is to find a suitable threedimensional carrier for the chondrocyte culture. After serial laboratory tests we used the tissue fibrin glue Tissucol (Baxter, Austria) as a chondrocyte carrier. Very good cell viability and capability of cell migration and outgrowth was documented. Tests on cadavers and pigs demonstrated good healing effects

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after chondrocyte transplantation. The histological examinations described hyaline-like cartilage. This technique is indicated for deep chondral defects of type IILa. and IILb. according to the Noyes-Stabler classification (Noyes, 1989). Best results can be reached in isolated cartilage defects above 2 cm 2 on the weight-bearing area of the femoral condyle. Age of patients should be under 50 years, but it is always necessary to consider the biological status of the knee joint and degree of osteoarthritis development (Figs. 13 and 14). Our technique consists of the following phases: ® arthroscopic diagnostic of defect; ® arthroscopic sampling of cartilage for in vitro cultivation;

-X

^^P^?

Fig. 13. Chondrograft prepared for transplantation to the defect.

Fig. 14. Chondrograft transplantation to the defect.

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• transport of samples to tissue bank; • enzymatic isolation, in vitro cultivation of chondrocytes; • graft formation (combination of fibrin glue with chondrocytes); quality control; and • transplantation (chondrograft agglutination in defect). A suitable defect was diagnosed during arthroscopy, during which we assessed the size of the lesion and obtained cartilage samples for cultivation. (300-500 mg from the margin of medial femoral condyle). Cartilage was transported in cold salt solution with antibiotics. Samples of cartilage were cleaned and cut into small pieces under laminar air flow hood conditions and digested enzymatically using trypsin and collagenase. The chondrocyte suspension was inoculated into flasks and cultured in an incubator at 37°C in a C 0 2 atmosphere. Culture medium exchange was provided every 48 hours. Proliferation of chondrocytes was monitored using inverted microscopy. Successful primocultivation resulted in the development of cell monolayers. The required number of cells (5-10 million of cells per ml) was obtained via several subcultivations. The following methods for quality control were used: (a) Determination of cell quantity; (b) Determination of cell viability via trypan blue staining. Viability between 90-95% was demonstrated; (c) Determination of proliferative activity was tested using growth quality control. A small part of chondrograft was placed into a Petri dish and incubated in culture medium. Migration and outgrowth of chondrocytes was documented (Fig. 15); (d) The morphological characteristics were studied by: • light microscopy (haematoxylin-eosin staining); • electron microscopy (transmission and scanning). A good stage of cellular organelles and production of extracellular matrix with protocollagenic fibrils was found in all cases; and • Immuno-fluorescence microscopy (using monoclonal antibodies against vimentin) (Fig. 16);

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Fig. 15. Light microscopy — migration and outgrowth of chondrocytes from the chondrograft.

Fig. 16. Imnumo-fluorescence microscopy — monoclonal antibodies against vimentin.

(e) We did not perform cell DNA authentication or isoenzymatic analysis. We used the fibrin tissue glue Tissucol (Baxter, Austria) as a three-dimensional carrier for the chondrocyte culture because of the excellent viability of chondrocytes, and their migration

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ability to Tissucol surface was documented. Before surgery the chondrocyte suspension was mixed with fibrin glue and applied into special form. The advantage was that the size and thickness of the graft was readily adapted according to cavity size and deepness. The prepared chondrograft was transported to the operating room, reshaped and transplanted into the defect. Fixation was realised via agglutination with fibrin glue (Figs. 17 and 18).

Fig. 17. Pre-operative picture of defect.

Fig. 18. Applied chondrograft.

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3.5.1. Results Our study included 14 patients treated with the technique of cultivated chondrocytes in Tissucol (10 males, four females). Average age in the study group was 35.0 years (range: 19-50 years). The injury was caused by trauma in 12 cases (acute: four cases, chronic: eight cases) and two cases were diagnosed as osteochondritis dissecans. Duration of clinical symptoms before surgery was 0-650 days. Average size of cartilage defect was 2.7 cm 2 , reaching from 1.7-4.5 cm 2 . The chondral defect was localised on the weight bearing area of the medial femoral condyle in 10 cases, and on the weight bearing area of the lateral condyle in four cases. We observed concomitant injuries of the soft knee: • • • •

rupture of anterior crucial ligament in nine patients; lesion of medial meniscus in four patients; lesion of lateral meniscus in three patients; and patellar chondromalacia up to degree Il.a. in four patients.

Monotrauma of cartilage on the weight bearing area was observed in four cases. In the remaining 11 cases there was a combination of chondral defects with the above injuries. Chondrograft application was provided from the open approach. We have not observed any serious clinical complications during the postoperative period. Reactive synovitis with exudation was documented in 4 cases (28%). Clinical symptoms disappeared after application of nonsteroid antiflogistics in four weeks. We eliminated any mechanical loading for eight weeks and full weight-bearing was allowed after 12-16 weeks. Intensive rehabilitation was an obligatory part of postoperative care. Follow-up of patients was provided five and 12 months after surgery (Table 4). The preoperative Lysholm knee score was 49.3 points; 5 months after surgery it rose to 76.5 points; and 12 months after surgery reached 81.0 points. Second look arthroscopy was provided in four patients 3-5 months after chondrograft implantation. In two patients, very

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Table 4. Defects treated with autologous chondrografts, Lysholm score before surgery, five and 12 months after surgery. Number Lysholm Lysholm Lysholm

of patients, n score before surgery score 5 months after surgery score 12 months after surgery

14 49.3 76.5 81.0

good and complete healing of the graft was documented, and in two cases partial chondrograft degeneration (30% of graft area) was documented. The graft surface was evaluated according to the cartilage repair assessment system (ICRS -— cartilage score). The average ICRS cartilage score was 8.0 points (almost normal graft surface). During second-look arthroscopy, samples for conventional light and electron microscopy were obtained. Microscopic controls documented presence of hyaline-like cartilage in the healing defect (presence of typical spherical chondrocytes, extracellular collagenous filaments, formation of typical isogenetic cellular groups) (Figs. 19-21). In cases of graft degeneration

Fig. 19. Conventional light microscopy — samples obtained by second look arthroscopy.

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/

Fig. 20. Transmission electron microscopy — samples obtained bysecond look arthroscopy.

Fig. 21. Scanning electron microscopy — samples obtained by second look arthroscopy.

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(fissuration) found during second look arthroscopy we documented neovascularisation of reparative tissue, with the presence of fibroblast-like cells. 4. Conclusions All our documented studies here present the wide range of reconstructive surgery options for knee joint cartilage. Former methods, such as subchondral bone penetration, are replaced by new methods that present better options for articular surface reparation. These former methods are still used, but their indications are becoming strictly selective. Their use is dictated by the character of cartilage defect, by the degree of biological age of the knee, and by body weight and other factors. Treatment of chondral defects by autologous cultivated chondrocytes in Tissucol presents a new original therapeutic option (Visna, 1999; 2002; Adler, 2002). Outcomes of the described chondrograft are promising, and this procedure presents a prospective and beneficial method in cartilage surgery. 5. Summary Chondral and osteochondral defects play an important role in knee surgery. Knee trauma is often followed by premature development of osteoarthritis due to limited reparative processes in the cartilage. Today's diagnostic possibilities and progress in arthroscopic techniques promote the early diagnostics and exact classification of osteochondral defects. The prognosis of these injuries is improved by early treatment. The paper presents therapeutic ways for treatment of cartilage injuries and the adjacent part of the subchondral bone. A new possible method to solve deep chondral defects on the weight-bearing area of the knee is mosaicplasty and autologous cultivated chondrocytes in Tissucol. 6.

Acknowledgment

Work was supported by GRANT IGA MZ CR No. 5972-3.

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7. References ADLER, J., KOMARKOVA, J., HORKY, D., VISNA, P. et al. (2002). Cultured autologous chondrografts for the treatment of cartilage defects, Proc. 26th Annual Meeting American Association of Tissue Banks, Boston, Massachusetts, p. 83. BERT, J.M. (1993). Role of abrasion arthroplasty and debridement in the management of osteoarthritis of the knee, Rheumat. Dis. Clin. North Am. 19, 725-739. BOBIC, V. (1996). Arthroscopic osteochondral autograft transplantation in anterior cruciate ligament reconstruction: A preliminary clinical study, Arthroscopy 3, 262-264. BRITBERG, M., LINDAHL, A., NILSSON, A., OHLSSON, C. and ISAKSON, O. (1994). Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, New Engl. J. Med. 331(14), 889-895. BUTLER, J.C. and ANDREWS, Jr. (1988). The role of arthroscopic surgery in the evaluation of acute hemarthrosis of the knee, Clin. Orthop. 228, 150-152. EWING, J.W. (1990). Arthroscopic treatment of degenerative meniscal lesions and early degenerative arthritis of the knee, In: Basic Science and Arthroscopy, J.W. Ewing, ed., Raven Press, New York, pp. 137-145. FLYNN, J.M., SPRINGFIELD, D.S. and MANKIN, H.J. (1994). Osteoarticular allografts to treat distal femoral osteonecrosis, Clin. Orthop. 303, 38-43. HANGODY, L., SZIGETI, I., KARPATI, Z. and SUKOSD, L. (1996). New method for treatment of serious localised cartilage damage in the knee joint, Mayer Verlag, Osteo. Int. 1-7. IWASAKI, M., NAKAHARA, H., NAKATA, K., NAKASE, T. and KIMURA, T. (1995). Regulation of proliferation and osteochondrogenic differentiation of periosteum-derived cells by

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transforming growth factor-beta and basic fibroblast growth factor, /. Bone Joint Surg. 77A(4), 543-553. JOHNSON, L.L. (1986). Arthroscopic abrasion arthroplasty historical and pathologic perspective. Present status, Arthroscopy 2, 54-69. NOYES, F.R. and STABLER, C.L. (1989). A system for grading articular cartilage lesions at arthroscopy, Am. J. Sports Med. 17, 505-513. PETERSON, L., MINAS, T., BRITBERG, M., NILSSON, A., SJOGREN-JAHNSSON, E. and LINDHAL, A. (2000). Two- to 9-year outcome after autologous chondrocyte transplantation of the knee, Clin. Orthop. 374, 212-234. PRIDIE, K.W. (1959). A method of resurfacing osteoarthritic knee joint, /. Bone Joint Surg. 41B, 211-228. STEADMAN, J.R., RODKEY, W.G. and RODRIGO, J.J. (2001). Microfracture: Surgical technique and rehabilitation to treat chondral defects, Clin. Orthop. 391, 362-369. VISNA, P., ADLER, J., NESTROJIL, P., POKORNY, V. and SELUCKY, J. (1999). Soucasne moznosti feseni chondralnich a osteochondralnich defektu kolene, Rozhledy v chirurgii 78(6), 259-264. VISNA P., PASA, L., HART, R., FOLVARSKY, J., ADLER, J. and HORKY, D. (2002). Treatment of deep cartilage defects in the knee by mosaicplasty combined with autologous cultivated chondrocytes in Tissucol, Proc. European Trauma Congress, Vienna, Austria, p. 173. WAKINATI, S., GOTO, T., PINEDA, S.J., YOUNG, G.R. and MANSOUR, J.M. (1994). Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage, /. Bone Joint Surg. 76A(4), 579-592.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

18 KNEE JOINT LIGAMENT ALLOPLASTY WITH TENDON GRAFTS STERILISED WITH GASEOUS ETHYLENE OXIDE

V.I. SAVELIEV, LA. KUZNETSOV, A.V. KALININ, A.A. BULATOV a n d LA. SOLODOV Russian Research Institute of Traumatology a n d Orthopaedics n a m e d after R.R. V r e d e n Baikov Str. 8, 195427 St. Petersburg, Russia

1. Introduction Transplantation of allogenic tendons is quite extensively used in Russia to treat patients with locomotive system injuries and diseases. This has become possible due to experimental research, favourable clinical outcomes, and certainly due to tissue banks that have extended the possibilities of reconstructive surgery. This is confirmed by the data found in the monograph of Nikitin and colleagues (1994) summing up the results of this kind of surgery in 1,363 cases during the 25-year period (19691994). The transplantation material for all these patients was supplied by the Tissue Bank of the Russian Research Institute of Traumatology and Orthopaedics named after R.R. Vreden. This bank possesses vast experience in procuring different biological transplants with the help of chemical sterilising agents, and in particular, with ethylene oxide, which has been used for this purpose since 1974. The number of tendon allografts sterilised 455

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Table 1. The number of allogenic tendon grafts delivered to different medical institutions. Medical institutions

Number of grafts

Russian Research Institute of Traumatology and Orthopaedics named after R.R. Vreden

3,232

Medical institutions of St. Petersburg

2,977

Medical institutions of Russia Total

2,281 8,490

with ethylene oxide given out to different medical institutions according to their requests, is shown in Table 1. 2. The Experience in Russia The biological material is procured in a morgue without aseptic precautions following the main rules of donor selection with subsequent sterilisation with gaseous ethylene oxide. The decision on the possibility of tissue removal is based on the history of the donor's death, and the results of objective examination of the corpse, autopsy and additional laboratory tests. Tissues are not procured in the presence of active infection; oncological diseases; chronic infectious diseases (hepatitis, HIV, syphilis, etc.); a number of autoimmune diseases; hormonal disturbances; and toxic agents in dangerous concentrations. Besides that, tissues from donors with degenerative-neurological dysfunctions (dementia, etc.) are not used if the donors have been on hormonal therapy (pituitary growth hormone, prolonged steroid therapy). The same applies to other doubtful cases. The donor's age is not a direct contraindication, but in transplantation of allogenic tendons (as well as of other supportive tissues) it is considered an important selection criterion. It is important to adhere to the rule according to which women should be under the age of 50 and men, under the age of 55 years.

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To preserve the tissues; useful qualities, they are procured within 12 hours of the donor's death if the corpse is maintained under room temperature (18 ± 5°C)/ or within 24 hours if it is kept in a fridge. The tissues from each donor are processed and packed separately to exclude the danger of cross-contamination. Before their sterilisation, allogenic tendons are placed into polyethylene containers consisting of two bags — one (larger) containing the graft, and the other (smaller), a test-object or a piece of the same tissue for bacteriologic control of sterilisation quality. The following procedure of postmortem tendon graft procurement is used. The skin is incised over the dorsum of the lower leg and foot. The line of the incision runs from the basic phalange of the fifth toe to the nail bed of the great toe, and from there — superior along the medial surface of the lower leg as far as the internal femoral condyle. The skin is separated along the whole area of the incision to reveal the fascia with the underlying tendons; the fascia and the tendon sheaths are then incised. Four tendons of the long toe extensor are freed and sectioned at the site of their transition into muscle tissue as well as at the level of their attachment to the basic and middle phalanges of the II-V toes. The great toe extensor tendon is isolated with two incisions going along it on the dorsum of the foot. After that, its muscle part is cut. An osseous plate at the site of the tendon insertion is cut with a chisel, or sawn out. In this way a small osseous fragment is left at the end of the graft, which helps to hold the tendon in place after its transplantation. To detach the tendon of the long flexor, the skin incision runs from the insertion of the Achilles tendon along the foot surface as far as the head of the IV metatarsal bone; turns medially reaching the great toe, and then continues along it as far as the tip of the distal phalange. The flap on the foot is separated. The tendon is freed of the surrounding tissues and sectioned. Its distal end is removed with an osseous plate from the distal phalange. The tendons of mm. gastrocnemius posterior and peroneus longus are uncovered with the help of an incision going from the lateral malleolus through the sole of the foot to

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458

the base of the first metatarsal bone, and transsectioned at the sites of their insertion. The Achilles tendon is removed through the incision running 10-15 cm superior from the calcaneal tubercle along the posterior surface of the lower leg. The tendon is cut in the place of its transition into the muscle part and near the calcaneal tubercle. It is possible to remove it with a fragment of the calcaneus. The method of gaseous sterilisation is as follows. After placing the sealed grafts into a chamber type steriliser (Fig. 1) the air is evacuated from it until the pointer of the manovacuum meter reaches the point of minus 1 kgc/cm 2 . Then, ethylene oxide is let into the chamber in the quantity necessary to increase the pressure up to -0.5 kgc/cm 2 that corresponds to the sterilising dose. After that, through a bacterial filter, carbon dioxide is introduced, increasing the pressure in the chamber up to plus 1 kgc/cm 2 .

System of pumping out air and used gas

System of regulation (instrumental panel)

System of introduction of gaseous ethylene oxide and gas-deflegmater

System of temperature control

System of energy supply

System of pressure control within the chamber Fig. 1. Functional charts of units (chambers) for sterilisation with gaseous ethylene oxide.

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Sterilisation time amounts to 1.5 hours under a temperature of 37°C, or two hours under the room temperature. After that, the gas is pumped out within one hour with a vacuum pump. When pumping has been completed, the chamber is filled again through a bacterial filter, with carbon dioxide or ordinary air, until the normal atmospheric pressure is established. The chamber is opened, the containers with their content are removed, hermetically sealed and placed into a refrigerator under the temperature regimen of -20°C. Before their clinical usage, sterilised tendon allografts should be obligatorily thawed in saline solution under room temperature. Bacteriologic testing of grafts procured in this way to prove their sterility, is repeated thrice in standard microbiologic media: immediately after their removal; after the end of their sterilisation, and selectively in the process of storage. Besides that, sterility testing is performed with washing-out directly in the operating room before the graft transplantation. Trials on experimental animals conducted earlier, and in our days (Tetyushkin, 1974; Jelezny, 1978; Saveliev and Rodyukova, 1992; Solodov, 2002) with transplantation of various biologic grafts sterilised with gaseous ethylene oxide in the described way, did not reveal any principal differences in the fate of sterilised and non-sterilised (control) allografts, which thus allowed their recommendation for clinical application. The authors' practical experience are based on 385 operations of transplantation of allogenic tendons sterilised with ethylene oxide performed for knee ligament injuries at the Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden, from 1988 to 1995. Two kinds of grafts were used: a "single" tendon of m. peroneus longus with an osseous fragment out of the base of the first metatarsal bone, and a socalled "double" tendon consisting of the tendons of the long flexor and the long extensor of the great toe with a common osseous fragment out of the base of its distal phalange (Figs. 3 and 4).

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Fig. 4. An allograft from the tendons of mm. flexor et extensor hallucis longus with an osseous fragment.

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The number of men with knee ligament allogenic reconstruction was twice as large as the number of women (68% and 32% respectively). More than half (54%) of these patients were under the age of 30; over three-quarters (77%) of all patients were younger than 40 years, and the number of patients on the right side of 50 amounted to 93%. In approximately one half of the patients (196 or 51%) it was necessary to reconstruct only the anterior cruciate ligament. As to the discussed series of findings as a whole, the anterior cruciate ligament needed reconstruction in the overwhelming majority of cases — in 96% of all operations (368 patients). In contrast, restoration of only the posterior crucial ligament was performed just in 1% of cases (5 patients); on the whole, including the operations of its reconstruction performed together with restoration of one or both collateral ligaments, and in cases when both crucial ligaments were injured, it was repaired in 14% of cases (56 patients). The collateral ligaments (one or both) were reconstructed with the help of sterilised allogenic tendons in a considerable number of the injured — in 170 cases or 44% of operations. In the majority of them (164 cases, or 43%) the collateral ligaments were restored along with plasty of one or both cruciate ligaments. Isolated plasty of one or both collateral ligaments was performed only in six cases, or 2%. Among 385 patients who had undergone knee ligament restoration with allogenic tendon grafts, no complications were seen during the early postoperative period, during which problems may be provoked by the transplanted material. In some cases within the first week after the surgery, subfebrile temperature and a moderate shift of leukocyte count to the left were registered, which was in complete agreement with the extent of the surgery. No septic complications were registered in this group of patients. In 14 (3.6%) out of 385 patients who had undergone knee ligament plastic restoration, additional operations on the ligamentous apparatus of the same joint were considered necessary.

Knee Joint Ligament Alloplasty with Tendon Grafts Sterilised

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In 11 of those (2.9%) with operations on the same knee earlier, there was a recurrence of instability. In two patients (0.5%) additional alloplasty of formerly untreated ligaments was performed. In one patient (0.3%) another operation on the same knee joint was done to dissect the bursa formed in the region of the osseous fragment of the allograft on the external surface of the lateral femoral condyle. Out of the named 11 patients operated upon for the recurrence of instability of earlier reconstructed ligaments, seven (1.8%) injured the same joint again within the period of one to three years. In four cases there was no history of trauma, and knee instability was noted in the process of regaining the main range of motion after the completion of plaster immobilisation. In one of them (a 32-year old male patient) plasty with "double" allogenic tendon graft was undertaken for the old injury of the anterior cruciate and tibial collateral ligaments of the knee joint. Nearly 2.5 months after this, in the process of movement training, signs of instability and aseptic synovitis (ascertained by aspiration with subsequent bacteriologic seeding) were found. This was the reason for diagnostic arthroscopy, which on the background of marked synovial membrane reaction could not reveal the presence of any allogenic tendon tissue in the region of the cruciate ligaments. It was interpreted as manifestation of tissue incompatibility, resulting in complete resorption of the allogenic graft. In three other cases the recurrence of knee instability was not accompanied by this type of reaction on the part of the tissues of the joint, and was probably caused by technical errors during the surgery. In seven out of these 11 patients with instability recurrence after ligament restoration with allogenic tendons, repeated plasty was performed. Tendon allografts were used for revision surgery only if there was a reliable history of repeated knee injury with absence of complications which might be caused by the transplanted allogenic material. In four other patients revision plasty of the ligaments was performed using autologous tendons.

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Thus, 385 patients treated at the Russian Research Institute of Traumatology and Orthopaedics named after R.R. Vreden had their knee ligaments restored by way of transplantation of allogenic tendon grafts sterilised with gaseous ethylene oxide. In 96% of these operations the anterior cruciate ligaments was reconstituted: in 44% — one or both collateral ligaments; in 14% — the posterior cruciate ligament. The following rule was followed: to reconstruct all cruciate and collateral ligaments, a complete rupture of which was diagnosed before the operation or during it. The overwhelming majority of knee ligament reconstructions were done for their old injuries; only 7.5% of patients were operated within the first month after the accident. Nearly one half (44.9%) of these operations was accompanied by resection of one or both menisci. The most common accompanying pathology in such patients is deforming arthrosis of the affected knee joint. Stability of the knee joints after reconstruction of their ligaments with allogenic tendons sterilised with gaseous ethylene oxide, was re-evaluated in 86 (22.3%) out of 385 patients in four to 11 years after the surgery (the medium length of follow-up amounting to eight years). Diagnostic arthroscopies done five to eight years after anterior cruciate ligament alloplasty, showed that the allogenic tendon had been completely replaced by the host's tissues. The regenerate formed in this way looked like an anterior cruciate ligament, and possessed similar mechanical characteristics. It was covered with a synovial-like lining and demonstrated arthroscopically visible vascular ingrowth (Fig. 5). Based on the described clinical data, it may be concluded that substitution of knee joint ligaments with allogenic tendons sterilised with gaseous ethylene oxide is a worthy method of surgical treatment of patients with this kind of injury. Allogenic tendons treated in the described way are easily accepted by hosts, this being proved by the absence of septic complications in the discussed series of findings. The rate of unfavourable late results found by re-testing of 86 patients was rather low,

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Fig. 5. A remodelled "double" tendon allograft, seven years after the operation. Arthroscopy.

amounting to 8.1% (group D on the IKDC scale). It should be emphasised that the rate of patients in whom the state of their knee joints was considered normal or almost normal (groups A and B on the IKDC scale) reached 69.8%, which is comparable with the outcome of alternative methods of knee ligament reconstruction, and completely satisfies the present clinical prerequisites. Thus, the achieved results, especially in the follow-up period, speak in favour of the high efficacy of clinical applications of allogenic tendon grafts sterilised with ethylene oxide. 3. References JELEZNY, P.A. (1978). Bone plasty operations with bioplastic material sterilised with gaseous ethylene oxide. Auto-abstract of M.D. Candidate Dissertation, Novosibirsk, 21 p. NIKITIN, G.D., Kornilov, N.V., Linnik, S.A. and EFIMOV, V.N. (1994). Allotendoplasty in Treatment of Muscle, Tendon and Ligament Injuries. St. Petersburg, 256 p.

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SAVELIEV, V.I. and PODYUKOVA, E.N. (1992). In: Osseous Tissue Transplantation. Nauka, Novosibirsk, 220 p. SOLODOV, LA. (2002). Application of tendon allografts sterilised with ethylene oxide for the sake of reconstruction of the knee joint ligamentous apparatus. Auto-abstract of M.D. Candidate Dissertation, St. Petersburg, 17 p. TETYUSHKIN, M.T. (1974). Chamber sterilisation of tissue grafts with gaseous ethylene oxide. Auto-abstract of M.D. Candidate Dissertation, Barnaul, 18 p.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

19 NEW APPROACHES TO COMPARATIVE EVALUATION OF ALLOGENIC AND AUTOLOGOUS BONE TRANSPLANTS PROCURED IN VARIOUS WAYS

A.V. KALININ, V.I. SAVELIEV a n d A.A. BULATOV Russian Research Institute of Traumatology a n d Orthopaedics, n a m e d after R.R. V r e d e n Baikov Str. 8, 195427 St. Petersburg, Russia

1. Introduction Any research work dealing with comparative evaluations of different ways of bone tissue conservation, starts with the choice of an experimental model. The latter should correspond to the aims and the goals of the research, as well as provide for standardisation of conditions in the process of experiment conduction. These conditions are rather difficult to achieve due to both external and internal factors. The external factors include peculiarities of operative technique with various degrees of tissue trauma, and security of graft fixation, or purulent complications. With acquired experience and model improvement the external factors may be under control. But it is more difficult to overcome the undesirable influence of intrinsic factors such as age, gender, individual and constitutional peculiarities of donors and hosts. The most reasonable decision under these conditions is to conduct such kinds of research on genetically similar lines of 467

468

A.V. Kalinin, V.I. Saveliev & A.A. Bulatov

experimental animals. But inbred animals, especially large ones, are still too expensive to be used in everyday practice of the majority of experimental laboratories. It should be emphasised that experimental models used at present for the sake of studying bioplastic characteristics of bone transplants, do not allow us to take into consideration the influence of all, or at least the majority of the enumerated factors. As a rule they are based on procurement of transplantation material from several animals-donors and the transplantation to other animal-recipients, into osseous defects created in various skeletal sites (Einhorn et ah, 1984). The transplanted grafts usually differ in their size, form and composition, which in combination with the absence of a standard method of graft fixation, has certain influences over the processes of bone regeneration, their speed and quality. These very reasons may explain existing disagreement about the term of consolidation and remodelling of osseous grafts (including demineralised bone) preserved in various ways. Besides that, the known experimental models are labour consuming, and need for their implementation plenty of animals — a number of them being killed without surgery due to the need of a great amount of transplantation material. In 1967 Saveliev devised an original model for comparative studies of bone transplant evolution. According his suggestion several grafts (from two to six) received from one animal-donor were transplanted to another animal-host (both of them being dogs). Rib fragments were used as transplantation material; they were placed in rib defects created in experimental animals by rib resection, both on the left and the right sides of the chest (Fig. 1). Thus the suggested model allowed us to standardise the transplantation material, and eliminate most of the unfavourable factors described above. The expenditure of animals decreased to a considerable degree because several operations were done on one and the same dog. In contrast to other models all manipulations on one animal, including its euthanasia, were performed at the same time — which excluded the influence of time factor.

New Approaches to Comparative Evaluation of Allogenic

DOG «A»

469

DOG «B» >

Fig. 1. A schematic drawing of the operations. Allotransplantation — transplantation of rib fragments from Dog "A" to Dog "B" after their sterilisation and conservation in different ways.

And finally, this model allowed one-stage orthotopic transplantation of bone grafts identical in their form, size and structure, to that of an animal-recipient. The following operation technique was used. Under intravenous thiopental anaesthesia, hair was cut in the area of the greatest convexity of the rib cage on the left and right sides in a strip 10-15 cm long. Due to the danger of injuring the pleura in the process of rib excision with subsequent pneumothorax development, dogs were usually intubated so that lung ventilation could be started if necessary. The skin was incised parallel to the spine. Then the soft tissues were cut transversely to the ribs. The periosteum was incised along the rib at the necessary length, and detached from it with a raspatory. After that a rib fragment was resected with cutting forceps. The intramedullary canals of the remaining parts of the ribs were widened with an awl. One end of the pin with the graft mounted on it was inserted into the vertebral end of the rib at a depth of 1-1.5 cm. After that, with the help of strong clamps the stumps of the rib were separated as far as possible to allow us to insert the other

470

A.V. Kalinin, V.I. Saveliev & A.A. Bulatov

end of the pin into the sternal rib stump, and the stumps were approximated to be in contact with the ends of the graft. The intercostal muscles were stitched with continuous sutures, followed by stitching of m. latissimus dorsi. The dog was extubated. The animals did not need any special care after the operation. They were sacrificed under general anaesthesia in due time, and macrospecimens were removed. The latter were studied macroscopically, roentgenographically (in two views), histologically, and in other ways. Later Saveliev (1978) devised a new version of the experimental model, according to which rib grafts removed from one side of the chest after their treatment with sterilising or preserving agents, were transplanted into rib defects created on the other side of the chest of the same animal (Fig. 2). Thus the first version was concerned with allogenic, and the second one — with autologous bone plasty. The application of the second version considerably increased the reliability of the achieved results, because the transplantation material was

DOG «C»

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>

Fig. 2. A schematic drawing of the operations. Autotransplantation — transplantation on the left side of the chest of the Dog "C" of rib fragments excised earlier on the right side, sterilised and preserved in different ways.

New Approaches to Comparative Evaluation of Allogenic

471

in complete conformance with individual characteristics of the host's body. In other words, in contrast to the first variant there was no disagreement between the antigenic uniform transplantation material and the non-uniform host bed. Alongside this, the follow-up period became considerably shorter: the longest follow-up time not exceeding six months in contrast to one to two years in the transplantation of allogenic bone. Elimination of difficulties caused by the necessity to reproduce similar experimental conditions, and possibilities of adequate control provided by a fresh (not preserved) rib fragment, resulted in the fact that all peculiarities of reparative osteogenesis found in experiments on transplantation of osseous tissue (preserved in different ways and for various periods of time), could be interpreted solely with respect to their influence. It is also worth mentioning that not a single test model used nowadays in experiments on bone transplantation, allows us to use the same amount of bone with uniform structure, volume and form as the one which is being described. In attempts to improve the second version of the discussed model, various ways of graft fixation were studied, which resulted in elaboration of the original method consisting in securing grafts with flat spear-like intramedullary rods made out of pins usually used in trauma and orthopaedic surgery. This rod securely joined the ends of the resected ribs, obliterated motion at their junction with the graft, and prevented graft displacement ("twisting") around the rib axis. In addition to that, it decreased the force acting at the central part of the graft, especially at the height of its remodelling which helped to prevent fractures and pseudarthroses. This spear-like rod appeared especially useful for holding demineralised bone grafts, which are difficult to stabilise with other means. In addition to fixation method improvements it was suggested that we dissect a rib fragment 2 - 3 m m smaller than the graft; this allowing us to achieve some compression along the line of bone junction. All these suggestions helped to improve conditions for grafts and favoured their substitution with new bone.

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A.V. Kalinin, V.I. Saveliev & A.A. Bulatov

The described versions of the model were used by the authors of this article in the study of bioplastic characteristics of allogenic and autologous bone grafts preserved in various ways. 2. Material and Methods The first experimental series (allotransplantation) included 45 operated dogs; the second one (autotransplantation) consisted of 38 animals. In the first series, rib fragments up to 35 mm in length were received from mature dogs-donors. Into the defects created on both sides of the rib cage of dogs-recipients, six grafts were placed: three on each side. Five of them represented allogenic material: the first graft was procured under sterile conditions and preserved by freezing at -20°C; the second one was preserved in 0.5% formalin solution; the third and the fourth were demineralised in 2.4 N and 1.2 N HC1 solution respectively;

a

b e

d

e

f

g

Fig. 3. Rib X-rays after allotransplantation of the following fragments: (a) intact rib; (b) demineralised in 1.2 N HC1 solution; (c) procured under sterile conditions and preserved by freezing at -20°C; (d) demineralised in 2.4 N HC1 solution; (e) preserved in 0.5% formalin solution; (f) autograft (reference); (g) sterilised with gaseous ethylene oxide, and preserved by freezing.

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473

the fifth graft was sterilised with gaseous ethylene oxide and preserved by freezing (Saveliev, 1971). The sixth (control or reference) graft was autologous; it was taken out during the operation and positioned into the defect. Fixation was achieved with metal pins. The time of graft preservation before their transplantation didn't exceed one month. All in all, 270 grafts were transplanted. The animals were sacrificed under general anaesthesia at one, three, six, nine and 12 months after the operation, and macrospecimens were removed. The latter were examined with macroscopic, roentgenographic (Fig. 3) and histologic (Fig. 4) methods.

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A.V. Kalinin, V.I. Saveliev & A.A. Bulatov

Histologic specimens were stained with hematoxylin-eosin and according to van Gieson. The second experimental series was aimed at studying the bioplastic characteristics of osseous autografts preserved by freezing, with formalin and by demineralisation. On the left side of the dog's chest, fragments of three ribs were resected subperiosteally, and autografts 35 mm in length were prepared out of them. The defects formed in this way were left unfilled. The wound was completely closed in layers. The rib fragments were thoroughly freed of periosteal remnants and preserved by freezing under the temperature of -20°C, in 0.5% neutral formalin and by demineralisation in 2.4 N HC1 solution with subsequent washing in sterile saline solution, and keeping in 70° alcohol under +4°C. The length of graft preservation depending upon the experimental conditions, amounted to one, three and six months. After that period of time another operation was performed on the contralateral (right) side of the chest. It consisted in rib resection (leaving an intact rib in-between) and substitution of these defects with preserved grafts. Four rib grafts were transplanted to each dog; one of them — "fresh" (removed during the surgery) — served as the control (reference). It should be pointed out that the most crucial moment of the operation consisted in detachment of the pleural sheet of the periosteum from the internal side of the rib with a raspatory. While doing this it is possible to injure the pleura. Pneumothorax developing under these circumstances is dealt with in a usual way: the pleura is sutured, and air is pumped out of the pleural cavity. The dog is immediately intubated, and artificial lung ventilation is started. Intubation may be performed before the operation. Our experience shows that this complication is rare and doesn't affect the transplantation outcome. No animals died during the surgery. The dogs were sacrificed in 15, 30, 90,180 and 270 days. The removed macro-specimens were examined with macroscopic, roentgenographic (Fig. 5) and histologic (Fig. 6) methods. Histologic specimens were stained with hematoxylin-eosin and according to van Gieson.

475

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/

/ t t

Fig. 5. X-rays of the ribs after allotransplantation. Autografts: (a) intact rib, (b) preserved in 0.5% formalin solution; (c) sterilised with gaseous ethylene oxide and preserved by freezing; (d) demineralised in 2.4 N HC1 solution; (e) "fresh", removed and transplanted during the operation.

:i

n .jgw

a.

•'*' c.

Fig. 6. Nine months after the operation. Histotopogramms of ribs with transplanted autografts: (a) sterilised with gaseous ethylene oxide and preserved by freezing at -20°C; (b) preserved in 0.5% formalin solution; (c) demineralised in 2.4 N HC1 solution; (d) "fresh", excised and transplanted during the operation.

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3. Results Our experiments on allotransplantation showed that in one month after the surgery, washed, de-proteinised allografts and autografts were seen in X-rays as shadows with distinct outlines, with the density approaching that of the host's ribs. The space between the ends of the grafts and the rib stumps was clearly identified. The demineralised graft could not be visualised at this time, but the osseous bed of the host demonstrated a marked periosteal reaction. Periosteal growth was also seen at the ends of the ribs with unsubstituted defects. In three months the washed graft looked less dense; its contours became irregular. The ends of the graft and the rib stumps were connected by periosteal bone callus. The shadow of the autograft (reference) was as dense as the host's rib. The ends of transplanted autologous bone were smooth; periosteal bone growth was clearly seen. In the place of the demineralised graft an osseous regenerate could be defined, the form and the structure of its shadow resembling the roentgenographic shadow of an intact rib. In the area of unfilled defects, end plates of sclerotic osseous tissue appeared on the rib stumps. Six months after the transplantation of demineralised bone and autologous tissue (reference) good osseous regenerates with an organotypic structure were formed. Roentgenographic and histologic studies showed active periosteal and endosteal bone formation, especially marked on the side of the pleural sheet of the periosteum. The demineralised graft was more intensely replaced than the autologous one (reference), the latter still demonstrating on histological specimens the presence of particles of old bone deprived of osteocytes in the bulk of the osseous regenerate. Bone formation, in response to washed grafts sterilised with ethylene oxide, was less marked. The weakest osteogenic reaction accompanied transplantation of deproteinised bone. The shadow of the graft endured resorption and fragmentation nearly along its whole length. No worthy osseous regenerate was formed. At the site of the defect left unfilled after the operation, there appeared a scar band joining the sclerotic rib stumps.

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In the second experimental series, the following stages of evolution of grafts and receiving beds were noted. On the 15th day, besides inflammatory changes, mild periosteal and endosteal reactions were observed — mainly on the side of the terminal areas of the receiving bed, as well as phenomena of osteoclastic graft resorption. Narrow fissures were seen between the grafts and the rib stumps. The demineralised graft was not visible in X-rays. By the 30th day primary osseous union appeared between the osseous bed and "fresh" (i.e., removed during the operation), frozen and formalinised grafts. Periosteal reparation was more marked than two weeks earlier, especially at the internal (pleural) surface of the grafts. The outline of their ends was not clearly seen in X-rays; histologically, newly formed osseous tissue of cancellous or lamellar character was defined in the area of the union. After 90 days, at the site of transplantation — predominantly from the side of the pleural surface — one could find organised osseous regenerates joining the ends of the rib stumps and incorporating remodelling grafts (Fig. 2). Autologous bone preserved in formalin needed more time for remodelling in comparison with frozen and "fresh" tissue. Demineralised bone demonstrated the most complete substitution by this time. At this term on histologic specimens of each regenerate, one could distinctly visualise a well pronounced cortical layer and an intramedullary cavity filled with a dense net of newly formed trabeculae with the presence of myeloid and fatty bone marrow. Fragments of old bone were found within the bulk of trabeculae. After 180 days roentgenographic and histologic examinations demonstrated further resorption of graft particles embedded in osseous tissue conjoining the ends of the resected ribs. By the 270th day practically complete anatomical restitution of rib integrity was achieved; their structure did not differ from intact ribs. The site of transplantation could be defined only by the presence of metallic rods. Histologically, in the depth of the new bone formed after formalinised graft transplantation, its small fragments enduring remodelling were still seen.

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4. Discussion The experiments have shown that demineralised rib fragments possess high bioplasic activity. In most of the animals, after a short time (3-6 months) after alloplasty there appeared new bone with an organotypic structure. After transplantation of frozen and formalinised grafts, nine to 12 or even more months were needed for complete restoration of rib integrity. Judging by the speed of new bone formation and remodelling after transplantation of both allogenic and autologous osseous tissue, demineralised grafts were the best, followed by frozen and formalinised material. But around frozen and formalinised grafts, as compared with demineralised ones, denser osseous tissue was always formed, although it happened much later. The results achieved in the present study allow us to evaluate the role of various components of osseous tissue in the processes of reparation. First of all, they show that the ground substance in autografts has a positive influence over transplantation outcomes in contrast to mineral elements, which, being present in transplants, hinder their assimilation. Resorption and utilisation of the mineral basis of the bone demand additional energetic and temporal expenditures on the part of the host's body. Comparing the results of morphologic examination of autologous and allogenic grafts preserved in one and the same way, one can note similarities as well as differences. Similarities consist in necrobiosis of the grafts; their infiltration with cellular elements; and resorption and synchronous (at best) substitution with newly formed osseous tissue. Differences are concerned both with reparation tempo, and quality of reparation. Studying morphologic remodelling of bone auto- and allotransplants, we came to the persuasion that the peculiarities of reparative osteogenesis depend in many respects upon the biological type of osseous tissue. Our findings showed that the reasons why reparation processes didn't proceed at a similar speed lay in the fact that they had important qualitative manifestations in their essence. Thus, a characteristic histologic

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feature of the early period following bone allotransplantation consisted in formation around the graft of fibrillar connective tissue without any participation of osteogenic cells of the receiving bed, which usually didn't occur in cases of autotransplantation. Besides that, one saw lesser activity of osteogenic elements of the osseous bed; weak endosteal bone formation; and prevalence of resorption processes over restorative activity, especially in the middle part of allografts. Allogenic and autologous transplantations differed also in such aspects as an increase in new osseous tissue amount, resorption speed of fatty bone marrow, mineral substances and collagen. The causes of these and many other differences are not yet known. It is interesting to point out that transplantation of demineralised autologous bone gave better results in comparison with allografts treated in the same way, although in both cases the transplantation material consisted mainly of the ground substance. Hence, it may be concluded that the host's body accumulates its own proteins at a greater speed, and that autologous protein possesses greater osteoinductive abilities in comparison with foreign matter. To our mind, these differences might be explained on the basis of either dissimilar antigen activity of allogenic and autologous collagen, or its structural (molecular) dissimilarity. Interesting findings were received in the process of comparative study of reparation processes after transplantation of autologous bone preserved by freezing, in weak solutions of formalin and by means of demineralisation. Before the start of the experiments it was supposed that these factors would cause certain biochemical and morphological changes in the graft, due to which the latter might be deprived of its advantages. But in reality the situation was different. An autologous osseous graft preserved by freezing at -20°C after being transplanted to its host, gave practically the same results as the one freshly produced during the operation. Better bioplastic qualities in comparison with allotransplantation were demonstrated by autologous bone kept in 0.5% formalin solution for three months, or treated

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with HC1. Truly, here as in earlier described cases, remodelling of formalinised grafts was slightly slower in comparison with frozen ones, and that of demineralised grafts, slightly faster. The experiments on autotransplantation lend another confirmation to the fact that viability of transplantation material was not the only, and moreover not the principal, prerequisite of success of grafting in clinical practice. Thus, with the help of the improved experimental model the following clinically important findings have been received: • Demineralised bone is a highly promising transplantation material suitable for clinical application. • The methods of biologic tissue preservation in 0.5% formalin solution and by freezing do not exclude each other from the point of view of their clinical application, but remodelling of formalinised bone is more slow. • Viability of isolated bone grafts has no decisive influence over transplantation outcomes. In conclusion it should be stated that the comparative evaluation of bioplastic characteristics of bone grafts based on the original experimental model has confirmed its definite usefulness and informational value. It allows us to eliminate the influence of immune, species-linked and other factors, to standardise in this way, experimental conditions, and to receive reproducible data. 5. References EINHORN, T.A., LANE, J.M., BURSTEIN, A.H., KOPMAN, C.R. and VIGORITA, V.J. (1984). The healing of segmental bone defects induced by demineralised bone matrix, /. Bone Joint Surg. 66-A, 274-279. SAVELIEV, V.I. (1967). Chemical sterilisation of tissue grafts and their usage in plastic surgery. Auto-abstract of M.D. Doctor Dissertation. Omsk, 30 p.

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SAVELIEV, V.I. (1971). Gaseous sterilisation of tissue grafts and a stationary appliance for this purpose, Ortopedia, Travmatologia i Protezirovanie 2, 76-78. SAVELIEV, V.I. (1978). An experimental model for bone graft comparative study. In: New Methods of Prevention, Diagnosis and Treatment in Orthopedic Diseases. Leningrad, pp. 136-141.

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SECTION V: CARDIOVASCULAR GRAFTS

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

20 CRYOPRESERVATION OF PORCINE AORTIC VALVE: OPEN STATUS OF THE AORTIC LEAFLETS RESULTS IN INCREASED MATRIX GLYCOSAMINOGLYCANS STRUCTURAL MAINTENANCE

LUCA DAINESE, GIANLUCA POLVANI, MARILENA FORMATO, 1 ANNA GUARINO and PAOLO BIGLIOLI Department of Cardiac Surgery - University of MilanItalian Homograft Bank (BIO) Centro Cardiologico Monzino, IRCCS Via Parea 4, 20138 Milan, Italy d e p a r t m e n t of Physiological, Biochemical and Cellular Sciences, University of Sassari Via Muroni 25, 07100 Sassari, Italy

1. Introduction In cryopreserved allograft valves, extracellular matrix damage may result in increased valve structural failure and calcification (Legare et ah, 2000; Mitchell, 1998). Processing conditions may adversely influence the integrity of the extracellular matrix in the cryopreserved aortic leaflets. Extracellular matrix is the main

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component of the flexion zone of the leaflet, and are submitted to the bending stresses that appear in vivo to be higher in diastole (Deck, 1988). This avascular matrix is semifluid, deformable and rich in glycosaminoglycans (GAGs) and collagen fibers synthetised and secreted by connective tissue cells. The tendency of such allografts to calcify following transplantation could be associated with changes in specific GAGs level and distribution (Shon, 1994). The present investigation was undertaken to study the effects of different cryopreservation procedures (referred to as "open" and "closed" position) on aortic leaflet glycosaminoglycan content and composition. 2. Materials and Methods Sixteen aortic porcine aortic valves were obtained from a local slaughterhouse immediately after sacrifice, and immediately placed into a transport medium (cold balanced salt solution — Eurocollins at 4°C). Mean cold ischemia time was 1 hour + 20 minutes. The aortic valves were dissected and placed in a small container in 100 ml of a low dose of antibiotic solution (RPMI 1640 with L-Glutamine tamponated to a pH of 7.2-7.4; cefoxitin 240/ig/ml, lincomycin 120/jg/ml, polymyxin BlOO/ig/ml, vancomycin 50 ^g/ml) for 24 hours at 4°C. Eight aortic valves were placed in closed position (group 1) and eight in open position (group 2) using a prolene 6-0 stiches in Hemofreeze Bag (Fig. 1) with cryopreservation solution (100 ml RPMI 1640 with L-Glutamine tamponated to a ph of 7.2-7.4 with 10% DMSO) than were cryopreserved in a programmed freezer (Kryo 10-16 series III, Planer London) that lowered the temperature + 1°C per minute down to a temperature of -80°C. Finally the allograft was mantained in liquid nitrogen vapours (-180°C). After 48 hours of storage, the specimens were thawed, throroughly rinsed with culture medium (100 ml RPMI 1640 with

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L-Glutamine tamponated to a ph of 7.2-7A ) at 4°C, deprived of the adventitia layer, and then analysed. Three different zones were cut and separately processed: aortic wall; zone of flexion of aortic valve; and its leaflet. After peeling and segregation of the three different types of tissue, their wet weight was determined. The methodology used for isolation and characterisation of GAGs includes delipidation, proteolytic digestion, anionexchange chromatography, ethanol precipitation and acetate cellulose electrophoresis. Tissues were fixed with 20 volumes of acetone at 4°C for 24 hours, and delipidated with 20 volumes of chloroform: methanol (2:1, volume/volume) at 4°C for 24 hours. The final defatted tissue was obtained by drying this material at 60°C. The final weight, called dry defatted tissue (DDT) weight, was about 32.671 ± 4.97%, 23.58 + 3.44% and 8.603 + 1.61% of the wet weight for aortic wall, zone of flexion and leaflet, respectively. 2.1. Extraction of total G A G s DDTs (100 mg) were rehydrated for 24 hours at 4°C in 0.1 mol/ L sodium acetate, pH 6.0, containing 5 mmol/L cysteine and 5 mmol/L EDTA. Papain (0.3 U / m g DDT) was then added to the mixture, which was incubated at 56°C for 24 hours under mild agitation. The digest was clarified by centrifugation (9,000 g, 20 minutes at 4°C) and the residue washed. 2.2. Purification of G A G s Digest supernatant and washing were combined and loaded on a DEAE-cellulose column (0.7 x 6 cm, 2.3 mL), equilibrated with 50 mmol/L sodium acetate, pH 6.0. The column was then washed with 50 mL of the same buffer and eluted with a twostep salt gradient (0.55 and 1.0 mol/L NaCl). Fractions of 1 mL were collected. These were assayed for hexuronate content by the method of Bitter and Muir (1972), using glucuronolactone as

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a standard. To minimise individual differences GAGs content was normalised to the corresponding aortic wall content. Fractions containing GAGs were pooled and precipitated with four volumes of absolute ethanol. The mixture was left overnight at 4°C, and the precipitate was separated by centrifugation, washed twice with ethanol and diethyl ether, and then dried. 2.3. Acetate cellulose electrophoresis GAGs composition was determined by discontinuous electrophoresis according to Cappelletti et al. (1979). GAGs identification was performed treating aliquots of the samples with specific eliminases, as previously described (Cherchi et al, 1994). The specificity and the efficiency of enzyme treatments were checked on standard GAGs under the same experimental conditions. GAGs composition was expressed as relative percentages by densitometry scanning of Alcian Blue stained srtrips performed with a Scan Analysis program (Thunder Scan-Biosoft). 2.4. Statistics Variables are reported as mean ± standard deviation total numbers and relative frequencies. For comparison groups, Student's T-test and the [chi]2 were used. A P < 0.05 was considered statistically significant. The were performed with SPSS Microsoft software.

(std) or between value of analyses

3. Results The aortic wall and the two leaflet selected areas under study were significantly different in their GAGs composition: electrophoretic data, combined with those from the degradation of GAGs with specific eliminases, indicate possible structural dissimilarities in GAGs chains depending on their topographic localisation.

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Anion-exchange chromatography on DEAE-cellulose of free GAGs allowed us to separate them on the basis of both their density charges. The qualitative nature of GAGs eluted by the two step chromatographic procedure of the three selected areas were quite different. GAGs from aortic wall eluting at a lower salt concentration (0.55 mol/L NaCl) consisted of hyaluronan (HA) and heparan sulfate (HS), while GAGs eluting at a higher salt concentration (1 mol/L NaCl) consisted of chondroitin 4 / 6 sulfate (CS) and dermatan sulfate (DS). Interestingly, GAGs from the zone of flexion of an aotic valve eluting at lower salt concentration, contained an additional component whose mobility was intermediate with respect to standard DS and CS. This faster migrating band contained material degradable by Condroitin-ABC and -AC lyases; but Streptotnyces hyaluroluticus Hyaluronidase resistant; therefore, it was identified as an undersulfated CS. GAGs from this area eluting with higher salt concentration showed the same composition of that from the aortic wall, also showing similar relative percentages of CS and DS. Table 1. Total aortic leaflet and leaflet flexion zone GAGs content (ug hexuronate/mg DDT). Leaflet O p e n position N = 8 mean Closed position N = 8 mean Test f

Leaflet flexion z o n e

4.595 ± 1.788

2.396 ±

0.47

5.95 + 2.3

1.671 +

0.34

Ns

0.03

Values are mean + std. Contents are given in micrograms of hexuronate per milligram of dry defatted tissue (DDT). Each value is normalised for aortic wall content. Total hexuronate content was calculated by adding the two-step chromatographic fraction contents. Each determination was performed in duplicate.

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Table 2. GAGs qualitative analysis (%) of aortic homograft leaflet flexion zone (± 1 standard deviation).

Open aortic valve Closed aortic valve

HS

HA

DS

CSs

CS

6.65 7.54

26.57 25.86

16.08 14.19

32.43 35.45

18.27 16.97

HS = heparn sulfate. DS = dermatan sulfate. HA = hyaluronan. CSs = slow chondroitin sulfate. CS = chondroitin sulfate. The GAGs electrophoretic patterns obtained from the leaflet were qualitatively similar to that obtained from its zone of flexion, but higher relative percentages of HA were detected. The most interesting result of this study was obtained from comparing the total GAGs content of the same areas under different cryopreservation procedures (Table 1). Our data suggest that cryopreservation in the closed position produces a significant reduction in GAGs level, although relative percentage remain unchanged (Table 2). 4. Discussion The aim of this study was to design an alternative approach to the preparation of homograft aortic valves that have better performance. Since loss or alteration in extracellular macromolecular components, such as GAGs, could affect the structural and functional integrity of the aortic valve homograft, and probably also modulating its calcific degeneration (Legare et al, 2000; Mitchell, 1998), we focused on the effects of different cryopreservation procedures on GAGs content and composition. The processes of antibiotic sterilisation and cryopreservation were found to maintain cellular viability of the heart valves that are structurally preserved at time of implantation (Legare et al,

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2000; Goffin et al, 1997; Mitchell et al, 1998; Shoen et al, 1999; Tominaga et al, 2000; O'Brien et al, 1987). Also, cellular viability and preservation of the collagen framework and intercellular matrix components seem to guarantee long term cryopreserved heart valve function by playing a role in the prevention of matrix mineralisation (Goffin et al, 1997; Kim, 1976). The collagen network provides the major structural basis for long term performance (Shoen et al, 1999), and cellular matrix is the main component of the stress area on the aortic leaflets' flexion zone. The aortic leaflet attachments to the aortic wall are subjected to different pressure stress in vivo during cardiac cycle, and the pressure stress is higher during diastole. This zone consists of avascular matrix which is mainly composed of concentrated GAGs (Deck et al, 1988; Shon et al, 1994; Mitchell et al, 1998; O'Brien et al, 1987). This matrix is semi-fluid, deformable and rich in proteoglycans (PGs), GAGs and collagen fibres synthetised and secreted by connective tissue cells (Deck et al, 1988). It is well known that content and distribution of PGs and GAGs throughout the blood vessel wall is variable (Whight, 1989) and could influence a number of physiological and pathological conditions of cardiac and vascular tissue (Cherchi et al, 1990; Klezovitch et al, 2000). Also, the tendency of cryopreserved allografts to calcify following transplantation could be associated with changes in specific GAGs level and distribution (Goffin et al, 1997; Mitchell et al, 1995; Schoen et al, 1999). The characterisation of GAGs from different areas of aortic valve homograft allows us to detect pronounced differences in GAGs distribution. The differences in GAGs composition between leaflet areas and the aortic wall indicate, not only the relative levels of each type of GAGs, but also, as in the case of CS, their sulphation degree, thus suggesting unusual biochemical pathways for some of these extracellular components in valve selected areas. The presence of undersulphated CS chains in the extracellular matrix of leaflets is not related to sterilisationcryopreservation-thawing treatments, as assessed by examining fresh porcine aorta valves (data not shown).

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The detection of undersulfated CS has never been described before in vascular tissue, and possibly it is a peculiarity of valve tissue. Therefore, further investigations are needed to structurally and functionally characterise this type of glycosaminoglycan, as well as to isolate the native proteoglycan carrying it. It has been reported that there is no significant alteration in the content, molecular size or distribution of PGs and GAGs in properly cryopreserved porcine aortic tissue (Shon et al, 1994). In contrast, our results suggest that in "closed" cryopreserved aortic valves at the level of the leaflet flexion zone there is a significant reduction in GAGs content compared with the same area cryopreserved in the "open" position. Some authors (Deck et al, 1988; Clark et al, 1971; 1974; Thubrikar et al, 1979; 1986) have reported that the valvular leaflet rotation, accomplished by folding the leaflet along the insertion axis to the aortic wall, indicates that the deformation required for the movement occurs in a limited area of the leaflet itself. This was considered to be related to the ultrastructure of the leaflets whose the main component is represented by a semi-fluid matrix enclosed between two fibrous tissue plates. Collagen microfibrils and the fibres in the fibrous plate are interconnected and suspended in a hydrated GAG-rich matrix that relieves the folding stress thanks to GAGs, synthesised and secreted quickly in the leaflet attachment area where there is higher protein turnover (Deck et al, 1988). The patterns of non-calcific structural damage in bioprosthetic valves are consistent with a molecular modification of collagen and a progressive and marked depletion of GAGs (Vyavahare et al, 1999). Both these structural deteriorations appear to be stress-induced in vitro (Vyavahare et al, 1999) and could act synergically with calcific degeneration in vivo (Armiger, 1998). In aortic valve homografts, the viable cells should have the potential to produce, repair and remodel their extracellular matrix. However, there is strong evidence for an incomplete restoration of cell functionality, and a progressive decline in cellularity (Mitchell et al, 1995; O'Brien et al, 1987; Armiger, 1998; Messier et al, 1999).

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Our results suggest that in 'closed' cryopreserved aortic valves, there is a significant reduction in GAG content at the level of the leaflet flexion zone compared with the same area of cryopreserved valve in the 'open' position. GAGs depletion could related either to a diffusion process (depending on the surface area and pressure gradient that is higher in a 'closed' valve), or to a sort of extraction effect (Messier et al, 1999). The nonselective removal of GAGs from a commissural matrix could be favoured by the absence of an intact endothelium, combined with the proteolytic action induced by cryopreservation. In fact, dead and dying cells in the allograft would certainly release lysosomal hydrolytic enzymes that alter extracellular macromolecules, including PGs and GAGs (Riddle et al, 1984). As reported by Schoen (1997) a decisive aortic leaflet mechanical element is represented by the extraordinary architecture formed by the extra-cellular fibril matrix subjected to continuous re-modelling by the connective cells. Several studies (Legare et al, 2000; Mitchell et al, 1998; Shon et al, 1994; Thubrikar et al, 1986) were carried out on the structural characteristics of leaflets and on their relationships with the functional properties of cells making up the valvular leaflets. Special attention is given to the presence of cells that make up the cusp interstitial matrix. It has been confirmed that aortic valvular leaflets can modify their structure as a function of haemodynamic stress, that there is a rearrangement process modulated by cellular components present in the extra-cellular matrix (Schoen et al, 1999) and that this responsiveness is essential to the vitality and durability of valvular cusps. The relevance of GAGs maintenance for valve functionality is confirmed by the findings of decreased levels in explanted bioprosthetic heart valves (Mako et al, 1997), as well as in rheumatic and aged valves (Baig et al, 1978; 1979; Torii et al, 1965). Moreover, to improve the mechanical properties of bioprosthetic valves, it has been recently proposed that chemical stabilisation of extracellular GAGs (Lovekmp et al, 2001) be performed. Also, in tissue-engineered valves, the structural

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integrity and biomechanical profile ultimately depend on proper extracellular formation, which has been demonstrated as predominantly GAGs (Lovekamp et al, 2001; Sodian et al, 2000; Hoerstrup et al., 2000). Interestingly, a new approach proposed to optimise replacement heart valve characteristics is an acellular, unfixed porcine aortic valve-based construct, in which the connective tissue "normal matrix" is completely preserved. The extracellular matrix integrity is proposed as the basis for recellularisation with host cells (Goldstein et al., 2000). Nowadays it is clear that the aortic valve is a "composite structure" able to change its elastic modulus as a function of stress. GAGs are important extracellular matrix components on which a functional valve depends. Quantitative and qualitative analysis of total GAGs from aortic cryopreserved valves in the open or closed position, suggest that alterations could be produced following the cryopreservation procedure. The "upside down cryopreservation" of aortic valves in the open position maintain a greater quantity of GAGs that are important for better preservation of extracellular matrix components. These findings could have important implications in terms of designing cryopreservation protocols to prolong homograft durability. 5. Summary 5.1. Purpose The present investigation was undertaken to study the effect of cryopreservation on glycosaminoglycans (GAGs) content on aortic leaflets cryopreserved in the closed and open positions. Mechanical properties of extracellular matrix are critically important to maintain the long term durability of allografts. 5.2. Methods and results Sixteen porcine aortic valves were obtained immediately after sacrifice. Mean cold ischemia time was 1 hour ± 20 minutes. The

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aortic valves were dissected and placed in a small containers in 100 ml of a low dose of antibiotic solution for 24 hours at 4°C. Eight aortic valves were cryopreserved in the closed position (groupl) and eight in the open position (group 2). After 48 hours of storage, the specimens were thawed, and three different zones were separately processed: aortic wall, leaflets flexion zone and leaflets. The methodology used for isolation and characterisation of GAGs includes delipidation, proteolytic digestion, anion-exchange chromatography, ethanol precipitation and acetate cellulose electrophoresis. The three selected areas under study were significantly different in their total GAGs content. The closed position produces a significant reduction in GAGs level in the leaflets' flexion zone. The qualitative nature of GAGs of the three selected areas were also different. The electrophoretic patterns obtained from the leaflet zone of flexion showed a higher relative percentage of hyaluronan (HA). 5.3. Conclusion Quantitative analysis of total GAGs from aortic valves suggests that alterations could be produced following the cryopreservation procedure. Electrophoretic data indicate structural dissimilarities in GAGs chains related to topographic localisation. Cryopreservation of the aortic valve in the open position can better preserve the cellular matrix of leaflets. 6. References AMIGER, L.C. (1998). Postimplantation leaflet cellularity of valve allografts: Are donor cells beneficial or detrimental?, Ann. Thorac. Surg. 66, 233-235. BAIG, M.M., DAICOFF, G.R. and AYOUB, E.M. (1978). Comparative study of acid mucopolysaccharide composition of rheumatic and normal heart valves in men, Circ. Res. 42(2), 271-275.

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BAIG, M.M. (1979). Acid mucopolysaccharides of congenitally defective, rheumatic, and normal human aortic valves, Am. J. Pathol. 96(3), 771-780. BITTER, T. and MUIR, H.M. (1972). A modified uroni acid carbazole reaction, Annal. Biochern. 4, 330-334. CAPPELLETTI, R., DEL ROSSO, M. and CHIARUGI, V.P. (1979). A new electrophoretic method for the complete separation of all known animal glycosaminoglycans in a monodimensional run, Annal. Biochern. 99, 311-315. CHERCHI, G.M., FORMATO, M., DEMURO, P. et al. (1994). Modifications of low density lipoprotein induced by the interaction with human plasma glycosaminoglycan-protein complexes, Acta Biochern. Biophys. 1212, 345-352. CHERCHI, G.M., COINU, R., DEMURO, P. et al. (1990). Structural and functional modifications of human aorta proteoglycans in atherosclerosis, Matrix 10, 362-372. CLARK, R.E. and BUTTERWORTH, G.A. (1971). Characterisation of the mechanics of human aortic and mitral valve leaflets, Surg. Forum 2, 134-139. CLARK, R.E. and FINKE, E.H. (1974). Scanning and light microscopy of human aortic leaflets in stressed and relaxed states, /. Thorac. Cardiovasc. Surg. 67(5), 792-804. DECK, D., THUBRIKAR, M.J., SCHNEIDER, P.J. et al. (1988). Structure, stress, and tissue repair in aortic valve leaflets, Cardiovasc. Res. 22, 7-16. GOFFIN, Y.A.H., DE GOUVEIA, R.H., SZOMBATHELYI, et al. (1997). Morphologic study of homograft valves before and after cryopreservation and after short-term implantation in patients, Cardiovasc. Pathol. 6, 35-42. GOLDSTEIN, S., CLARKE, D.R., WALSH, S.P., BLACK, K.S. and O'BRIEN, M.F. (2000). Transpecies heart valve transplant:

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Advanced studies of a bioengineered xeno-autograft, Ann. Thorac. Surg. 70, 1962-1969. HOERSTRUP, S.P., SODIAN, R., DAEBRITZ, S.7 WANG, J., BACHEA, E.A., MARTIN, D.P., MORAN, A.M., GULESERIA, K.J., SPERLING, J.S., KAUSHAL, S., VACNTI, J.P., SCHOEN, F.J. and MAYER, J.E. (2000). Functional living trileaflet heart valves grown in vitro, Circulation 102(suppl. Ill), 111-44—111-49. KIM, K.M. (1976). Calcification of matrix vescicles in human aortic valve and aortic media, Federation Proc. 35, 156-162. KLEZOVITCH, O., FORMATO, M., CHERCHI, G.M. et al. (2000). Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan, /. Biol. Chem. 275, 18913-18918. LEGARE, F.J., LEE, T.D.G. and ROSS, D.B. (2000). Cryopreservation of aortic valves results in increased structural failure, Circulation 102(suppl. Ill), 75-78. LOVEKAMP, J. and VYAVAHARE, N. (2001). Periodate-mediated glycosaminoglycan stabilisation in bioprosthetic heart valves, /. Biomed. Mater. Res. 51, 478-486. MAKO, W.J., CALBRO, A., RATLIFF, N.B. and VESELY, I. (1997). Loss of glycosaminoglycans (GAGs) from implanted bioprosthetic heart valves, Circulation 95, 1-155. MESSIER, R.H., BASS, B.L., DOMKOWSKI, P.W. and HOPKINS, R.A. (1999). Interstitial cellular and matrix restoration of cardiac valves after cryopreservation, /. Thorac. Cardiovasc. Surg. 118, 36-49. MITCHELL, R.N., JONAS, R.A. and SHOEN, F.J. (1995). Structure-function correlations in cryopreserved allograft cardiac valves, Ann. Thorac. Surg. 60, 108-113. MITCHELL, N.R. (1998). Pathology of explanted cryopreserved allograft heart valves: Comparison with aortic valves from

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orthotopic heart transplants, /. Thorac. Cardiovasc. Surg. 115, 118-127. O'BRIEN, M.F., STAFFORD, E.G., GARDNER, M.A.H. et al. (1987). A comparison of aortic valve replacement with viable cryopreserved allograft valves, with a note on chromosomal studies, /. Thorac. Cardiovasc. 94, 812-823. O'BRIEN, M.F., STAFFORD, E.G., GARDNER, M.A.H. et al. (1987). The viable cryopreserved allograft aortic valve, /. Card. Surg. 2(suppl), 153-167. RIDDLE, J.M., JENNINGS, J.L., STEIN, P.D. et al. (1984). A morphological overview of the porcine bioprosthetic valve before and after its degeneration, Scanning Electron Microscopy 1, 207-214. SHOEN, F.J. (1997). Editorial: Aortic valve structure-function correlations: Role of elastic fibers no longer a stretch of the imagination, /. Heart Valve Dis. 6, 1-6. SCHOEN, F.J. and LEVY, R.J. (1999). Tissue heart valves: Current challenges and future research perspectives, /. Biomed. Mater. Res. 47, 439-465. SCHOEN, F.J. (1991). Pathology of bioprostheses and other tissue heart valve replacements. In: Cardiovascular Pathology, 2nd Ed., M.D. Silver, ed., Churchill Livingstone, New York, pp. 15471605. SHON, Y.H. and WOLFINBARGER, L. Jr. (1994). Proteoglycan content in fresh and cryopreserved porcine aortic tissue, Cryobiology (Apr), 31(2), 121-132. SODIAN, R., HOERSTRUP, S.P., SPERLING, J.S., DAEBRITZ, S., MARTIN, D.P., MORAN, A.D., KIM, B.S., SCHOEN, F.J., VACANTI, J.P. and MAYER, J.E. (2000). Early in vivo expedience with tissue-engineered trileaflet heart valves, Circulation 102(suppl. Ill), III, 22-29.

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THUBRIKAR, M., BOSHER, P. and NOLAN, S.R (1979). The mechanism of opening of the aortic valve, J. Thorac. Cardiovasc. Surg. {77), 6, 863-870. THUBRIKAR, M., AOUAD, J. and NOLAN, S.P. (1986). Comparison of the in vivo and in vitro mechanical properties of aortic valve leaflets, J. Thorac. Cardiovasc. Surg. 92, 29-36. TOMINAGA, T., KITAGAWA, T., MASUDA, Y. et al. (2000) Viability of cryopreserved semilunar valves: An evaluation of cytosolic and mitochondrial activities, Ann. Thorac. Surg. 70, 792-795. TORII, S., BASHEY, R.I. and NAKAO, K. (1965). Acid mucopolysaccharide composition of human-heart valve, Acta Biochem. Biophys. 101(3), 285-291. VYAVAHARE, N., OGLE, M., SCHOEN, F.J., ZAND, R., GLOECKNER, D.C., SACKS, M. and LEVY, R.J. (1999). Mechanisms of bioprosthetic heart valve failure:fatigue causes collagen denaturation and glycosaminoglycan loss, /. Biomed. Mater. Res. 46, 44-50. WHIGHT, T.N. (1989). Cell biology of arterial proteoglycans, Arteriosclerosis 9, 1-20.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

21 PATHOLOGIC CHANGES OF THE CRYOPRESERVED CAROTID ARTERY AND JUGULAR VEIN IMPLANTED AT THE CANINE CAROTID ARTERY

HAN-KI PARK, YOUNG-HWAN PARK, CHEE-SOON YOON, 1 SHI-HO KIM,2 SAM-YOON LEE,3 SANG-HYUN LIM, JONG-HOON KIM, JONG-CHUL PARK, DONG-WOOK HAN, SANG-HO CHO and BUM-KOO CHO Yonsei Cardiovascular Research Institute, Cardiovascular Hospital, Yonsei University College of Medicine, Seoul, 2 Konyang University College of Medicine, Daejun, 2 Donga University College of Medicine, Busan, 3 Wonkwang University College of Medicine, Iksan, Korea

1. Introduction Patients who do not have sufficient autologous vascular graft material, will, when they have to take re-operations for coronary artery or peripheral artery occlusive diseases, need another source of vascular grafts. Prosthetic vascular grafts that are less than 6 mm in diameter are commercially available now, but their patency rate is not satisfactory. Recently, great advances 501

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has been achieved for development of tissue engineered vascular grafts, but more research is required before they can be applied clinically. Homografts can be used in those cases. However, fresh homograft is not always available. Cryopreservation provides long-term preservation of human tissue and guarantees relatively good cell viability of the tissue. Cryopreserved human arteries and veins can be used in treatment of patients who do not have sufficient autologous graft material. To evaluate the adequacy of cryopreserved artery and vein as a small diameter vascular graft, we studied the cellular viability of cryopreserved and fresh vascular grafts, and the pathologic changes after implantation in a canine model. 2. Materials and Methods Four types of vascular allografts (cryopreserved carotid artery, fresh carotid artery, cryopreserved jugular vein, and fresh jugular vein) were implanted into the canine carotid artery, and pathologic changes were studied after one, two and three months. 2.1. Experiment animal Sixteen adult mongrel dogs weighing from 15 to 20 kg were used. Four dogs were used as carotid artery and jugular vein donors for cryopreservation. Twelve dogs received arterial (n = 6) or venous graft (n = 6) implantation into bilateral carotid arteries. Because fresh jugular vein grafts were harvested from arterial grafts of the recipient dogs, those dogs lose one jugular vein. We observed for development of neurological deficit for a week after harvesting of one side jugular vein. We proceeded with the experiment, because these dogs showed no definite neurologic deficit. For the experiment, anesthesia was induced by intravenous injection of ketamine (5 mg/kg) and xylazine (0.2-1 mg/kg), and

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maintained with halothane inhalation. The experimental protocols were concordant with the Guidelines and Regulations for Use and Care of Animals in Yonsei University. 2.2. Procurement and cryopreservation of carotid artery and jugular v e i n The carotid arteries and jugular veins for cryopreservation were harvested in sterile conditions. The harvested jugular veins and carotid arteries were prepared into the required 3 to 4 cm in length. The harvested arteries and veins were submerged in the medium (RPMI-1640/ Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) containing the antibiotics (Cefoxitin, 240/ig/ml; Lincomycin, 120/ig/ml; Polymyxin, 100/ig/ml; Vancomycin, 50 /xg/ ml) at 4°C for 12 hours. The media was replaced with freezing solution, consisting of RPMI-1640 with 10% fetal calf serum and 10% dimethylsulfoxide (DMSO). The arteries and veins in this solution were transferred into plastic bags and were chilled to -80°C in a computerised freezer, by decreasing the temperature at 1°C per minute. The frozen arteries and veins were stored in vapours of liquid nitrogen at -196°C for one month before implantation. 2.3. Implantation of arteries and v e i n s The cryopreserved arteries and veins were thawed in saline solution at 37°C. The cryoprotectant solution was then removed by dilution with 0.5, 0.25, and 1.0 mol/L solutions of DMSO in DMEM. Each animal was operated on as one of a pair. We performed graft implantation into two dogs at the same time, so that fresh allograft artery and vein harvested from Animal I and II could be transplanted from one to the other. In each pair, the dogs were randomly assigned as I and II. A unilateral jugular vein was harvested from Animal I, and a carotid artery was harvested

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from Animal II for fresh allografts. Then, Animal I received implantation of the prepared cryopreserved artery and fresh artery grafts on its right and left carotid artery. Animal II received venous grafts in the same manner. As a result, six dogs received arterial grafts, and the other six dogs received venous grafts. Before the vessels were clamped, heparin (100 unit/kg) was injected. Anastomosis between the graft and native carotid arteries was performed by continuous stitches of Prolene 6/0. Antibiotics were administered intravenously for a week postoperatively. No immunosuppressive medication was used. One, two and three months later, two dogs with arterial grafts, and two dogs with vein grafts, were sacrificed, and the implanted grafts were retrieved for pathologic evaluation. 2.4. Cellular viability test Just before implantation, we took some samples from the grafts and investigated the viability of endothelial cells and whole cells dissociated from the arteries and veins. The Griffonia simplicifolia agglutins-duorescein isothiocyanate (GSA-FITC) and propidium iodide (PI) double staining method in combination with flowcytometry, were employed to determine the viability of endothelial cells and whole cells (Park, 2000). Both endothelial and whole cellular viability of fresh vascular allografts was higher than that of cryopreserved ones. (Table 1, Fig.l) 2.5. Pathologic evaluation The explanted grafts were fixed in 10% buffered formaldehyde and embedded in paraffin. A pathologist examined the stained sections of the explanted grafts under the microscope. Hematoxylin-eosin, trichrome, and van Gieson elastin stains were used. The patency and the luminal narrowing of the grafts were measured. Inflammatory cell infiltration in vascular wall, intimal

505

Pathologic Changes of the Cryopreserved Carotid Artery

Table 1. Cell viability (%). Vein

Artery

Duration

Fresh

Cryopreserved

Fresh

Cryopreserved

Whole Endothelial Whole Endothelial Whole Endothelial Whole Endothelial cell cell cell cell cell cell cell cell 3 months

79.4 73.8

53.7 54.3

95.9 97.6

93.0 92.5

68.2 70.6

56.4 57.9

89.0 95.5

85.8 92.3

2 months

76.3 88.8

65.5 75.3

90.3 93.8

89.4 91.0

78.8 85.2

70.1 77.1

92.7 93.0

90.2 80.7

1 months

79.5 80.7

75.8 78.3

86.1 97.6

80.0 96.3

80.6 82.1

66.5 81.7

90.1 95.8

83.3 91.5

Mean

79.8 ±5.1

67.2 ±11.1

93.5 ±4.6

90.4 + 5.6

77.6 ±6.7

68.3 ±10.1

92.7 + 2.8

87.3 ±4.8

1UU.U]

P = .0006 H

90.0"

.0002

P = 002 P-

80 0" >. 70.01 60.0> =3 so.ou 40.0' 30.O 20.0 IO.O-

nrv WholeCell

Endothelial Cell

Whole Cell

Artery

Endothelial Cell Vein

| ~ | Cryopreserved

• Fresh Fig. 1. Cell viability test.

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Fig. 2, Microscopic finding (xlOO) of fresh (left) and thawed cryopreserved canine carotid artery (right). The structure of the arterial wall is the same as that of the normal artery.

Table 2. Luminal narrowing of transplanted carotid artery and jugular vein at different time intervals after transplantation. Duration of implantation

Transplanted graft

Animal I

Animal II

1 month

Cryopreserved artery Fresh artery Cryopreserved vein Fresh vein

Patent Patent Occluded 80% stenosis

90% stenosis 60% stenosis Occluded 80% stenosis

2 months

Cryopreserved artery Fresh artery Cryopreserved vein Fresh vein

Occluded Patent Occluded 20% stenosis

Patent Patent 10% stenosis 10% stenosis

3 months

Cryopreserved artery Fresh artery Cryopreserved vein Fresh vein

Patent Patent 50% stenosis 20% stenosis

Occluded 20% stenosis 50% stenosis 50% stenosis

Pathologic Changes of the Cryopreserved Carotid Artery

507

hyperplasia and any significant abnormalities were evaluated and compared with a normal carotid artery. (Fig. 2) 3. Results 3.1. Patency of grafts The overall patency rate of vascular allografts was poor. Among 24 vascular allografts implanted for up to three months, five grafts were totally occluded, and seven grafts had 50% or higher luminal narrowing (Table 2). Among the six specimens of cryopreserved arterial grafts, three were obstructed with thrombi, and the others showed patent lumen. Just one of six fresh artery grafts showed 60% obstruction of lumen, and the others were patent. No fresh venous grafts were totally obstructed. Three of them showed luminal narrowing over 50%, and all of them showed various degrees of lymphocyte infiltration. Cryopreserved venous grafts showed the worst luminal patency among the four kinds of grafts. Three of six grafts showed total obstruction, and the others showed significant luminal narrowing due to thrombosis and intimal hyperplasia. The patency rate was affected by the type and preservation methods of the grafts. The patency of a fresh graft was better than that of cryopreserved grafts. The vein graft showed better patency than the arterial graft. Fresh arterial grafts showed the best patency rate, while cryopreserved venous grafts showed the worst patency rate. During the observation period of three months, the patency rate did not increase. 3.2. Lymphocyte infiltration More diffuse and dense lymphocyte infiltration in the vascular wall was observed in fresh artery and vein grafts than cryopreserved ones. The lymphocyte infiltration was most

H.-K. Park et al.

508

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i ^ _^5rwf ' "" \"

Fig. 3. Microscopic finding (xlOO) of an implanted cryopreserved allograft carotid artery at one month (upper), two months (middle), and three months (lower). (Upper left) There were no specific findings except adventitial fibrosis. Lumen was patent. (Upper right) Lumen was narrowed 90% by old thrombus. A little infiltration was found. (Middle left) The lumen was completely obstructed by old organised fibrotic thrombosis. Calcification was found. The hemosiderin was infiltrated mainly in media (yellow colour). (Middle right) Lymphocyte infiltration was mild in media and adventitia. Mild smooth muscle degeneration was found. The lumen (X200) was patent. (Lower left) The lumen (X50) was patent as dilated but within normal levels. There was no abnormal finding in HE, Elastic Trichrome stain. There was no significant finding but lymphocytes were infiltrated in intima, media and adventitia. (Lower right) The lumen was obstructed by organised fibrous thrombosis (95%), lymphocyte infiltration and media's elastic layer were normal.

509

Pathologic Changes of the Gyopreserved Carotid Artery

prominent in the specimen after one month of implantation, and decreased as duration from implantation increased. However, there was no significant difference in the amount of lymphocyte infiltration between arterial and venous grafts. (Figs. 3-6)

it

-if ^

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•

^

.

t*rv^.^

Fig. 4. Microscopic finding (X100) of an implanted fresh allograft carotid artery at one month (upper), two months (middle), and three months (lower). (Upper left) Lumen was patent, and there was no thrombus. The lymphocyte was diffusely infiltrated in media and adventitia. (Upper right) The lumen was obstructed 60% by the organised thrombus. (Middle left) The lumen was patent as normal. Endothelial cells were found in moderate numbers, and smooth-muscle degeneration in media was found. (Middle right) The findings were normal and the lumen was patent. (Lower left) We can find very focal intimal hyperplasia (mild). The lumen was patent. (Lower right) Recent thrombosis and mild intimal hyperplasia was found eccentrically. The lumen was narrowed 20% and there was no thrombus.

H.-K. Park et al.

510

W-

=*»•• •

\^

^

.if

Fig. 5. Microscopic finding (xlOO) of an implanted cryopreserved allograft jugular vein at one month (upper), two months (middle), and three months (lower). (Upper left) The lumen was almost closed by recent and old thrombus. The lymphocyte was infiltrated in media and adventitia. (Upper right) The lumen was completely obstructed by old and recent thrombus. Minor calcification and lymphocyte infiltration were found in media and adventitia. (Middle left) The lumen was completely obstructed. We found old organised fibrotic thrombus with calcification. The vessel wall was normal. (Middle right) The lumen was narrowed 10% by recently formed thrombus. The abscess was found in adventitia but media was normal. (Lower left) The lumen was narrowed 50% but patent. Because intimal hyperplasia occurred eccentrically and the recent organising thrombosis was formed. The elastic tissue in media was normal. There was no elastic fibre and collagenous fibrous tissue in the intimal hyperplasia and the thrombosis. (Lower right) The lumen was narrowed 50%. The lumen was patent. The lesion resulted from circumferential thrombosis and intimal hyperplasia. The fibrous tissue was proliferated.

511

Pathologic Changes of the Cryopreserved Carotid Artery

%

•"*W

4?? r

Fig. 6. Microscopic finding (xlOO, x200) of an implanted fresh allograft jugular vein at one month (upper), two months (middle), and two months (lower). (Upper left) The lumen was almost closed 80% by recent and old thrombus. The lymphocyte was moderately infiltrated in media and adventitia. Calcification was also found. (Upper right) The lumen was closed 80% by recent thrombus. Lymphocyte infiltration was moderate. (Middle left) The vessel was dilated and recently formed thrombus was found as 20% narrowed lumen. The vessel was normal. (Middle right) The lumen was narrowed 10%. There was recently formed thrombus and abscess in adventitia. The media was normal. The endothelial cell was not found. (Lower left) The lumen was narrowed 20% but patent. The vessel was dilated. (Lower right) The lumen was narrowed 50% but patent.

512

H.-K. Park et al.

3.3. Intimal hyperplasia Various degrees of intimal hyperplasia were found in the luminal surface of cryopreserved and fresh vascular grafts in three months, and this contributes to the luminal narrowing. However, this feature was not found in the specimen of one month. There was no definite difference in intimal hyperplasia among different types of grafts. (Figs. 3-6) 3.4. Other abnormal microscopic findings Focal calcification was found in some specimens, but this finding was not related to the type of grafts and duration of implantation. 4. Discussion If patients do not have sufficient autologous vascular graft material, when they have to take re-operations for coronary artery or peripheral artery occlusive diseases, they need another source of vascular grafts. Prosthetic vascular grafts that are less than 6 mm in diameter are commercially available now, but their patency rate is not satisfactory. Recently, great advances has been achieved for the development of tissue engineered vascular grafts, but more research is required before results can be applied clinically. Homografts can be used in those cases, but fresh homograft is not always available. Cryopreservation provides long-term preservation of human tissue, and guarantees relatively good cell viability of the tissue. Cryopreserved human arteries and veins can be used in treatment of patients without sufficient autologous graft material. However, the clinical outcome of cryopreserved saphenous vein grafts as a conduit for coronary artery bypass grafting or limb salvage surgery, is still unsatisfactory. (Gelbfish, 1986; Laub et al., 1992; Posner et al., 1996) Our experiment showed similar low patency for the cryopreserved vein graft. Among the six

Pathologic Changes of the Cryopreserved Carotid Artery

513

cryopreserved jugular vein grafts, five were totally obstructed or showed significant luminal narrowing. For cryopreserved arterial grafts, the patency rate was higher than that of cryopreserved vein. Cryopreserved arterial grafts are being used for peripheral arterial bypass grafting, and provide a safer and more effective treatment for mycotic aneurysms and infected vascular pros theses than conventional prostheses. (Vogt et al, 1998) Many clinical reports prove the effectiveness of homograft implantation for eradication of infection in patients with infective endocarditis. (Lytle, 2002; Vogt and Turina, 1997; Sabik et al, 2002) The architecture of the artery and vein is not changed by cryopreservation. This finding is consistent with other experiments (Brockbank, 1990). Overall patency of fresh artery and vein graft was better than that of cryopreserved artery and vein. And the cellular viability of fresh vascular allograft was higher than that of cryopreserved ones. With this result, we assume that endothelial cells may play an important role in maintaining the patency of implanted vessels. It was demonstrated that loss of anticoagulant activity complicates the problems of the exposure of a thrombogenic subendothelial matrix to blood in implanted cryopreserved veins. (Bambang et al, 1995) Even though fresh allografts had a higher patency rate, regarding the immunologic reaction, the fresh arterial and venous graft demonstrated unfavourable results with higher mononuclear cell infiltration. Cryopreserved allografts showed less infiltration of immunocompetent cells. This supports the conclusion that fresh allografts are more antigenic than cryopreserved grafts. It is reported that vein allograft failure is in part mediated by rejection. (Carpenter and Tomaszewski, 1997) The fact that patency of cryopreserved vascular graft was improved by immunosuppression confirms the role of immune reaction in the graft failure (Carpenter et al., 1997; Deaton et al., 1992; Posner et al., 1996). It was demonstrated that homograft valves rapidly lose their cellular components and normal tissue architecture (Koolbergen et al, 2002). This also indicates the possible immunogical reaction on the homografts. Methods to reduce the

514

H.-K. Park et al.

antigenicity without deterioration of the endothelial cell function should be developed. According to the result of this short-term animal experiment, the patency of the fresh artery graft was higher than that of cryopreserved arteries. However, fresh artery induces more immune cell infiltration in its vascular wall, so long-term animal experiment is required to demonstrate the long-term patency rate. Even though fresh artery has superior long-term patency, it has a limitation to availability when patients need allografts. This is why cryopreserved grafts still have a role to play. This study does have some limitations. The results showed relatively consistent pathologic findings for the different types of graft, but the number of experimental animals was too low to reveal the statistical difference between the cryopreserved and fresh graft, and between artery and vein. Larger studies are necessary to confirm these results. And the duration of implantations in this experiment is not long enough for the evaluation of chronic immune reaction. In conclusion, the patency of cryopreserved arterial and venous allografts is lower than that of fresh arterial and venous grafts. When cryopreserved arteries and veins are compared, the artery seems to be a better allograft for a small diameter vascular conduit. Fresh vascular allografts were more antigenic than cryopreserved grafts, and this may be related to the viability of endothelial cells. If the role of the endothelium in transplantation of cryopreserved allografts is clearly identified, and on this basis, if the patency rate is improved, then cryopreserved vascular grafts will provide another option for doctors who need vascular grafts for patients. 5. Summary To evaluate the adequacy of the cryopreserved artery and vein as a small diameter vascular graft, we studied the cellular viability of cryopreserved and fresh vascular grafts, and the pathologic changes after implantation in a canine model.

Pathologic Changes of the Cryopreserved Carotid Artery

515

Cryopreserved carotid artery and fresh artery were implanted at the carotid artery, and cryopreserved jugular vein and fresh vein were implanted at the carotid artery in each dog. The grafts were pathologically studied before and after one, two and three month later of implantation. The architecture of artery and vein was not changed by cryopreservation method only. The cryopreserved artery and vein showed lower a patency rate than fresh artery and vein. Arterial graft patency was higher than that of venous grafts in both cryopreserved and fresh grafts. Lymphocyte infiltration was more prominent in fresh grafts than cryopreserved grafts. After implantation, intimal proliferation progressed as duration from implantation increased. In conclusion, the patency of cryopreserved arterial and venous allografts is lower than that of fresh arterial venous grafts. Fresh vascular allografts were more antigenic than cryopreserved grafts. When cryopreserved arteries and veins are compared, the artery seems to be a better allograft for a small diameter vascular conduit. If the role of the endothelium in transplantation of cryopreserved allografts is clearly identified, and on this basis, if the patency rate is improved, the cryopreserved vascular graft will provide another option for the doctors who need vascular grafts for patients. 6. A c k n o w l e d g e m e n t This study was supported by a grant (HMP-98-G-2-052) of the HAN (Highly Advanced National) Project, Ministry of Health & Welfare, R.O.K. 7. Reference BAMBANG, L.S., MAZZUCOTELLI, J.P. MOCZAR, M , BEAUJEAN, F. and LOISANCE, D. (1995). Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells, /. Thorac. Cardiovasc. Surg. 110, 998-1004.

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BROCKBANK, K.G.M., DONOVAN, T.J., RUBY, S.T., CARPENTER, J.F., HAGEN, P.O. and WOODLEY, M.A. (1990). Functional analysis of cryopreserved veins, Preliminary report, /. Vase. Surg. 11, 94-102. CARPENTER, J.P. and TOMAZEWSKI, J.E. (1997). Immunosuppression for human saphenous vein allograft bypass surgery: A prospective randomised trial, /. Vase. Surg. 26, 32-42. DEATON, D.W., STEPHENS, J.K., KARP, R.B., GAMLIEL, H., ROCCO, F., PERELMAN, M.J., LIDDICOAT, J.R., GLICK, D.B. and WATKINS, C.W. (1992). Evaluation of cryopreserved allograft venous conduits in dogs, /. Thorac. Cardiovasc. Surg. 103, 153-162. GELBFISH, J., JACOBOWITZ, I.J., ROSE, D.M., CONNOLY, M.W., ACINAPURA, A.J., ZISBROD, Z., LIM, K.H., CAPPABIANCA, P. and CUNNINGHAM, J.R. (1986). Cryopreserved homologous saphenous vein: Early and late patency in coronary artery bypass surgical procedures, Ann. Thorac. Surg. 42, 70-73. KOOLBERGEN, D.R., HAZEKAMP, M.G., HEER, E., BRUGGEMANS, E.F., HYUSMANS, H.A., DION, R.A.E. and BRUIJIN, J.A. (2002). The pathology of fresh and cryopreserved homograft heart valves: An analysis of 40 explanted homograft valves, /. Thorac. Cardiovasc. Surg. 124, 689-697. LAUB, G.W., MURALIDHARAN, S., CLANCY, R., ELDREDGE, W.J., CHEN, C , ADKINS, M.S., FERNANDEZ, J., ANDERSON, W.A. and MCGRATH, L.B. (1992). Cryopreserved allograft veins as alternative coronary artery bypass conduits: Early phase results, Ann. Thorac. Surg. 54, 826-831. LYTLE, B.W., SABIK, J.F., BLACKSTONE, E.H., SVENSSON, L.G., PETTERSSON, G.B., and COSGROVE III, D.M. (2002). Reoperative cryopreserved root and ascending aorta replacement for acute aortic prosthetic valve endocarditis, Ann. Thorac. Surg. 74, 1754s-1757s.

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PARK, J.-C, SUNG, H.-J., LEE, D.H., PARK, Y.H., CHO, B.K. and SUH, H. (2000). Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow cytometry, Artif. Organs 24, 829-833. POSNER, M.P., MAKHOUL, R.G., ALTMAN, M., KIMBALL, P., COHEN, N., SOBEL, M., DATTILO, J. and LEE, H.M. (1996). Early results of infrageniculate arterial reconstruction using cryopreserved homograft saphenous conduit (CADVEIN) and combination low-dose systemic immunosuppression, /. Am. Coll. Surg. 183, 208-216. SABIK, J.F., LYTLE, B.W., BLACKSTONE, E.H., MARULLO, A.G., PETTERSSON, G.B. and COSGROVE, D.M. (2002). Aortic root replacement with cryopreserved allograft for prosthetic valve endocarditis, Ann. Thorac. Surg. 74, 650-659. VOGT, P.R. and TURINA, M.I. (1997). Management of infected aortic grafts; development of less invasive surgery using cryopreserved homografts, Ann. Thorac. Surg. 67, 1986-1989. VOGT, P.R., ROCCA, H.B., CARREL, T., SEGESSER, L.K., RUEF, C , DEBATIN, J., SEIFERT, B., KIOWSKI, W. and TURINA, M.I. (1998). Cryopreserved arterial allografts in the treatment of major vascular infection: A comparison with conventional surgical techniques, /. Thorac. Cardiovasc. Surg. 116, 965-972.

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

22 CELLULAR THERAPY FOR HEART FAILURE: A REVIEW OF SKELETAL MYOBLAST TRANSPLANTATION INTO INFARCTED MYOCARDIUM

DORIS A. TAYLOR* *Center for Cardiovascular Repair University of Minnesota BSBE 7-105 312 Church Street 5E Minneapolis, MN 55455 SITARAM EMANI, MATTHEW ELLIS and RICHARD B. THOMPSON Departments of Medicine and Surgery Duke University Medical Center Box 3345, DUMC Durham, NC 27710

1. Introduction As cellular and tissue engineering strategies progress, we continue to search for the best way to address the clinical syndrome of congestive heart failure (CHF). One of the most promising areas of recent research is cellular transplantation to repopulate injured myocardium. With the publication of French surgeon Philippe Menashe's results involving transplantation of autologous skeletal myoblasts into infarcted myocardium in a 519

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patient undergoing simultaneous coronary artery bypass, cellular therapy for heart failure has taken the all-important step from bench to clinic (Menasche et al, 2001). Cell transplantation has become a reality, and patients should, if it proves safe and efficacious, at last be able to benefit from 15 years of intensive research in this field. Medical options for the treatment of acute myocardial infarction and congestive heart failure have been developed and refined over the past several decades. Yet, only a few have shown a significant impact, and even then only in selected patients. Medical treatment includes the use of beta-adrenergic receptor blockers, angiotensin converting enzyme inhibitors, and diuretics. In end-stage clinical settings, adrenergic agonists and peripheral vasodilators such as dobutamine, milrinone, and digoxin are often used. These treatments are temporising, and only prevent further progression of heart failure. Furthermore, these current medical regimens only symptomatically treat CHF; none address the underlying pathology that leads to decreased myocardial performance. Patients who are refractory to medical management receive surgical intervention. The only treatment currently available that allows for near normalisation of patient activity is cardiac transplantation. Yet, patients with severe CHF may have to wait several years prior to receiving definitive treatment with this modality, due primarily to scarcity of donor organs. After the transplant, all patients are burdened with the need for longterm immunosuppression. Despite these intensive regimens of immunosuppression, all grafts eventually succumb to chronic rejection. As a bridge to cardiac transplantation, left ventricular assist devices are implanted in a subset of patients. However, these devices are invasive, often require long-term hospitalisation, and in general are not used for a long period of time. Given the limitations of the current therapeutic repertoire, many investigators have been evaluating the use of novel cellular and molecular means for heart failure therapy. One such potential

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therapeutic modality is the use of autologous skeletal myoblasts to repopulate the injured myocardium (Chiu et at, 1995). 2. History Using differentiated, intact skeletal muscle to improve myocardial performance is not a new idea. In fact, in the early 1980s, preclinical data were gathered demonstrating auto transplantation of skeletal muscle grafts onto myocardium (Sola et at, 1996). Similarly, both preclinical and clinical data have been obtained in the past 15 years with dynamic cardiomyoplasty, in which skeletal muscle is wrapped around the myocardium; and aortomyoplasty, in which skeletal muscle is wrapped around the aorta (Acker, 1999; Ninami et at, 1996; Lee et at, 1991; Kass et at, 1995). In addition, essentially parallel skeletal muscle ventricles have been constructed which can perform cardiac-like work without direct myocardial contact (Ninami et at, 1996). Of the techniques using differentiated skeletal muscle to augment cardiac function, dynamic cardiomyoplasty has been the most widely studied. The latissimus dorsi muscle is used to encircle the heart and provides an extrinsic source of cardiac work. Initial reports in selected patients with congestive heart failure demonstrated symptomatic improvement as measured by the NYHA class. However, this symptomatic improvement has not consistently been associated with improvements in either myocardial performance or survival rate (Acker, 1999). Several studies have shown some improvement in myocardial remodeling and wall stress in animal models; however, improvements in ejection fraction or contractility have not been definitively shown (Lee et at, 1991; Kass et at, 1995). Although the results have been disappointing, pre-clinical evaluations of skeletal muscle ventricles and dynamic cardiomyoplasty have shown that differentiated skeletal muscle is capable of performing cardiac-like work when conditioned (with repetitive stimuli) prior to use (Kass et at, 1995; Sreter et at, 1973). In general, the failure of dynamic cardiomyoplasty has been

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attributed to differences in mature skeletal muscle biology as compared to myocardium, which will be described later. Although, dynamic cardiomyoplasty has not become widely practiced clinically, evidence from several studies indicates that chronically stimulated skeletal muscle acquires features that increase its suitability for cardiac work (Sreter et al, 1973). These data, which demonstrated that skeletal muscle can adapt to perform cardiac-like work, coupled with knowledge that immature skeletal muscle cells easily repair injured peripheral muscle, has fostered research into the use of these skeletal muscle progenitor cells for myocardial regeneration. One of the earliest reports of successful injection of skeletal muscle satellite cells into injured myocardium was by Chiu et al., 1995. Shortly thereafter, the utility of cell transplantation to improve myocardial performance was demonstrated (Taylor et al, 1997). Since that time, the dramatic increase in the understanding of the process of cell differentiation, and the therapeutic potential of stem cells, has increased enthusiasm for myoblast transplantation in models of cardiovascular disease. Although multiple cell types might appear to be beneficial, autologous skeletal myoblasts offer several specific advantages over other cell types. First, skeletal muscle is abundant and accessible in the body; muscle can be harvested without causing functional disability. Second, since skeletal myoblasts are autologous, they do not require implantation of a foreign material, and hence do not require any form of immunosuppression. Finally, skeletal muscle is the only extracardiac tissue that performs significant amounts of work by a contractile mechanism similar to that of the heart. An understanding of these considerations led to skeletal myoblasts becoming one of the first cell types to be investigated for use in cellular cardiomyoplasty (Taylor et al, 1998). 3. Choice of Cell Type Recently, as interest in cell-based therapies has increased, several cell types in addition to skeletal myoblasts have been proposed

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to treat cardiovascular diseases — adult, neonatal or fetal cardiocytes, bone marrow-derived stem cells and embryonic stem cells (Leor et al, 1996; Klug et al, 1996; Orlic et al, 2001). The seemingly obvious choice for repopulating injured myocardium is cardiocytes. Fetal, neonatal and adult cardiomyocytes have been utilised by several investigators, and have been reported to improve myocardial performance (Leor et al, 1996; Sakai et al, 1999). The advantages of utilising cardiocytes include potential for electromechanical coupling, and fatigue resistance. The disadvantages include poor to no tolerance to an ischaemic environment, and the need for immunosuppression (if allogeneic cardiocytes are used). Embryonic and bone marrow-derived stem cells have also been used with varying degrees of success (Klug et al, 1996; Orlic et al, 2001; Tomita et al, 1999). The major advantages of these approaches include the potential for cells to differentiate into cardiomyocytes if placed in the appropriate environment. However, these types of cells are so sensitive to the environment in which they are placed, that they may just as readily differentiate into fibroblasts if placed in scar tissue, as suggested by Wang et al, 2000. Data from Orlic et al, suggest that lineage negative (Lin") c-kit p o s subpopulations of stem cells can be derived and supplied in numbers sufficient to repopulate infracted regions of myocardium with cardiocytes and vascular structures (Orlic et al, 2001). Although this is an extremely exciting development, these data have only been obtained in the mouse heart, where the infarct size is exceedingly small. In fact, attempts to reproduce those data in non-human primates have failed. Furthermore, the functional impact of these cells has not been rigorously evaluated. Translating these initial results into a larger, more physiologically relevant model of human cardiac disease, is the critical next step in evaluating these types of stem cells. The potential of utilising a multipotent stem cell for cardiac repair is intriguing, but our current understanding of stem cell biology is not presently sufficient to allow widespread clinical usage.

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Also of interest is new controversial research suggesting that myocardium may be able to repair itself using the body's own progenitor cells. This novel concept of cardiac chimerism has been the subject of several recent high profile publications (Quaini et al., 2002; Glaser et al., 2002). These two studies challenge the conventional dogma that the heart is unable to repair itself. At issue is the degree to which self-repair can occur, and whether both vascular smooth muscle cells and cardiomyocytes are capable of such repair. Quaini et al., report up to 30% of transplanted myocardium being regenerated within 28 days after allotransplantation by cardiac-derived stem cells originating from the recipient (Quaini et al., 2002). In contrast, Glaser et al., report the presence of infiltrating host cells comprising 5.8% of the vascular smooth muscle cells, but comprising none of the cardiomyocytes (Glaser et al., 2002). With the striking contrast of data, it is likely the answer lay somewhere in between, with the degree of chimerism depending on several factors, including methods used to identify "chimeric cells"; the limitations of those measurements; the timing of the observations; and the criteria by which chimerism is identified (Taylor et al., 2002). Nonetheless, this represents a very interesting area for further research. Cellular therapy involving skeletal myoblasts is not limited to autologous cells — several other types of skeletal myoblasts have been utilised for cellular cardiomyoplasty in animal models, including neonatal (allogeneic or syngeneic), and xenogeneic cells. Utilising non-autologous sources for myoblasts has the distinct advantage of not requiring the patient to donate tissue. However, these sources have several disadvantages. They include the need for immunosuppression, and the lack of clear evidence that these non-myogenic alternate sources differentiate into a cell type capable of contractile function and activity in the myocardium. The fiber source of skeletal myoblasts may also play an important role in the results of cellular cardiomyoplasty. The regenerative capacity of skeletal muscle measured in vivo after muscle injury, as well as the in vitro growth rate of skeletal

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myoblasts, differs between muscle types (Pavlath et al, 1998; Qu et al, 1998). In animal models, we have had best results using myoblasts taken from slow-twitch fatigue-resistant muscle fibers (soleus). 4. Physiologic Considerations Cardiac myocardium differs from skeletal muscle in several important ways. Cardiac muscle lacks the ability to effectively regenerate following myocardial injury. This is primarily due to the inability of cardiac myocytes to proliferate beyond the neonatal period and the demonstrated lack of cardiac stem cells. Skeletal muscle, on the other hand, is characterised by the presence of satellite cells which, after skeletal muscle injury, proliferate to regenerate functional skeletal muscle. Since adult skeletal muscle can be conditioned to perform cardiac-like work, recent attempts at regenerating functional myocardium after injury have focused on cardiomyoplasty using skeletal myoblasts. Having considered the theoretical advantages and disadvantages of having skeletal muscle incorporated into cardiac myocardium, we hypothesise that it would be advantageous to incorporate some of the desirable qualities of both skeletal muscle and cardiac muscle into a single cell type. Cells with the ischemia-resistance of skeletal muscle as well as the work capability of myocardium, would be ideal. Since skeletal myoblasts are undifferentiated cells and do not possess qualities of differentiated skeletal or cardiac myocytes, but normally mature to form muscle, we anticipated that injection of these myoblasts into a cardiac environment would direct their differentiation, perhaps even into a modified phenotype. Histologic data from several laboratories seems to indicate this is possible (Chiu et al, 1995; Taylor et al, 1998). However, conflicting data from other investigators indicate that the cells retain their skeletal muscle phenotype (Murry et al, 1996). In either case, we and others propose that it may be possible to alter the myoblast phenotype by genetic manipulation, to yield myoblasts capable of differentiation into cardiac-like myocytes.

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5. Differentiation Pathways It is generally accepted that cells committed to one pathway of differentiation cannot transdifferentiate to express characteristics of cells committed to another lineage. However, this idea is being challenged in a number of areas; more recently it has been proposed that immature skeletal myoblasts are more plastic than originally thought, as with other undifferentiated cells (Lee et al, 2000). The potential for such undifferentiated precursor cells to undergo multi-lineage differentiation depending upon the microenvironment is conceivable, but controversial. Mounting evidence suggests that this is possible when skeletal myoblasts are injected into the cardiac environment, but the molecular and genetic signals leading to differentiation in this situation are not completely understood. Certain transcription factors direct the ultimate phenotype of progenitor cells undergoing differentiation. MyoD is the master switch that directs skeletal myogenesis in skeletal myoblasts. Overexpression of MyoD in various non-myogenic cell lines has been shown to activate transdifferentiation into the skeletal phenotype (Choi et al, 1990; Weintraub et al, 1989). A master switch or transcription factor capable of inducing cardiac myogenesis from progenitor cells has not yet been identified. Future identification of transcription factors capable of directing cardiac differentiation may provide an ideal cell for cardiac repair. To date, myoblasts appear to be the first generation's most viable candidate. Electrical integration is a critical component of successful cell-based cardiac repair. To date, it remains an area of concern, although pre-clinical data have not suggested pro-arrhythmia potential of cell transplantation. Early clinical data suggest myoblasts may generate a transient period of ventricular tachycardia, but the mechanism of the effect is unclear. In contrast, preclinical data suggest that myoblasts can effectively couple with cardiomyocytes. For example, skeletal myoblasts cocultured with cardiomyocytes express gap junction proteins n-cadherin and connexin 43, and display electromechanical coupling with

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cocultured cardiomyocytes (Reinecke et al„ 2000). This suggests that preconditioning by coculture with cardiomyocytes could be utilised to alter myoblast phenotype prior to injection. 6. Method of Delivery The most studied method of transplanting myoblasts into an area of myocardial injury is direct intramyocardial injection. This can be accomplished via direct myocardial exposure (e.g., thoracotomy) and injection via a needle introduced through the epicardium. Alternately, myoblasts can be introduced through a percutaneous injection device, and injected into the endocardium. Epicardial injections performed under direct visualisation allow the surgeon to direct placement of myoblasts into areas of obvious myocardial injury. Moreover, infiltration of the injured regions with infusate may be visually confirmed. We have found that the use of a purse string suture around the site of needle insertion prevents retrograde leakage of myoblasts into the thoracic cavity following injection into injured myocardium. Technical considerations for myoblast infusion include extent of infiltration required, number of injection sites, gauge of needle used, and orientation of the needle at the time of injection. Theoretically, infiltration of myoblasts into injured myocardium (as far as its border) with normal myocardium may promote intercellular communication and continuity between normal myocardium and the resultant graft. Conversely, such bridging may promote electrical instability of the border zone and be pro-arrhythmic. This has not been studied experimentally, but may prove to be an important consideration during cellular cardiomyoplasty. Devices continue to be developed to assist in percutaneous myoblast delivery. Current systems consist of a catheter that can be introduced into the arterial system via a peripheral vessel, advanced into the left ventricular cavity, and manipulated under either fluoroscopic or echocardiographic guidance. A protruding needle can be positioned in the infarcted myocardium at variable

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Fig. 1. Percutaneous endoventricular injection catheter (Courtesy of Bioheart Inc.)

Fig. 2. (Top) Direct surgical delivery allows tangential cell grouping. (Bottom) Catheter-based systems are limited to perpendicular cell delivery.

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depths, and cell delivery can be performed (Fig. 1). Since the needle utilised in most prototypes of such devices is fairly short, the injection is limited by wall thickness, and a limited number of cells can be delivered at each spot in the myocardium. Thus, multiple areas of the myocardium must be infiltrated with cells using this technique in order to repopulate the scar (Fig. 2 Bottom). Furthermore, delivery of cells by this method yields populations of cells oriented in a perpendicular fashion to the endocardium; surgical delivery yields a tangential cell grouping. The efficacy of cell delivery using catheter techniques has not yet been demonstrated, but several versions of the devices are currently being tested. Anecdotal reports from the Dutch group indicate functional improvement utilising catheter based delivery (Surruys et ah, personal communication). Intracoronary delivery of myoblasts has been demonstrated in the normal myocardium of rabbits, and results in perivascular distribution of myoblasts. However, the engraftment and efficiency of myoblast delivery using this technique is low when compared to direct injection techniques. Initially, injected cells lodge in the coronary capillaries. One week following intracoronary delivery of cells, myoblasts can be found in the myocardial interstitium not only in the capillary beds (Robinson et ah, 1996) Moreover, the gross appearance of engrafted cells using this technique differs from that resulting from direct injection. The former results in perivascular infiltration of myoblasts, whereas the latter yields a distinct functional plug of engrafted cells (Taylor et ah, 1997). Currently, improvement in contractility has only been proven after direct injection of myoblasts, not after intracoronary delivery. While future development of this technique may prove it to be efficacious, one theoretical limitation is the presence of coronary occlusive disease affecting the distribution of cells infused using this method, as areas of poor perfusion (i.e., infarct scar) may not receive adequate numbers of cells to augment contractility. More recently a transvenous catheter approach has been proposed. Devices being investigated would

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provide the benefit of a tangential injection, but work is early, and reports are anecdotal. Attempts at optimising cell engraftment, and the ability of skeletal myoblasts to augment cardiac function, have included the use of various scaffold materials. Gel foam impregnated with myoblasts can be implanted into the heart, and cells grown on biodegradable polymers can be sewn onto the epicardial surface of the heart (Li et al., 1999). Experience with the use of such techniques is limited, but is an area of active investigation. Additional considerations with regard to the technique of cell transplantation include extent of the injection into the normal myocardium, and whether to inject cells into the core of the scar versus the periphery of the scar. Several studies have suggested that reducing graft isolation from the surrounding normal myocardium may improve graft integration and electromechanical coupling (Reinecke et al., 1999). Theoretically, cells injected into the periphery of the scar may experience improved vascular perfusion from the surrounding myocardium and the ingrowth of vessels, but balancing this benefit with the potential hazards of electrical coupling in this area is an important consideration. 7. Timing of Delivery Progressive changes in tissue composition occur following myocardial infarction. Within the first 24 hours, cardiocyte necrosis and apoptosis occur, which are accompanied by infiltration of inflammatory cells. Around seven days after myocardial infarction, the scar begins to remodel with the influx of macrophages into the myocardium. Over the course of the next several weeks, the area of infarction is replaced by highly vascularised granulation tissue, eventually forming a fibrous scar. The ability for injected cells to engraft in scar tissue may depend upon the timing of injection. If injected too early after a myocardial infarction, cells may be subjected to elimination by the inflammatory reaction. If the scar is allowed to mature prior to injection, decreased vascularity may be encountered. Functional benefit, in

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the absence of myogenesis, may be minimal under these circumstances, although early clinical data suggest improvements can occur. 8. Cell Culture Technique Of critical importance to the clinical success of cellular cardiomyoplasty using autologous skeletal myoblasts will be the ability to grow and expand biopsy derived cell populations. Ideally, a biopsy of a muscle with a high concentration of satellite cells is used. In animal studies our lab uses the soleus muscle, but in clinical use other muscles may afford similar successful cell populations. After the skeletal muscle biopsy is obtained, the tissue is transported on ice in DMEM to the cell culture facility. For rabbits and pigs, 100 mg-200 mg of soleus muscle is sufficient to begin the process. In patients 5-10 g is used. To isolate skeletal myoblasts, tissue is dissected mechanically, washed in phosphate buffered saline, resuspended, and plated in Dulbecco's Modified Eagle's Medium containing 20% horse serum (growth medium). Tissue fragments are triturated daily to prevent adherence to the dish. After three days, tissue samples are removed, and the cells are replaced into fresh growth culture medium. Myoblasts are expanded in vitro for 7-14 days on culture plates and are passaged as necessary to maintain cell density of less than 70% confluence as assessed by microscopy. This is because, after reaching approximately 70% confluence, skeletal myoblasts begin to differentiate into skeletal myotubes, and if allowed to continue beyond this confluence, will fuse to form myofibrils. At the time of injection, myoblast colonies will have undergone three or four passages. Prior to transfer, the myoblasts are harvested from culture plates with trypsin and ethylenediaminetetraacetic acid. Trypsin is inactivated by the addition of serum. Recovered myoblasts are washed three times to remove serum, resuspended in a minimal volume of normal saline, and stored on ice (typically not more than 20 min.) until delivery into the myocardium.

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Similar techniques have been used to grow human skeletal myoblasts. The expansion time of 7-14 days remains a potential limitation as clinical trials move forward, but progress in tissue banking techniques may overcome this hurdle. 9. Cardiac Function It is clear that autologous skeletal myoblasts improve systolic and diastolic function when injected into injured myocardium. Injection of autologous skeletal myoblasts into chronically infarcted hearts improves diastolic properties, as measured by the relationship between left ventricular pressure and strain, and left ventricular pressure and end-diastolic segment length. Changes in diastolic compliance appear to occur early after cell transplantation (Atkins et at., 1999). Regional systolic function as measured by preload recruitable stroke work (PRSW) relationships show that myocardial contractility decreases significantly after cryoinfarction, but is regained as early as three weeks after cell transplantation (Taylor et at., 1998). The functional effect of autologous skeletal myoblast transfer into rabbit heart after cryoinfarction is seen in Fig. 2. In contrast, injection of another cell type — dermal fibroblasts — into infarcted myocardium, showed improvement in diastolic performance without improvement in contractility (Hutcheson et at., 2000). Though it is clear that autologous skeletal myoblasts improve function, the mechanism by which they work remains unclear. Several prevailing theories include a conservative view that the benefit seen is compensation of surrounding myocardium. Some believe that the functional benefit is limited to an improved compressibility of the scar tissue, and that injected cells will never be in electrical communication with existing myocardium. The most radical and promising of the theories is that these injected myoblasts become capable of synchronous contractile activity with the rest of the heart. Some unpublished regional function data from our lab shows that prior to injection of cells, regional stroke work across the infarct is negative, indicating a

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dyskinetic aneurysm. After cell injection a dramatic conversion to positive stroke work occurs, indicating synchronous contraction with surrounding myocardium. (Fig. 3) 10. Resultant Cell Type Evidence from several different laboratories indicates that the cell type resulting from implantation of undifferentiated skeletal

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Fig. 4. (A) Hematoxylin and eosin staining (x4 magnification before reduction) of an injected heart containing engrafted myotubes within approximately 75% of a transmural cryoinjury in a typical animal two weeks after transplantation. Open and closed arrows depict regions marked with similar arrows and displayed at higher magnification in B and C, respectively. (B) Higher power (x20 magnification before reduction) hematoxylin and eosin staining of a section at the periphery of a typical cryolesion, showing isolated normal myocardium at the left, the sharp demarcation of the injury, and myogenin-negative cells (arrow). (C) Higher power (x20 magnification before reduction) hematoxylin and eosin staining of the myotubes corresponding to the region of the same heart marked by the closed arrow in Fig. A (reprinted with permission from Ann. Thor. Surg. (1999), 67, 127 (Fig. 2).

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myoblasts into the cardiac environment resembles that of both cardiocyte and skeletal muscle cells. The phenotype of cells injected into the cardiac milieu resembles that of a slow-twitch muscle fiber similar to cardiac myocytes. Phospholamban, which is usually expressed in cardiac cells and slow-twitch skeletal muscle, can be found in myoblasts injected into the cardiac microenvironment (Robinson et ah, 1996). The slow-twitch isoform of myosin heavy chain is also induced in myoblasts after engraftment into the cardiac environment (Murry et ah, 1996). Various markers are used to determine cell type. These include intracellular enzymes such as cardiac troponin T and fast skeletal troponin T, the presence of connexin 43, desmin (suggesting the presence of gap junctions), and cardiac specific transcription factors such as GATA 4 and NKx-2.5. All of these markers have been found in transplanted autologous sketelal myoblasts. In addition, histologic examination of cell grafts reveals centrally located nuclei and the presence of intercalating disks, both being features of cardiac myocytes. However, not all investigators have been able to demonstrate the presence of intercalating disks after myoblast transplantation into normal or injured myocardium. This discrepancy may be due to species variation or conditions under which myoblasts are cultured. When undifferentiated skeletal myoblasts are injected into areas of myocardial injury, they differentiate into two populations in vivo (Atkins et al., 1999). (Fig. 4) Cells present in the centre of the injured myocardium resemble skeletal muscle, and express myogenin. A second population of cells located at the periphery of myocardial injury resembles immature cardiomyocytes, which are myogenin negative with centrally located nuclei. 11. Evidence for Electromechanical Coupling Evidence suggests that myoblasts injected into the cardiac microenvironment begin to express components of desmosomes including gap junctions, indicating functional electromechanical coupling. Grafts resulting from injection of skeletal myoblasts

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Fig. 5. Electron micrograph of transplanted skeletal myoblasts showing intercalated discs labelled (i) (reprinted with permission from Nat. Med. 4(8), 932 (Fig. 3).

into cryoinjured myocardium display the presence of intercalating disks on electronic microscopy and gross histologic staining (Atkins et al, 1999). (Fig. 5) When delivered via the arterial circulation, myoblasts engrafting in both normal and injured myocardium display staining for connexin 43 and evidence for gap junctions between cells at sites of host and donor cell interface (Marelli et al, 1992). 12. Limitations of Myoblast Cell Transfer in Patients As we move forward in clinical trials involving myoblast transplantation there are a number of potential limitations that could affect clinical outcomes. Pre-clinical data has been very promising, and anecdotal clinical experiences have been similarly positive, but many considerations ranging from cell culture techniques to arrhythmias must be addressed. One potential consideration as clinical myoblast transplantation becomes more prevalent is that myoblasts isolated from certain populations of individuals may not be capable of similar differentiation and metabolic activity. Thus, the results described above may vary from patient to patient depending upon the

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quality of harvested myoblasts. Several encouraging studies have shown that the quantity and in vitro proliferative capability of skeletal myoblasts is not decreased significantly with aging as was previously thought (Grounds et ah, 1998). Patients with ischaemic heart disease and coexistent chronic peripheral vascular disease may experience muscular atrophy, and the quality of myoblasts obtained from such patients is unknown and will likely be different from relatively healthy patients. Further investigation into the regenerative capacity of myoblasts isolated from individuals with heart failure and advanced age are required. Of paramount importance as we move forward in clinical trials is the demonstration of patient safety after myoblast transplantation. Most frequently discussed is the potential for lethal arrythmia caused by aberrant electrical pathways formed where previously no electrical activity had been seen. Along these lines, anecdotally, Menashe et al., reported ventricular tachycardia in four out of 10 patients who underwent cell transfer with CABG. The arrythmias reported in these patients all occurred at 2-4 weeks after transplant. Cells with heterogeneous electrical and conduction properties are hypothetically predisposed to the development of reentry and ventricular arrhythmias, but little has been published in preclinical data relating to this phenomenon. Another often overlooked issue is cell culture technique. Current cell culture techniques utilise serum extracts from various species. The skeletal myoblasts require serum to proliferate in vitro, and often, minute amounts of serum are included in the injectate despite meticulous cell washing. The use of serum during cell culture raises concerns of cross-species contamination and hypersensitivity reactions, especially internationally where there is less control over serum, and more chance for infectious organisms such as bovine spongiform encephalopathy (BSE). Biodistribution of cells injected into the cardiac microenvironment using any delivery method has been poorly defined. Methods of tracking injected cells are limited to gender specific

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markers and radioactive isotope labelling. Cells delivered into the heart by intravascular delivery depend upon migration across the vascular endothelium, and a significant portion may recirculate in the vascular system following injection. A certain percentage of cells injected locally into the cardiac microenvironment by direct injection may enter into the systemic circulation and embolise to peripheral organs. The extent and consequences of dissemination of skeletal myoblasts to extracardiac organs require detailed investigation. Autologous skeletal myoblasts are expected to have a low risk of neoplastic degeneration, when compared to other potential cell types. However, concerns of tumorigenicity must be addressed prior to the clinical use of cell transplantation, especially if genetically engineered myoblasts are to be used and biodistribution remains uncertain. 13. Future Directions One of the most promising attempts at augmenting skeletal myoblast engraftment and optimising function is co-injection with growth factors. During embryonic cardiac development, expression of certain members of transforming growth factor (BMPs 1 and 2) are essential for differentiation of embryonic mesoderm into cardiac muscle (Lough et al, 1996; Schultheiss et al, 1997). Concurrent injection of such growth factors, or genetic manipulation of skeletal myoblasts to constitutively secrete these growth factors, may assist differentiation of skeletal myoblasts into cardiac myocytes. The use of anti-inflammatory agents to optimise cell survival after transplantation has not been shown in cardiomyoplasty. However, evidence from myoblast transfer into skeletal muscle has shown that genetically engineered myoblasts expressing an inhibitor of the cytokine IL-1 or transforming growth factor beta 1 result in improved cell survival (Qu et al., 1998; Merly et at., 1998).

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14. Prospects for Clinical Use Skeletal myoblast transplantation is being considered as a novel therapeutic modality in various clinical settings, including myocardial regeneration, vascular disease, and avascular necrosis of the femoral neck. Its application as a novel therapeutic modality in cardiovascular disease is attractive, since numerous studies in animal models suggest that implantation of autologous skeletal myoblasts augments cardiac function and improves survival. The ultimate goal is to identify patients with cardiac dysfunction following transmural myocardial infarction, and to perform cell transplantation prior to the development of cardiac remodelling and decompensation. The best method of cell delivery in these patients awaits determination with both noninvasive techniques (percutaneous intracoronary or percutaneous intramyocardial injection) and direct invasive techniques offering individual benefits. Further demonstration of safety and efficacy in humans in randomised clinical trials is required prior to its widespread application in patients following myocardial infarction. Small clinical trials are currently underway in Europe and the United States, most focusing on cell transplant as an adjunct to coronary bypass surgery. However the changes in cardiac function in this setting may not reflect efficacy of myoblast transplantation, since results are confounded by the simultaneous coronary revascularisation. Another exciting trial underway is aimed at terminally ill patients with congestive heart failure secondary to ischaemic heart disease who require cardiac transplantation. More frequently, these patients are undergoing implantation of a left ventricular assist device as a "bridge" to transplantation. Myoblast injection into infarcted myocardium at the time of LVAD implantation may be performed, and efficacy may be measured from haemodynamic parameters and the ability to wean the patient from the assist device. In patients ultimately undergoing cardiac transplantation, engraftment and histologic appearance of implanted skeletal myoblasts may be evaluated.

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15. Conclusions Transplantation of autologous skeletal myoblasts is emerging as a novel therapeutic strategy with demonstrable engraftment and improvement in myocardial function. Concerns of longterm safety must be addressed prior to widespread application in patients with heart failure and ischaemic heart disease. Further progress in cell culture and delivery techniques will facilitate its transition into the clinical arena. Elucidation of the molecular mechanisms behind myoblast differentiation and the development of genetically engineered skeletal myoblasts may translate into improved graft survival and function. The challenge for the next decade is to determine the appropriate patients, timing and methods of cell delivery, and cell culture techniques that will optimise results of myoblast transplantation. 16. A c k n o w l e d g m e n t Bryce H. Davis, B.S.E. This work was supported in part by N I H / NHLBI awards to DAT including HL-5798; HL-63346 and HL63703. 17. References ACKER, M A . (1999). Dynamic cardiomyoplasty: At the crossroads, Ann. Thorac. Surg. 68(2), 750-755. ATKINS, B.Z., HUEMAN, M.T., MEUCHEL, J.M., COTTMAN, M.J., HUTCHESON, K.A. and TAYLOR, D A . (1999). Myogenic cell transplantation improves in vivo regional performance in infarcted rabbit myocardium, /. Heart Lung Transplant. 18(12), 1173-1180. ATKINS, B.Z., LEWIS, C.W., KRAUS, W.E., HUTCHESON, K.A., GLOWER, D.D. and TAYLOR, D A . (1999). Intracardiac transplantation of skeletal myoblasts yields two populations of striated cells in situ, Ann. Thorac. Surg. 67(1), 124-129.

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CHEN, C.Y. and SCHWARTZ, R.J. (1996). Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription, Mol. Cell Biol. 16(11), 6372-6384. CHIU, R.C., ZIBAITIS, A. and KAO, R.L. (1995). Cellular cardiomyoplasty: Myocardial regeneration with satellite cell implantation, Ann. Thorac. Surg. 60(1), 12-18. CHOI, J., COSTA, M.L., MERMELSTEIN, C.S., CHAGAS, C , HOLTZER, S. and HOLTZER, H. (1990). MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes, Proc. Natl. Acad. Sci. USA 87(20), 7988-7992. CLEAVER, O.B., PATTERSON, K.D. and KRIEG, P.A. (1996). Overexpression of the tinman-related genes XNkx-2.5 and XNkx-2.3 in Xenopus embryos results in myocardial hyperplasia, Development 122(11), 3549-3556. GLASER, R., LU, M.M., NARULA, N. et al. (2002). Smooth muscle cells, but not myocytes of host origin in transplanted human hearts, Circulation 106, 17-19. GROUNDS, M.D. (1998). Age-associated changes in the response of skeletal muscle cells to exercise and regeneration, Ann. N.Y. Acad. Sci. 854, 78-91. HUTCHESON, K.A., ATKINS, B.Z., HUEMAN, M.T., HOPKINS, M.B., GLOWER, D.D. and TAYLOR, D.A. (2000). Comparison of benefits on myocardial performance of cellular cardiomyoplasty with skeletal myoblasts and fibroblasts, Cell Transplant. 9(3), 359-368. KASS, D.A., BAUGHMAN, K.L., PAK, P.H., CHO, P.W., LEVIN, H.R., GARDNER, T.J., HALPERIN, H.R., TSITLIK, J.E. and ACKER, M.A. (1995). Reverse remodeling from

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cardiomyoplasty in human heart failure. External constraint versus active assist, Circulation 91(9), 2314-2318. KLUG, M.G., SOONPAA, M.H., KOH, G.Y. and FIELD, L.J. (1996). Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts, /. Clin. Invest. 98(1), 216-224. LEE, J.Y., QU-PETERSEN, Z., CAO, B., KIMURA, S., JANKOWSKI, R., CUMMINS, J., USAS, A., GATES, C , ROBBINS, P., WERNIG, A. and HUARD, J. (2000). Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing, /. Cell Biol. 150(5), 1085-1100. LEE, K.F., DIGNAN, R.J., PARMAR, J.M., DYKE, CM., BENTON, G., YEH, T. Jr., ABD-ELFATTAH, A.S. and WECHSLER, A.S. (1991). Effects of dynamic cardiomyoplasty on left ventricular performance and myocardial mechanics in dilated cardiomyopathy, /. Thorac. Cardiovasc. Surg. 102(1), 124-131. LEOR, J., PATTERSON, M., QUINONES, M.J., KEDES, L.H. and KLONER, R.A. (1996). Transplantation of fetal myocardial tissue into the infarcted myocardium of rat. A potential method for repair of infarcted myocardium? Circulation 94(9 Suppl), 11332-336. LI, R.K., JIA, Z.Q., WEISEL, R.D., MICKLE, D.A., CHOI, A. and YAU, T.M. (1999). Survival and function of bioengineered cardiac grafts, Circulation 100(19 Suppl), 1163-69. LOUGH, J., BARRON, M., BROGLEY, M., SUGI, Y., BOLENDER, D.L. and ZHU, X. (1996). Combined BMP-2 and FGF-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm, Dev. Biol. 178(1), 198-202. MARELLI, D., DESROSIERS, C , EL-ALFY, M., KAO, R.L. and CHIU, R.C. (1992). Cell transplantation for myocardial repair: an experimental approach, Cell Transplant. 1(6), 383-390.

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MENASCHE, P., HAGEGE, A.A., SCORSIN, M., POUZET, B., DESNOS, M., DUBOC, D., SCHWARTZ, K., VILQUIN, J.T. and MAROLLEAU, J.P. (2001). Myoblast transplantation for heart failure, Lancet 357, 279-280. MERLY, F., HUARD, C., ASSELIN, I., ROBBINS, P.D. and TREMBLAY, J.P. (1998). Anti-inflammatory effect of transforming growth factor-betal in myoblast transplantation, Transplantation 65(6), 793-799. MURRY, C.E., WISEMAN, R.W., SCHWARTZ, S.M. and HAUSCHKA, S.D. (1996). Skeletal myoblast transplantation for repair of myocardial necrosis, /. Clin. Invest. 98(11), 25122523. NIINAMI, H., GREER, K., KOYANAGI, H. and STEPHENSON, L. (1996). Skeletal muscle ventricles: Another alternative for heart failure, /. Card. Surg. 11(4), 280-287. ORLIC, D., KAJSTURA, J., CHIMENTI, S., JAKONIUK, I., ANDERSON, S.M., BAOSHENG, L., PICKEL, J., MCKAY, R., GINARD, B.N., BODINE, D.M., LERI, A. and ANVERSA, P. (2001). Bone marrow cells regenerate infarcted myocardium, Nature 5(410), 701-705. PAVLATH, G.K., THALOOR, D., RANDO, T.A., CHEONG, M., ENGLISH, A.W. and ZHENG, B. (1998). Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities, Dev. Dyn. 212(4), 495-508. QU, Z., BALKIR, L., VAN DEUTEKOM, J.C., ROBBINS, P.D., PRUCHNIC, R. and HUARD, J. (1998). Development of approaches to improve cell survival in myoblast transfer therapy, /. Cell Biol. 142(5), 1257-1267. QUAINI, F., URBANEK, K., BELTRAMI, A.P. et al. (2002). Chimerism of the transplanted heart, N. Engl. J. Med. 346, 5-15.

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REINECKE, H., MACDONALD, G.H., HAUSCHKA, S.D. and MURRY, C.E. (2000). Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair, /. Cell Biol. 149(3), 731-740. REINECKE, H., ZHANG, M., BARTOSEK, T. and MURRY, C.E. (1999). Survival, integration, and differentiation of cardiomyocyte grafts: A study in normal and injured rat hearts, Circulation 100(2), 193-202. ROBINSON, S.W., CHO, P.W., LEVITSKY, H.I., OLSON, J.L., HRUBAN, R.H., ACKER, M.A. and KESSLER, P.D. (1996). Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: Long-term survival and phenotypic modification of implanted myoblasts, Cell Transplant. 5(1), 77-91. SAKAI, T., LI, R.K., WEISEL, R.D., MICKLE, D.A., KIM, E.J., TOMITA, S., JIA, Z.Q. and YAU, T.M. (1999). Autologous heart cell transplantation improves cardiac function after myocardial injury, Ann. Thorac. Surg. 68(6), 2074-2080. SCHULTHEISS, T.M., BURCH, J.B. and LASSAR, A.B. (1997). A role for bone morphogenetic proteins in the induction of cardiac myogenesis, Genes Dev. 11(4), 451-462. SOLA, O.M., DILLARD, D.H., IVEY, T.D., HANEDA, K., ITOH, T. and THOMAS, R. (1985). Autotransplantation of skeletal muscle into myocardium, Circulation 71(2), 341-348. SRETER, F.A., GERGELY, S. and ROMANUL, F. (1973). Synthesis by fast muscle of myosin light chains characteristic of slow muscle in response to long-term stimulation, Nature 241, 1 7 19. TAYLOR, D.A., ATKINS, B.Z., HUNGSPREUGS, P., JONES, T.R., REEDY, M.C., HUTCHESON, K.A., GLOWER, D.D. and KRAUS, W.E. (1998). Regenerating functional myocardium: Improved performance after skeletal myoblast transplantation, Nat. Med. 4(8), 929-933.

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TAYLOR, D.A., HRUBAN, R., RODRIGUEZ, E.R. and GOLDSCHMIDT, P.F. (2002). Cardiac chimerism as a mechanism of self-repair: Does it happen and if so to what degree? pp. 2-4. TAYLOR, D.A., SILVESTRY, S.C., BISHOP, S.P., ANNEX, B.H., LILLY, R.E., GLOWER, D.D. and KRAUS, W.E. (1997). Delivery of primary autologous skeletal myoblasts into rabbit heart by coronary infusion: A potential approach to myocardial repair, Proc. Assoc. Am. Physicians. 109(3), 245-253. TOMITA, S., LI, R.K., WEISEL, R.D., MICKLE, D.A., KIM, E.J., SAKAI, T. and JIA, Z.Q. (1999). Autologous transplantation of bone marrow cells improves damaged heart function, Circulation 100(19), 247-256. WANG, J.S., SHUN, T.D., CHEDRAY, E., ELIOPOULIS, N., GALIPEAU, J. and CHIU, R.C.J. (2000). Marrow stromal cells for cellular cardiomyoplasty: The importance of microenvironment for mileau-dependent differentiation (abstract), Circulation 102(18 supp II), 683. WEINTRAUB, H., TAPSCOTT, S.J., DAVIS, R.L., THAYER, M.J., ADAM, M.A., LASSAR, A.B. and MILLER, A.D. (1989). Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD, Proc. Natl. Acad. Sci. USA 86(14), 5434-5438.

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SECTION VI: CORNEA GRAFTS

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

23 CORNEA TRANSPLANTATION STRATEGY — ORGAN CULTURE VERSUS COLD STORAGE

DAGMAR HAVRANOVA, JIRI ADLER, JANA KOMARKOVA, ANNA TEJKALOVA and EVA HLAVACKOVA Tissue Bank, University Hospital Brno Czech Republic EVA VLKOVA, HANA HRUBA and MONIKA HORACKOVA Ophthalmology Department, University Hospital Brno Czech Republic MAHMOOD FARAZDAGHI Tissue Banks International, Baltimore, USA

1. Introduction Since the introduction of keratoplasty, several methods have been developed for optimal preservation of donor corneas. The McCarey-Kaufman (MK) Medium introduced in 1974 is used for short-term storage up to four days at 4°C. Tissue preservation medium is used for intermediate-term storage for up to 14 days

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at 4°C. This method is widely used in the USA, where Optisol GS is the most popular storage medium. Organ-culture medium is used for long-term storage up to 42 days at 31°C in an incubator. This method is used mainly in Europe. However, some European eye banks also use hypothermic storage. According to the January 2002 European Eye Bank Association (EEBA) Directory, 30% of the European eye banks perform hypothermic storage at 4°C only; 48% perform organ-culture at 31°C only; and 22% use both methods of storage. There were 69 eye banks responding to the questionnaire. The quality of donor endothelium is a crucial factor in the success of a penetrating keratoplasty. Therefore, any method used for storage of donor corneas must maintain endothelial viability. This study compares two storage methods — intermediate-term hypothermic storage and long-term organ-culture — and their influence on corneal endothelium quality and the clinical outcome of transplantation. 2. Materials Cornea donors were selected according to criteria set by the European Eye Bank Association (EEBA), Eye Bank Association of America (EBAA) and International Federation of Eye and Tissue Banks/Tissue Banks International (IFETB/TBI). Donors were screened for Hepatitis B surface Antigen, HIV I, II antibodies, Hepatitis C antibodies and for Syphilis. Corneas were recovered within 18 hours post-mortem by the excision in situ technique and immediately placed in the glass vial with recovery medium (organ culture medium, Optisol® GS or MK medium). Organ-culture media were prepared by tissue bank personnel in the laminar airflow box, class 100. Culture medium contained Minimal Essential Medium (MEM) with Earl's salts, L-glutamine and 20 mM HEPES buffer (PAN), 2% Fetal Bovine Serum (PAN), gentamicin 40 mg/1 (LEK) and Amphotericin B 25 mg/1 (Bristol). Thinning medium had the same composition supplemented by 5% Dextran T 500 (Pharmacia). For hypothermic storage the

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Optisol GS (Bausch and Lomb) was used (2.5% Chondroitin Sulphate — Dextran Corneal Storage Medium containing gentamicin 100 mg/1 and streptomycin sulphate 200mg/l). 3. Storage Methods 3.1. Long-term organ-cultivation After recovery and quality evaluation, corneas were processed in the laminar airflow box, class 100. Surgical suture was tied to the scleral rim and to the rubber stopper; corneas were suspended in glass bottles with 60 ml organ culture medium and incubated at 31°C. The medium was checked daily for changes in colour and transparency to catch microbial or fungal growth. The medium was changed every 10-14 days according to the colour change indicating the metabolic activity and consumption of the nutrients. Mean storage time was 24 days. To thin the corneas before transplantation, they were incubated at 31°C in 30 ml glass vials completely filled with medium supplemented by Dextran. Thinning time varied from 17 to 24 hours. 3.2. Hypothermic storage After recovery, corneas were placed into the glass vials with Optisol GS that served also for storage at 4°C. Mean storage time was two days. Corneas in Optisol GS were transplanted without any additional manipulation. 4. Evaluation of the Corneal Endothelium 4.1. After recovery Endothelium was evaluated by light microscopy in recovery vials. A specular microscope (Konan KeratoAnalyzer) was used. Endothelial cells of the central part of the cornea were evaluated for their size, shape and density, and endothelial cell count was

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performed using the central method. The minimal endothelial cell count accepted for transplantation was 2,000 cells per mm 2 . 4.2. After transplantation Endothelium was evaluated in situ at the ophthalmology department over several time periods, particularly 1, 3, 6, 12, 24, 48, 72 and 96 months after transplantation. The Specular SP-1000 Topcon endothelial non-contact microscope was used. Endothelial cells of the central part of the cornea were evaluated for their size, shape and density. Endothelial cell count was performed automatically if density was greater than 800 cells per mm 2 , and manually if density was less than 800 cells per mm 2 . 5. Characteristics of Investigated Groups 5.1. Duration of the study The study started in 1997 and was completed in 2002. 5.2. Age and sex ratio, f o l l o w up period 90 patients (57 males, 33 females, 63%:37%), mean age 52.2 years (SD 21.5, range 16-86) were transplanted with organ cultured corneas and followed up for the period of 30.1 months (SD 19.1, range 3-54). 117 patients (65 males, 52 females, 56%:44%), mean age 53.7 years (SD 20.8, range 18-84) were transplanted with corneas stored in Optisol and followed up for the period of 31.5 months (SD 21.5, range 3-66). 5.3. Indications for transplantation Organ-culture group: keratoconus in 25 patients (27.8%), bullous keratopathy in 18 patients (20.0%), rejection in 17 patients (18.9%), spontaneous ulcer perforation in 9 patients (10.0%), keratitis in 7 patients (7.7%), vascularised leucoma in 5 patients

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(5.5%), descemetocoele in 4 patients (4.4%), dystrophy in 2 patients (2.2%), trauma in 2 patients (2.2%) and fibrosis of cornea in 1 patient (1.1%). Optisol group: keratoconus in 38 patients (32.5%), bullous keratopathy in 33 patients (28.2%), cornea rejection in 21 patients (17.9%), spontaneous ulcer perforation in 8 patients (6.8%), keratitis in 5 patients (4.3%), vascularised leucoma in 7 patients (6.0%), descemetocoele in 4 patients (3.4%) and dystrophy in 1 patients (0.9%). 5.4. Post-operative complications Organ-culture group: secondary glaucoma in 35 patients (38.9% of the group), endothelial rejection in 27 patients (30.0%), nonhealing epithelium in 12 patients (13.3%), graft infection in 9 patients (10.0%), graft vascularisation in 8 patients (8.9%), total cornea rejection in 8 patients (8.9%). Optisol group: secondary glaucoma in 46 patients (40.2% of the group), endothelial rejection in 30 patients (25.6%), non-healing epithelium in 13 patients (11.1%), graft infection in 11 patients (9.4%), graft vascularisation in 10 patients (8.5%), total cornea rejection in 9 patients (7.7%). 6. Results The quantitative data on endothelial cell density and endothelial cell loss are summarised in Table 1 and Fig. 1. Fig. 1 clearly illustrates considerable endothelial cell loss during the time after transplantation. If the endothelial cell count one month after transplantation is taken for comparison, and the results of the following measurements are expressed as a cell loss related to the cell count one month after transplantation, there is endothelial cell loss of 22.8% in both groups six months after transplantation. There is endothelial cell loss of 46% in the organ-culture group and 49.5% in the Optisol group 24 months after transplantation. The difference is not statistically significant (F-Test).

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Table 1. Post-operative endothelial cell density on patients transplanted with organ-cultured corneas and corneas stored in Optisol®. Organ culture Period after transplant (months)

Optisol

Endothelial cell density (cells/mm 2 )

Endothelial cell loss

1

2540 (SD 320)

3

Endothelial cell density (cells/mm 2 )

Endothelial cell loss (%)

0

2689 (SD 281)

0

2215 (SD 344)

12.8

2306 (SD 292)

14.2

6

1960 (SD 340)

22.8

2076 (SD 385)

22.8

12

1615 (SD 416)

36.4

1603 (SD 489)

40.4

24

1371 (SD 420)

46.0

1359 (SD 461)

49.5

(%)

0 Optisol • Organ-Culture 3000-

H 1500 .2 £

1000

o C

0)

500

0

1

3

6

12

24

months after transplantation Fig. 1. Post-operative endothelial cell density on patients transplanted with organ cultured corneas, and corneas stored in Optisol.

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7. Conclusion Two groups of patients transplanted either with organ-cultured corneas a n d / o r corneas stored in Optisol, were compared. The storage time for corneas can be extended by preservation in organ-culture medium. Post-operative survival of endothelial cells in both groups is comparable without any significant differences. Penetrating keratoplasty has similar outcomes in the first two post-operative years, in both groups. 8. Discussion As the adult human corneal endothelium has minimal regenerative capacity, the success of corneal transplantation depends mainly on having an adequate number of viable endothelial cells in the donor cornea. For that reason it is very important to preserve endothelial cell viability during storage. There is a correlation between the loss of endothelial cells and their preservation time (Pels and Schuchard, 1983). On the other hand, it was shown (Doughman et ah, 1974) that endothelial cells in organ culture have a capacity to regenerate defects in the monolayer. In our study, endothelium of organ-cultured corneas seemed to be more stable two years post-operatively, but the difference was not statistically significant. Optisol GS makes it possible to store corneas successfully for up to 14 days. It is a more frequently used preservation method without the high demands on handling. Long-term preservation at 31 °C has the following advantages: (a) There is time for surgery scheduling and Human Leucocyte Antigen matching if required. (b) Transportation at room temperature facilitates shipping of tissue over long distances. (c) Corneas with bacterial or fungal contamination can be identified and discarded before grafting. On the other hand, more handling and more laboratory equipment is required. Since both methods does not show any

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significant difference in clinical outcome of the transplantation it depends on the eye bank which method would prefer. 9. Summary Donor corneas were stored either in Optisol GS for up to 14 days, or in organ culture medium for up to 42 days. Slit lamp examination and endothelial cell examination using light microscopy was performed. The patients were investigated after cornea transplantation, and the endothelial cell count was evaluated at several time intervals. Two groups of patients transplanted with organ-cultured corneas and corneas stored in Optisol were evaluated. Clinical follow up ranged up to the three years. Both methods of cornea storage have their advantages and disadvantages, but both ensure a good quality harvest of endothelial cells. 10. A c k n o w l e d g e m e n t s The study was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic, grant number NK 7729-3. 11. References ANDERSEN, J. and EHLERS, N. (1986). Corneal transplantation using long-term cultured donor material, Acta Ophthalmol. 64, 93-96. ARMITAGE, W.J. and EASTY, D.L. (1997). Factors influencing the suitability of organ-cultured corneas for transplantation, Invest. Ophthalmol. Vis. Sci. 38, 16-24. DOUGHMAN, D.J., VAN HORN, D., HARRIS, J.E., MILLER, G.E., LINDSTROM, R. and GOOD, RA. (1974). The ultrastructure of human organ-cultured corneas. I. Endothelium, Arch. Ophthalmol. 92, 516-523.

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MCCAREY, B.E. and KAUFMAN, H.E. (1974). Improved corneal storage, Invest. Ophthalmol. 13, 165-173. MOLL, A.C., VAN RIJ, G., BEEKHUIS, W.H., RENERADEL DE LAV ALETTE, J.H.C., HERMANS, J., PELS, E. and RINKEL VAN DRIEL, E. (1991). Effect of donor cornea preservation in tissue culture and in McCarey-Kaufman medium on corneal graft rejection and visual acuity, Documenta Ophthalmologica 78, 273-278. PELS, E. and SCHUCHARD, Y. (1983). Organ-culture preservation of human corneas, Documenta Ophtalmologica 56, 147-153. PELS, E. and SCHUCHARD, Y. (1985). The effects of high molecular weight dextran on the preservation of human corneas, Cornea 3, 219-227. SPERLING, S., OLSEN, T. and EHLERS, N. (1981). Fresh and cultured grafts compared by post-operative thickness and endothelial cell density, Acta Ophthalmol. 59, 566-575. VAN DER WANT, H.J.L., PELS, E., OLESEN, B. and SPERLING, S. (1983). Electron microscopy of cultured human corneas, Arch. Ophthalmol. 101, 1920-1926.

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SECTION VII: SPERM BANKING

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

24 CHARACTERISATION AND DEPLETION OF MEMBRANE DETERIORATED HUMAN SPERMATOZOA AFTER CRYOPRESERVATION

SONJA GRUNEWALD, UWE PAASCH and HANS-JUERGEN GLANDER* Department of Dermatology/Division of Andrology University of Leipzig, Stephanstrasse 11 D-04103 Leipzig

1. Introduction Cryopreservation and thawing of semen samples is a frequently used technique in assisted reproduction, but is associated with significant damage to spermatozoa. Until now the pathophysiological mechanism underlying this process is poorly understood. Cryopreservation induces externalisation of phosphatidylserine (EPS) at the outer membrane (Glander and Schaller, 1999), which is known to occur early during the terminal phase of apoptosis in somatic cells (Martin et al, 1995). Using the specific binding of Annexin V conjugated microbeads (ANMB) to phosphatidylserine, cells showing EPS were depleted by magnetic activated cell sorting (MACS) (Despres et al., 2000). Investigation of

To whom correspondence should be addressed. 561

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S. Grunewald, U. Paasch & H.-J. dander

markers of apoptosis such as the Fas-receptor (CD95) and activation of cytosolic aspartate specific proteases (caspases) which transduce and execute apoptotic signals, were performed in the subpopulations of sperm (ANMB positive and ANMB negative ones). It was our aim to examine whether cryopreservationinduced membrane deterioration is associated with apoptotic features. 2. Materials and Methods Semen samples were provided by 30 healthy volunteers (donors) in accordance with ethical standard guidelines and written informed consent after sexual abstinence of three days. In addition, 10 semen samples from random patients of the infertility centre were used to investigate any side effects of the MACS system. The investigation of the classical semen parameters was performed according to WHO guidelines (WHO, 1999). The ejaculates fulfilled the following requirements: ratio of spermatozoa to leukocytes greater than 100:1; sperm concentration above 20 Mio/ ml; more than 30% of the spermatozoa with a normal morphological shape; and more than 50% appearing progressively motile. 2.1. Sperm preparation The semen samples were filtered through glass wool to remove the gelatinous masses, then diluted with human tubal fluid, HTF (Quinn et al, 1985), and washed twice with HTF (400 x g, 5 min). The supernatants were discarded and the pellets were used for further experiments. 2.2. Cryopreservation Aliquots were gradually cooled accordingly (McLaughlin et al., 1990) in Nicool LM10 (Compagnie Francaise de Produits Oxygenes) using TEST yolk buffer (Irvine Sci., Catalogue No. 9971) to achieve best vitality parameters of spermatozoa after cryostorage,

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as previously demonstrated (Glander & Schaller, 1999). The tubes were plunged into liquid nitrogen and stored at -196°C. The thawing was performed by incubation in a 37°C water bath for 5 minutes. 2.3. D e p l e t i o n of apoptotic spermatozoa by magnetic cell separation (MACS) Following preparation the spermatozoa were incubated with 100 [A ANMB at room temperature for 15 minutes. The ANMBlabelled spermatozoa are expected to be retained in the separation column, which is placed in a very strong permanent magnet, while the non-apoptotic spermatozoa pass through. After removing the column from the magnetic field, the retained fraction was eluted. 2.4. Detection of CD95 (Fas) on the sperm surface One hundred microlitres of the spermatozoal suspension were transferred to SuperFrost® Plus — slides by the cytospin technique and fixed with methanol-acetone 1:1. The incubation with 50 ill of the monoclonal mouse anti-human CD95 solution (DAKO, M3554, 1:25 in PBS) at room temperature for 60 minutes was followed by washing twice in PBS. Envision™, an alkaline phosphatase labelled polymer in conjunction with Fuchsin substrate-chromogen (DAKO K4018 and K0624, Germany) visualised the binding of the primary antibody. As control assays the spermatozoa were incubated with an unspecific monoclonal antibody (IgGl) from Immunotech (Cat.-Nr.: 571; Germany) as the primary antibody. 2.5. Detection of active caspases in vital spermatozoa Active caspases 1-9 were detected in living spermatozoa through the use of the carboxy fluorescein labelled caspase inhibitor

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FAM-VAD-FMK (carboxyfluorescein, FAM, derivate of benzyloxycarbonyl valylalanyl aspartic acid fluoromethyl ketone, zVADFMK). This cell-permeable and non-cytotoxic caspase inhibitor binds covalently to active caspases 1-9 (Ekert et a\., 1999). The fluorogenic substrate becomes fluorescent upon cleavage by the caspases (Vaux and Korsmeyer, 1999). The detection of activated caspases by the inhibitor was performed according to the instruction manual of the manufacturers of the fluorescein caspase (VAD) activity kit (CaspaTag™, S7300, Intergen Co., Oxford, England) with controls. 2.6. Evaluation of the M A C S technique regarding potential impacts on sperm The following examinations of patients spermatozoa performed before and after MACS:

were

2.6.1. Computer-aided sperm motion analysis (CASA) The Stroemberg-Mika cell motion analyser (Version 4.4, Mika Medical GmbH, Rosenheim, Germany) was applied for determination of sperm motility. 2.6.2. Monitoring of the acrosomal status The acrosome status was monitored by the binding of FITClabelled Pisum sativum agglutinin (Cross et ah, 1986) in a modification according to (Tesarik et ah, 1993). Evaluation was done using the epifluorescence microscope Jenamed (Carl Zeiss, Germany) to differentiate three fluorescence staining patterns: selective staining of the entire acrosome region; spermatozoa unstained in the acrosomal region; and staining in the post acrosomal region, which were regarded as acrosome-intact, acrosome-reacted and dead spermatozoa, respectively (Tesarik et al., 1993).

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2.6.3. Eosin staining As described in the WHO guidelines (WHO, 1999) the eosin staining was used to discriminate vital (eosin negative) from avital (eosin positive) sperm. 2.7. Statistical Analysis Data analyses were performed by the non-parametric Wilcoxon and Mann Whitney U-test for evaluation of the differences as appropriate for data type and distribution. The correlation coefficient (R) between various parameters was determined applying Spearman's rank sum test, utilising the statistical computer program STATISTICA 6.0 for Windows (StatSoft Inc., Tulsa, OK 74104, USA). Values are expressed as mean + standard error of the mean (SEM). P values of less than 0.05 were considered as statistically significant. 3. Results An enrichment of spermatozoa after cryopreservation with superior quality was the aim of magnetic cell separation (MACS). The efficacy of MACS separating spermatozoa without ability to bind Annexin V microbeads (ANMB") and spermatozoa binding these beads (ANMB + ) was evaluated in neat and cryopreserved semen samples. Analyses of the unlabelled sperm fraction (ANMB+) revealed a significantly lower percentage (p < 0.01) of sperm with apoptosis markers: only 0.6 ± 0.3% (X ± SEM) showed CD95 on their surface, whereas 40.6 + 6.7% of the remaining spermatozoa in the column (ANMB+) were CD95 positive (p < 0.01). The sperm count with detectable CD95 domain did not significantly differ between neat and cryopreserved semen in the non-separated samples, as well as in the analogous fractions after MACS (p > 0.05; Table 1).

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566

Table 1. Spermatozoa showing CD95 on their surface before and after immunomagnetic cell separation (MACS), mean ± SEM, Wilcoxon test:a-b p < 0.01; Mann-Whitney-U test:c'd-e p > 0.05.

Semen samples

CD95+ overall [%]

CD95+ in ANMB" [%]

CD95+ in ANMB+ [%]

Neat (n = 10) Cryopreserved (n = 10)

2.5 ± 0.5C 4.3±1.2C

0.1 + 0.1a'd 0.6±0.3b-d

48.9 ± 6.1a-e 40.6±6.7b-e

Table 2. Activated caspases in neat and cryopreserved spermatozoa before and after MACS, mean + SEM; n = 10 paired semen samples; Wilcoxon test (*p<0.01). MACS decreased significantly (a-bp<0.01) the percentage of neat and cryopreserved spermatozoa with active caspases 1-9.

Sperm characteristics Neat Cryopreserved

ANMB+ [%] 14.4 ±2.5 49.0 + 8.1*

Pan caspase"1" overall [%] a

21.8 ± 2.6 47.7 + 5.8*
Pan caspase"1" in ANMB" [%] a

9.2 ± 1.4 9.3 + 2.2b

Pan caspase"1" in ANMB + [%] 97.7 + 1.0 89.1 ± 2.3*

The correlation between CD95 and phosphatidylserine at the spermatozoal surface was found to be significantly positive (R = 0.84, p<0.01). After cryopreservation a significantly higher percentage of spermatozoa with active caspases was detectable compared to the neat semen samples (47.7 ±5.8% versus 21.8 ±2.6%; p < 0 . 0 1 ; Table 2). The MACS separation technique resulted in a significant depletion (p < 0.01) of spermatozoa having caspases activated within the ANMB - fractions: to 9.3 ± 2.2% in the cryopreserved spermatozoa and to 9.2 ±1.4% in the neat semen group. Indicating overabundant separation capacity, the percentage of

567

Depletion of Apoptotic Human Spermatozoa

caspase-positive sperm in the ANMB fraction did not significantly differ between neat and cryopreserved sperm (p > 0.05; Table 2). A significant positive correlation between the overall percentage of spermatozoa with activated Caspases 1-9 and those binding Annexin V was measured in the neat as well as in the cryopreserved semen sample group (Spearman rank sum: R = 0.89 [neat], R = 0.95 [cryopreserved]; p < 0.01), whereby cryopreservation increased both parameters (Figs. 1 and 2). There was no significant influence of the separation column and the magnetic field on the sperm functions of patients' semen samples (% spermatozoa before versus after passage; p > 0.05), such as progressive motile spermatozoa 32.5 ± 4.7% versus 31.5 ± 4.9%, spermatozoa with intact acrosome 16.7 + 2.0% versus 15.4 ± 1.9%, spermatozoa stainable by eosin 29.2 + 6.4%

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versus 29.6 + 6.7%. The passage through the column led to a sperm loss of 0.8 + 1.2%. 4. Discussion The term apoptosis defines a cell death programme, which is molecularly and morphologically distinct from necrosis. Intense research gave a remarkable insight into that signalling cascades through the network of pathways. While apoptosis is extensively described for somatic cells, there is sadly, a lack of data in male germ cells. Recent models of apoptosis include receptormediated pathways and intrinsic triggered apoptosis besides cytotoxic or stress-induced forms. Binding of ligands like Fas-L activates the death receptors (for example, CD95, Fas) resulting in trimerisation and coupling with adapter proteins to form a death-inducing signalling complex (DISC). The Fas-receptor (CD95) is a glycosylated cell surface molecule of about 45 to

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52 kDa (Walczak and Krammer, 2000). DISC transduces the apoptotic signal via activation of caspases. Caspases (cytosolic cysteine containing aspartate specific proteases, CP) are a family of highly specific proteases that contain an amino acid, cysteine, in their active sites. After proteolytic activation in a cascade (Thornberry and Lazebnik, 1998; Wolf and Green, 1999) their targets are cleaved after the amino acid aspartate (Alnemri, 1997). Depending on their function, two groups of caspases could be differentiated: initiator (CP 8, 9, 2, 10), and effector or downstream (CP 3, 6, 7) caspases. Caspase 8 was identified to be the most important initiator enzyme triggered by DISC (Kischkel et al., 1995). CP9 realises, together with many other regulators and transductors (for example, cytochrome c released from disintegrated mitochondria), intrinsic apoptosis. Both enzymes are highly effective activators of downstream caspases. CP3, the most important among them, executes the final disassembling of the cell by cleaving of a variety of cell structure proteins. On the other hand, activation of protein kinase C5 which is involved in the phosphatidylserine redistribution and endogenous endonucleases is known to be CP3 triggered (Frasch et al., 2000). Finally, the caspase-activated DNAse generates DNA strand breaks resulting in decreased male fertility (Enari et al., 1998; Zini et al., 2001). Therefore, the usage of spermatozoa that are about to absolve apoptotic processes, should be minimised in assisted fertilization techniques. After cooling biological membranes, a phase transition of the lipids in the membrane of spermatozoa takes place (Hammerstedt et al., 1990). This reordering of membrane components may lead to a loss of stability of the lipid bilayer (Schiller et al., 2000) and exposure of phosphatidylserine on the sperm surface, which occurs in nucleated cells during early phases of apoptosis (Fadok et al., 1992). The location of phosphatidylserine on the outer leaflet of the plasma membrane can be identified by the phosphatidylserine-binding ligand Annexin V, a 3 5 36 KD protein (van Heerde et al., 1995). The specifity of Annexin V-binding to phosphatidylserine has been demonstrated (Martin

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et ah, 1995) and was applied in our experiments for immunomagnetic separation of spermatozoa after cryopreservation. Both the death receptor CD95 and the activation of caspases 1-9 were predominantly found within the ANMB positive spermatozoa. Cryopreservation significantly raised the number of cells showing externalised phosphatidylserine. The elevation in CD95 positive cells after cryopreservation was not significant, but there was a marked increase of Pan-caspase positive sperm. The process of freezing and thawing increased the levels of spermatozoa presenting with those activated enzymes in the ANMB positive fraction only. This indicates induction, transmission and execution of apoptosis due to cryopreservation. Obviously, cryopreservation has had no significant impact on CD95. These results imply that apoptosis due to cryopreservation is not triggered by Fas-Ligand — Fas-Receptor (CD95) interaction, but involves the caspase cascade. The association between externalised phosphatidylserine and CD95 may represent a significance of the death receptor in different settings. Our investigations revealed a highly significant correlation between externalisation of phosphatidylserine and CD95 at the spermatozoal surface, as well as activation of Caspases 1-9 in the cytosol. The Annexin V-MACS technique was able to strongly reduce spermatozoa that exhibit these features of apoptosis, and might be a very useful tool to achieve vital, non-apoptotic spermatozoa in order to improve the fertilisation rates in assisted reproduction. Furthermore, the separation columns and their magnetic field did not exert any detectable effect on the spermatozoa in our experiments — a prerequisite for application in the clinical laboratory. MACS may be used not only for providing a high quality sperm fraction. The technique may also be applied to the evaluation of semen sample quality by detection of the ratio between ANMB" and ANMB+ — sperm fractions. Thus, the known feasibility and safety of the MACS enrichment procedure in patients with autologous transplantation of peripheral blood stem cells (Despres et ah, 2000) was confirmed by andrological examination methods.

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Taken together, the binding of superparamagnetic Annexin V-conjugated microbeads is an excellent method with which to deplete apoptotic spermatozoa from cryopreserved semen samples. 5. Summary Cryopreservation increases the rate of spermatozoa with decreased capability to fertilise oocytes. In order to optimise the fertilisation rates, a better understanding of the underlying biochemical process is necessary. Externalisation of phosphatidylserine at the outer membrane as a feature of apoptosis in somatic cells, was observed after cryopreservation. It was the objective of our study to evaluate apoptosis in terms of CD95 (Fas-receptor) on the sperm surface, and activated caspases in the cytosol of spermatozoa showing cryopreservation-induced membrane damage. Using the high affinity of Annexin V to phosphatidylserine 30, neat and cryopreserved semen samples were treated by immunomagnetic cell sorting (MACS) with Annexin V microbeads (ANMB) to deplete cells with deteriorated membranes from those cells with intact ones. Cryopreservation increased the number of cells with externalised phosphatidylserine and activated caspases, but not those having CD95 on their surface — indicating an important role of the caspase cascade in cryopreservation-induced apoptosis. The Annexin V-MACS was able to remove sufficient CD95-positive and caspase-positive spermatozoa from neat as well as from cryopreserved semen samples. There was no significant influence of the separation column and the magnetic field on the sperm functions, and a very slight sperm loss by the Annexin V-MACS. Therefore, this technique is an excellent method with which to eliminate spermatozoa at early apoptotic stages, from the cryopreserved semen samples, and might be a useful tool in future assisted reproduction programmes.

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6. References ALNEMRI, E.S. (1997). Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases, /. Cell Biochem. 64, 33-42. CROSS, N.L., MORALES, P., OVERSTREET, J.W. and HANSON, F.W. (1986). Two simple methods for detecting acrosomereacted human sperm, Gamete Res. 15, 213-226. DESPRES, D., FLOHR, T., UPPENKAMP, M., BALDUS, M., HOFFMANN, M., HUBER, C. and DERIGS, H.G. (2000). CD34 + cell enrichment for autologous peripheral blood stem cell transplantation by use of the CliniMACs device, /. Hematother. Stem Cell Res. 9, 557-564. EKERT, P.G., SILKE, J. and VAUX, D.L. (1999). Caspase inhibitors, Cell Death. Differ. 6, 1081-1086. ENARI, M., SAKAHIRA, H., YOKOYAMA, H., OKAWA, K., IWAMATSU, A. and NAGATA, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD, Nature 391, 43-50. FADOK, V.A., SAVILL, J.S., HASLETT, C , BRATTON, D.L., DOHERTY, D.E., CAMPBELL, P.A. and HENSON, P.M. (1992). Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognise and remove apoptotic cells, /. Immunol. 149, 40294035. FRASCH, S.C., HENSON, P.M., KAILEY, J.M., RICHTER, D.A., JANES, M.S., FADOK, V.A. and BRATTON, D.L. (2000). Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta, /. Biol. Chem. 275, 23065-23073. GLANDER, H.J. and SCHALLER, J. (1999), Binding of Annexin V to plasma membranes of human spermatozoa: A rapid assay

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for detection of membrane changes after cryostorage, Mol. Hum. Reprod. 5, 109-115. HAMMERSTEDT, R.H., GRAHAM, J.K. and NOLAN, J.P. (1990). Cryopreservation of mammalian sperm: What we ask them to survive, /. Androl. 11, 73-88. KISCHKEL, F.C., HELLBARDT, S., BEHRMANN, I., GERMER, M., PAWLITA, M., KRAMMER, P.H. and PETER, M.E. (1995). Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor, EMBO J. 14, 5579-5588. MARTIN, S.J., REUTELINGSPERGER, C.P., MCGAHON, A.J., RADER, J.A., VAN SCHIE, R.C., LAFACE, D.M. and GREEN, D.R. (1995). Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl, /. Exp. Med. 182, 1545-1556. MCLAUGHLIN, E.A., FORD, W.C. and HULL, M.G. (1990). A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semi-programmable freezer, Hum. Reprod. 5, 724-728. SCHILLER, J., ARNHOLD, J., GLANDER, H.J. and ARNOLD, K. (2000). Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy — Effects of freezing and thawing, Chem. Phys. Lipids 106, 145-156. TESARIK, J., MENDOZA, C. and CARRERAS, A. (1993). Fast acrosome reaction measure: A highly sensitive method for evaluating stimulus-induced acrosome reaction, Fertil. Steril. 59, 424-430. THORNBERRY, N.A. and LAZEBNIK, Y. (1998). Caspases: Enemies within, Science 281, 1312-1316.

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VAN HEERDE, W.L., DE GROOT, P.G. and REUTELINGSPERGER, C.P. (1995). The complexity of the phospholipid binding protein Annexin V, Thromb. Haemost. 73, 172-179. VAUX, D.L. and KORSMEYER, S.J. (1999). Cell death in development, Cell 96, 245-254. WALCZAK, H. and KRAMMER, P.H. (2000). The CD95 (APO-1/ Fas) and the TRAIL (APO-2L) apoptosis systems, Exp. Cell Res. 256, 58-66. WHO (1999). WHO Laboratory Manual for the Examination of Human Semen and Sperm — Cervical Mucus Interaction. Cambridge University Press. WOLF, B.B. and GREEN, D.R. (1999). Suicidal tendencies: Apoptotic cell death by caspase family proteinases, /. Biol. Chem. 274, 20049-20052. ZINI, A., BIELECKI, R., PHANG, D. and ZENZES, M.T. (2001). Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men, Fertil. Steril. 75, 674-677.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

25 A REPOSITORY SYSTEM FOR CRYOPRESERVED SEMEN SAMPLES AND TESTICULAR BIOPSIES EMBEDDED IN A WORKFLOW MANAGEMENT SYSTEM

CORNELIA THIEME, UWE P A A S C H a n d HANS-JUERGEN G L A N D E R D e p a r t m e n t of Andrology, University of Leipzig Stephanstrasse 11, 04103 Leipzig, G e r m a n y

1. Introduction High levels of quality standards, multi-disciplinary and multicentre co-operation are demanded in the management of patients in andrology (ESHRE Andrology Special Interest Group, 1998). Huge amounts of data originating from several workplaces and units are acquired during the diagnostic and treatment of infertile couples. In addition, cryopreservation of ejaculated spermatozoa and testicular biopsies as a standard therapeutic procedure, has to be organised in a reliable and safe way. Therefore, fast electronic recording systems and standardised data management tools would facilitate the treatment approaches in reproductive medicine (Evidence-Based Medicine Working Group, 1992). However, the paper-based medical record is commonly used for documentation in spite of the advantages of computerisation

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(Moritz et at, 1995). These situations originated from the lack of specialised software and interfaces which would allow the automated processing of information, and provide active support to control the process of diagnosis and treatment (Ludwig et ah, 2001). On the other hand, the advantages of computerisation for a repository is well known. Therefore, "standalone" data systems for those tasks in andrology are available. The disadvantage of these systems lies in the missing connection to all other clinical data. This type of data management limits the value and accessibility of that data (Paasch and Glander, 1997). The integration of all systems to a workflow supporting system is restricted due to the very basic specification of interfaces and standards of data exchange (HL7, Health Level 7, Internet: http://www.hl7.de/) used in health care. Therefore, new concepts of integration and workflow managment systems are required to support the process of medical care and repository management according to the latest guidelines, and to assure the highest level of quality (Mortimer and Fraser, 1996; Evidence-Based Medicine Working Group, 1992).

2. Methods A concept of an electronic database programme with an embedded repository system for cryopreservation has been developed from December 1995 to June 2002 (Winsperm®, WSP). 2.1. Software and database m o d e l Out of a long list of available products with their own strengths and weaknesses (Oracle, SQL-Server, Sybase, Informix, Interbase, Delphi, Paradox, Dbase, Approach, 4th Dimension, File Maker, FoxPro ...) the relational database management system (RDBMS) Microsoft® Access® (MSA) integrated in the software package Microsoft® Office® for Windows 9x/NT/2000® was chosen because of its wide distribution at andrology centres in Germany.

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MSA is a powerful RDBMS that incorporates a client-server architecture and allows the portability to the hard- and software used at the infertility centres. On the other hand, this system causes a few problems. Firstly, there is no downstream compatibility (for example, Access 2.0, 95 (7.0), 97 (8.0)). Secondly, it is difficult to set up a management system for real time processing of high amounts of data under remote area conditions. All data are stored in four backend systems, 114 tables, more than 100 reports and approximately 1,000 database fields. User Access is given by two FrontEnds: WSP Client and WSP Work. The first realises a structured data entry system while the latter gives unlimited access for scientific evaluation. The CryoBase is defined as a completely integrated repository system which offers all the features needed for the administration of cryopreservation-related data. The software development was organised according to the principles of evolutionary system development in co-operation with six university-based centres of andrology (Leipzig, Muenster, Marburg, Frankfurt, Munich and Jena (Floyd et al., 1997; Kushniruk, 2002). This type of development realised a close integration of the users from the outset, and resulted in an enhanced quality of process management during software development. 2.2. The w o r k f l o w — M o d e l The couple orientated view entails the integration of all logistic, administrative, evaluation and execution of diagnosis and treatment into a complete module within a electronic system. Computer systems designed to support those tasks are called electronic Workflow Management Systems (WMS). WMS are designed for fast and structured acquisition of data at the place and time at which they are produced. In addition, the quality of data is improved if the owner or producer of the data practises reliable methods for storage after-control (Hastedt-Marckwardt,

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1999). Electronic signatures and authorisation settings had to be implemented to achieve high quality results. Working environments supported by WMS are suitable for handling even large amounts of data — as is the case for repository systems for cryopreserved specimens — which are necessary for andrologists. An optimal solution offers the complete integration of those functions into the WMS. The first level of workflow management control is realised in WSP by 12 separate waiting room lists (WRL) according to the diagnostic and treatment regimen. The workflow tasks are generated at the moment of a patient's registration at the desk. However, at each point of consultation within the workflow process, new tasks can be created by the medical staff. First of all, the simple generation of WRL avoids the time consuming search for the recordset needed to start the next task. By clicking at one of the list entries out of the appropriate WRL, the recordset needed is instantly selected, and all data can be entered into several of the Task Office (TAO) data forms in a structured manner. After finishing the data entry, a double-click on the WRL item terminates the given workflow process and deletes the item from the WRL. The couple then can be notified for another WRL which ensures the proper adjustment of the whole process of diagnosis and treatment (Leymann, 1997). 2.2.1. The Control Centre The screen which is seen at the start of the system is called Control Centre (COC, Fig. 1). It forms a complete navigation and acquisition system for demographic data (patient navigator, PAN), the waiting room list (WRL) as a part of the workflow management system, and the access to all data forms by means of the Task Office. In addition, a centrally-placed menu bar houses icons of the CryoBase; the report manger (REM, Fig. 2); forms which display all important data of the couple at a glance; parameter settings; password change function, and the statistics as well as a quality control module.

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2.2.2. Patient navigator The upper third of the COC is defined as PAN. There the desired couple, in terms of demographic data as well as information about health care insurance, the general practitioner, the date of first admission and the main reason for consultation, can be easily selected. Almost all people in Germany who are insured for health care are equipped with an electronic smart card which stores demographic data provided by the insurance company. Therefore WSP is equipped with an smart card reader. Reading the insurance company smart card (ICC) is the probably best way to search for a male or a female partner within WSP. Alternatively, the database can be searched using the couple number, or by jumping to the first or final recordset. Finally, one can move throughout the database by incremental or decremental search of the couple number. Once the desired recordset of a couple is selected, all demographic data might be rapidly updated using the form demographic data. Also, the notices to general practitioners are accessible here. If the first name, name and date of birth entered by hand or smart card do not match with any entries for male or female demographics, a new recordset of demographic, data for a couple is created automatically by increasing the couple number. 2.2.3. Workflow management system To initiate a work flow the actual within the PAN, the selected couple must be simply notified for a WRL. WSP is developed with 12 WRL: spermatology, consulting room 1-5, psychologist, blood test, secretary, ultrasound, operation and genetic council. According to the nature of the WRL, the male alone, or both partners, might be notified separately. The workflow is then continued by selecting the appropriate WRL at the workplace. A single click on a WRL list entry selects the couple within the PAN and all data can be acquired using the TAO. The TAO also provides direct access to the cryopreservation repository system

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(CryoBase) via the form microsurgery and spermatology. Once all data are stored, the workflow is terminated by a double-click on the WRL list entry. List entries can be recovered even when deleted by accident. According to the process of diagnosis and treatment, a new WRL entry might be set. 2.2.4. Task Office According to the appropriate data relations and the process of data creation in diagnosis and treatment, the TAO of WSP provides the access to standardised forms for data entry (Fig. 3). At the time of data entry the data's correctness, completeness and plausibility is checked if possible. To avoid wrong entries because of misinterpretations, a small tip-text appears for each field if selected by the cursor. All forms are designed in a consistent manner, and consist of a head and a body. The head contains control buttons and fields to display the couple selected, in conjunction with optional buttons (for example, CryoBase at spermatology). 2.2.5. Data evaluation There are many ways to evaluate data in WSP. A variety of reports are supplied by the REM of WSP and its optional modules. Alternatively, queries might be used for scientific evaluation using WSP Work. In addition, the open database connectivity (ODBC) interface can be used by programmes equipped with it to directly extract the desired data out of WSP. 2.2.6. WSP Client Reports First, the report manager (REM, Fig. 2) allows a rapid printout of 51 differently designed reports of all areas of routine diagnosis and treatment. These reports can be previewed separately for women and men; instantly printed or exported to Microsoft® Word®. The address of the general practitioner and a second

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undersigning officer may be added optionally. The integrated repository system CryoBase and the module for Statistics are equipped with their own REM and together provide an additional 44 reports. Apart from routine documentation for patients, WSP is equipped to provide laboratory documentation (log files). Another tool for rapid communication is the set of two serial letter modules and an epicrisis writing system integrated into the central menu bar. The first serial letter module designed to create letters to referring colleagues; the second serial letter module is to send letters to patients; and the latter writing system is used to create rapid documentation. All modules are equipped with a SLL containing standard sentences. After those have been selected, free text entry is allowed to adapt the text to special needs. The same time of report creation possibilities is integrated into the histology form. 2.2.7. Query formulation in WSP Work The use of queries is the most powerful way for sorting, calculating, and organising data from any of the incorporated tables of the database. Queries produce a temporary collection of fields of the selected recordsets with the latest data every time they are run. Queries on this database can be independently designed by the users. A query assistant implemented in Microsoft® Access® allows users to create queries easily, without any knowledge of the Structured Query Language (SQL) which has to be used. Alternatively, the SQL-syntax may be used. 3. Cryopreservation Repository System — CryoBase Evidence based medicine in reproductive medicine comprises deep temperature cryopreservation in close conjunction with all other features of clinical procedures. Therefore it is not only necessary to be in charge of a sufficient repository system — a close connection to all other patient's data has to be established.

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Therefore WSP contains a completely integrated repository system called CryoBase. CryoBase consists of a standard repository system in conjunction with a powerful search engine and also the German version of a system for billing and dunning, as well as an automatic alert function (Fig. 4). The interrelation of the repository system with the workflow of WSP, starts at the level of data acquisition for ejaculates or microsurgery. Both forms are equipped with the appropriate button for the CryoBase. Therefore, not only data as used for the repository are accessible by the system — all related data to a frozen specimen can be extracted out of the system. Even if a sample has been used for therapeutic interventions, the interrelations are not deleted. This concept allows clinical and scientific data access at any time. Reports and contracts of the CryoBase (n = 15) can be created as known from the standard REM. In addition, samples for scientific purposes can also be administrated by that repository system. 4. Results WSP was first introduced at a single workplace in 1997. Real time networking and perfection of the WMS began in 1998. At present WSP has been migrated from Microsoft® Access® 95 over to Access 97 and then Access 2000. The computing power of a standard office computer is found to be sufficient to run WSP smoothly. Network configuration and data transmission bandwidths are very important to support (instead of hinder) the daily clinical work. To date, there are no reports from users about any instability of the software which is used an different platforms (Microsoft® Windows 9x/NT/2000®), network operating systems (Microsoft® Windows NT/2000 Server®) and network facilities (twisted pair, glass fibre). Compromised databases are often the result of disrupted communication to the server during the writing of recordsets. However, administrators are able to fix a damaged backend with the integrated repair

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module (Paasch and Glander, 1999a; Paasch and Glander, 1999b; Paasch et ah, 2000). WSP is at present used at six university centres in Germany, and another three centres are going to implement it. Up to autumn 2002, the DWH in our centre contains approximately 5,500 couples and approximately 40,000 clinical record sets. Almost 6,500 spermiograms with 1,460 related CASA results are stored in the proper relation to all other data within WSP. Time is saved in daily routine work by the usage of the three REM with 95 templates to create letters, reports, log files and statistics. Of additional advantage is the Statistics module which instantly provides the latest results of quality control by computing the so-called monthly mean. This module provides, in addition, the easiest way to detect errors due to wrong data entry. Presently, the 380 semen samples or testicular biopsies stored within the CryoBase are also related to all other information of the system. This close integration of a repository system into a workflow management system provides unique access to all data never before realised (to our knowledge). As a consequence, the system has been used to identify trends of classical spermiogram variables, differences in subpopulations of patients, and of testicular cancer (Paasch et ah, 2001). The German Society of Andrology (Deutschen Gesellschaft fiir Andrologie) has classified WSP as a sufficient tool for evaluation of epidemiological trends (Krause et ah, 2002). A German or English trial version can be obtained on request. 5. Conclusion WSP realises the first step toward an andrological DWH with an integrated, complete repository system for cryopreserved specimens. Electronic workflow management systems support the administrative and scientific decision-making at present, and will be an important fact of competition in future (Ludwig,

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2002). The integrated CryoBase achieves the close connection of all cryopreservation-related information in a repository system to any data of clinical and research origin stored in WSP. Furthermore the real time online administration capabilities help staff to achieve better internal quality control in the cryobank. 6. Summary Real time acquisition of all clinical and logistic data concerning cryopreserved semen or testicular tissue combined with "Datamining (DMG)" within a "Data Warehouse (DWH)" for andrology realises a step towards better handling and internal quality control of a cryobank. The DWH was setup on operational databases which was not available for andrological settings u p to the introduction of Winsperm® in 1997. This relational database management system accomplish a specialised workflow database to store, retrieve and evaluate all data of the samples cryopreserved in term of a repository system. Our objective is the presentation of the repository system CryoBase implemented in Winsperm®2003 (WSP), a specialised workflow database for standardized acquisition and evaluation of data of infertile couples as a prerequisite of an evidence based medicine according to the guidelines of the European Academy of Andrology and European Society of Human Reproduction and Endocrinology (EAA and ESHRE). 7. References ESHRE ANDROLOGY SPECIAL INTEREST GROUP (1998). Guidelines on the application of CASA technology in the analysis of spermatozoa, Hum. Reprod. 13, 142-145. EVIDENCE-BASED MEDICINE WORKING GROUP (1992). Evidence-based medicine. A new approach to teaching the practice of medicine, JAMA 268, 2420-2425.

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FLOYD, C , KRABBEL, A., RATUSKI, S. and WETZELL, I. (1997). Zur Evolution der evolutionaren Systementwicklung: Erfahrungen aus einem Krankenhausprojekt, Informatik-Spektrum 20, 13-20. HASTEDT-MARCKWARDT, C. (1999). Workflow-ManagementSysteme, Informatik-Spektrum 22, 99-109. KRAUSE, W., BEHRE, H.M. and NIESCHLAG, E. (2002). Bestandsaufnahme der Entwicklung der Andrologie in Deutschland, Reproduktionsmedizin 18, 89-91. KUSHNIRUK, A. (2002). Evaluation in the design of health information systems: Application of approaches emerging from usability engineering, Comput. Biol. Med. 32, 141-149. LEYMANN, F. (1997). Transaktionsunterstuetzung fuer Workflows, Informatik Forsch. Entw. 12, 82-90. LUDWIG, C.A. (2002). Qualitatsstandards fur das computerbasierte Patientendossier, Schweizerische Arztezeitung 82, 291-292. LUDWIG, C.A., BUERKI, L., ECKHARDT, A. and CAMEY, B. (2001). Strategiegesteuerte Informationssysteme fur das Gesundheitswesen, Swiss Medical Informatics 48, 6-9. MORITZ, V.A., MCMASTER, R., DILLON, T. and MAYALL, B. (1995). Selection and implementation of a laboratory computer system, Pathology 27, 260-267. MORTIMER, D. and FRASER, L. (1996). Consensus workshop on advanced diagnostic andrology techniques, Hum. Reprod. 11, 1463-1479. PAASCH, U. and GLANDER, H.J. (1997). Contribution to a standardised computer assisted sperm motion analysis, CASA (System Stroemberg Mika), Z. Hautkr. 72, 29-34. PAASCH, U. and GLANDER, H.J. (1999). Principle network architectures using the andrological database Winsperm 99.x, Andrologia 31, 383.

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PAASCH, U. and GLANDER, H.J. (1999). The andrological Database Winsperm® 99.x — Techniques of network architecture and remote support. 1st European symposium on Teledermatology, Jena 19-20 November 1999, Allergologie 22(11), 682-683. PAASCH, U., THIEME, C. and GLANDER, H.J. (2000). Winsperm — Elektronisches Datenmanagement in der Andrologie, /. Pert. Reprod. 10, 13-25. PAASCH, U., THIEME, C. and GLANDER, H.J. (2001). Analyses of sperm quality in large populations over decades by "Datamining" and "Datawarehousing" (Winsperm®). Andrology in the 21st Century, /. Androl. 22(Suppl), 188.

SECTION VIII: CRYOPRESERVATION

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Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

26 FINDING THE IDEAL FREEZING CURVE FOR TISSUES THROUGH INDIRECT THEMOPHYSICAL CALCULATION

YOUNG-HWAN PARK, WOONG-SUB YOON 1 , TAEK-SOO KIM1, CHEE-SOON YOON 2 , SHI-HO KIM3, HAN-KI PARK, JONG-HOON KIM, DONG-WOOK HAN, JONG-CHUL PARK, BUM-KOO CHO Department of Thoracic and Cardiovascular Surgery, Yonsei Cardiovascular Research Institute, Cardiovascular Hospital, Yonsei University College of Medicine 1 Yonsei Propulsion-Combustion Laboratory, Yonsei University College of Mechanical Engineering, Seoul 2 Department of Thoracic and Cardiovascular Surgery, Konyang University College of Medicine, Daejun 3 Department of Thoracic and Cardiovascular Surgery, Donga University College of Medicine, Busan, Korea

1. Introduction The human cardiac valve allograft is the choice replacement for a damaged cardiac valve due to valvular heart disease or degeneration. The cardiac valve can be harvested from braindeath donors, and tissue preservation is the most important process. These tissues must be treated by chemical agents, 593

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radiation and refrigeration. However, the duration of preservation is limited; therefore these valves must be frozen. The freezing method presents several problems. Firstly, the latent heat of fusion changes the crystal size of water during freezing, and will damage the cellular structure. Secondly, there is the problem of variability of crystal size below -80°C. Angel et al, reported his experience with the cryopreservation method. (Angel, 1976). Below -80°C, the frozen tissue will be stable in a so-called "glassy state". At present, most homografts are stored in liquid nitrogen at about -196°C. Computer-controlled freezing with liquid nitrogen solves the problem caused by the latent heat of fusion. The freezing programme for valved homografts is well known and is widely used in most tissue banks. This freezing programme will not be applied to the saphenous vein, carotid artery or tissue engineering products. So, studies are needed to determine new ideal freezing curves for various tissues, using a simulation programme. 2. Materials and Methods 2.1. Freezing process The physiochemical behavior of materials during freezing is shown in Fig. 1. In pure water (ABCDE) and aqueous solutions (A'B'C'D'E'), the chart shows the temperature change with time. Before the process of crystallisation, the temperature drops below the freezing temperature. This is the so-called super-cooling that provides the activation energy during the nucleation process as a non-equilibrium state. In the case of pure water, before crystallisation, several degrees of super-cooling are observed. When the water is frozen, air bubbles, particles or rough surfaces will be needed for nucleation. If these are absent, water will be super-cooled. After the development of small crystals, the freezing process occur rapidly. This phenomenon is observed in ampoule freezing, but not in a freezing solution. In aqueous solution, the starting temperature

Finding the Ideal Freezing Curve for Tissues

595

Time Fig. 1. Freezing curve.

of freezing (B') is higher than that of pure water (B) because the solute acts as the nucleation point. Because the freezing process of the freezing solution is very similar to that of the aqueous solution, and the concentration of the solution is high, supercooling is not observed. Freezing time is defined as the time interval during which the system begins the crystallisation process and releases the latent heat of the system. Modelling this interval is generally a difficult task, so some assumptions were made, and therefore the results may be inaccurate. 2.2. Thermophysical m o d e l i n g Here, the lumped thermal capacity model is defined: • • • • •

the interaction between the system and its surroundings; assume uniformity of temperature; no temperature gradient in the system; no heat transfer within the system; and quiescent environment.

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These assumptions are relatively simple, but they allow easy calculations. Freezing can be divided into three phases: cooling (above freezing), freezing, and cooling (below freezing). The whole freezing process of about 100 minutes was monitored every one second, and thermophysical responses were calculated over 6,000 divisions. For this purpose, we use the equations below to find solutions, which were used to calculate the time constant. During the cooling process, we used the lumped thermal capacity model, and during the freezing process, we assumed that system temperature was constant during the release of the heat of fusion, and after this was released, the freezing process was deemed to have been completed. Releasing heat of fusion was calculated as the summation of thermal flow in each step. 3. Equations 3.1. Cooling process Energy equation: dt : internal energy,

6': heat transfer,

0'v: heat generation.

dt For fixed mass (pV) dU - pVdu, du = CdT, VC* — = 6' p H dt PVc^

at

=

-hcA(T-Te).

Where, Te is the environment temperature. Solution of the temperature response assumes:

597

Finding the Ideal Freezing Curve for Tissues

(a) Bath (chamber) is large. (b) hcA/pVC is approximated by a constant value independent of temperature. The solution is: T-T T0-Te

= g-t/tc

tc =

pVc hrA'

For the direct calculation approach, every thermodynamic property of the system needs to be known before we can find

f

Start J I Density, Latent heat of fussion A Specific heat, Volume, Area, etc.

Read thermophysical properties

Input chamber temp, profile

Cooling process Depression of freezing point

Freezing process

Cooling process

Result out

(

End

")

Fig. 2. Algorithm for direct calculation.

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the thermal response, whereas the indirect approach needs only the time constant from experiments. 4. Direct Calculation Firstly, the programme reads thermophysical properties like density, latent heat of fusion, specific heat and volume, etc. Thereafter, the chamber temperature profile is input. The freezing process is divided into three phases. The first is prefreezing cooling; the second is the freezing process, and the third is the post-freezing cooling. The computer calculates the result and draws the freezing curve (Fig. 2). C Start i

'

Read experimental data • '

Polynomial regression analysis r •

Time constant calculation '' Thermal response calculation

Chamber temp, calculation

Sample temp, calculation

Result out

(^

End

^)

Fig. 3. Algorithm for indirect calculation.

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Finding the Ideal Freezing Curve for Tissues

5. Indirect Calculation After reading experimental data of specific tissue freezing, the data is approximately fitted with polynomial curves and the time constants are calculated (Fig. 3). Based on the indirect calculation theory, we developed the freezing curve programme. This programme froze four different tissue engineered products. We observed the cellular viability depending on different freezing curves and different freezing methods. 5.1. Results By the direct calculation method, samples of 3.6 cc ampoules, 180 cc saphenous vein, and 200 cc cardiac valve homografts were calculated (Figs. 4-6). There were several differences between the calculated freezing curve and the experimental curve. This

•

Direct Calculation Results (1) 3.6 cc ampoule (solvent)

lv-»8

mperat ure (deg)

—•—Chamber temp. I -•— Sample temp, f

"

J

A.A.L..

.^^

jj)

I

-

-SO

—•— Chamber temp. -•— Sample temp.

i

Si,

8 . -100

V-

E

-

-150

^ Time (min)

Experiment

Time (min)

Calculation

Fig. 4. Result of direct calculation for 3.6 cc ampoule. Left graph shows actual experiment data, and right graph is calculated.

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• Direct Calculation Results (2) 180 cc saphenous vein (solution) 50 — * - j - Chamber temp.

ure (deg]

* ! Sample temp.

h

[ ^EJ^ \ i 1 "\

1J

i-

40

60

80

100

120

Time (min)

Time (min)

Calculation

Experiment

Fig. 5. Result of direct calculation for 180 cc saphenous vein. Left graph shows actual experiment data, and right graph is calculated.

•

Direct Calculation Results (3) 200 cc cardiac homograft (solution) 1

•

I

'

'

'

! 0

V

:

— • — C h a m b e r temp. - • — Sample temp.

• r Chamber temp. —»-j- Sample temp.

-—».-_

^E^4-Q.

i :

-100

E

!

•

~

, , , i , , , i , , , i , 100

Time (min)

Experiment

120

100

120

Time (min)

Calculation

Fig. 6. Result of direct calculation for 200 cc cardiac homograft. Left graph shows actual experiment data and right graph is calculated.

601

Finding the Ideal Freezing Curve for Tissues

• Indirect Calculation Results (1) Inverse operation of the time constant —•—180cc -•— 200cc

insta nt(se

fi

o

CJ

310 4

:

.

•

f

-

\*

-

\^t)}% •

5000 0 -60

-40

-20

0

Sample temp, (deg)

Fig. 7. Inverse operation of the time constant by the indirect calculation method. This graph shows that the time constants from experimental data are different according to the size (volume) and the thermal properties of the tissues.

occurred because the factors working in the equations were incorrect or impossible to be calculated, and there were too many conditions to calculate. By the indirect calculation method, samples of 180 cc saphenous vein, 200 cc cardiac valve homograft were frozen by the usual homograft freezing curve, and the time constant was calculated. This time constant is the time required to reach the same temperature, and there are many differences between two samples (Fig. 7). This graph shows that the inverse operation of the time constant from the experimental data, is different according to the size (volume) and the thermal properties of tissues. Based on the time constant, temperature changes in the chamber with this time constant define the temperature change of the sample tissues (Fig. 8). In the left graph, the freezing curve is the cardiac homograft freezing curve, and if

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•

Indirect Calculation Results (2) Analogy (Time constant)

50 — • — Chamber temp. - • — Sample temp.

— • — Chamber temp. - • — sample torn p. ^

0

o g)

-;

g -50 3

-50

\ \

"3

i

Q.-100

E

-

-150

20

40

60

Time (mi

80

100

1 0

-200

20

40

60

80

100

12

Time (min)

Fig. 8. Example graph for showing analogy by time constant. Temperature change in the chamber with this time constant causes the temperature changes in the sample tissue. So two graphs are the same, depending on the time constant. In the left graph, the freezing curve is the cardiac homograft-freezing curve. If the chamber temperature profile is changed like the graph on the right, the sample temperature will follow as dotted line.

the chamber temperature profile is changed as shown on the graph to the right, the sample temperature will follow as a red line. For the linear temperature response of the sample tissue, the rate of change of the chamber temperature should be infinite (Fig. 9). It is impossible in nature. So the chamber temperature should be controlled in the range of not increasing sample temperature (Fig. 10). The right chamber temperature profile will prevent latent heat damage and reduce the fluctuation of crystal size with temperature variations. This computer programme reads the experimental data through a digitiser, and we can get the data at every turning point. If chamber data is to be changed, the computer calculates the sample temperature curve, and the sample data needs to be changed, the computer calculates the chamber temperature curve. This programme will be plotted

Finding the Ideal Freezing Curve for Tissues

603

• Indirect Calculation Results (3) Inverse calculation of the chamber temperature

— • — Sample temp. -•— Chamber lemp.

Sample temperature

:

Inverse "

Chamber temperature

X >

:

~ n 20

40

Time (min)

Fig. 9. Inverse calculation of the chamber temperature. For the linear temperature response of the sample tissue, the rate of change of the chamber temperature should be infinitely large.

• Indirect Calculation Results (4) —"— Chamber temp. ~*— Sample lemp.

20

40

60

Time (min)

100

120

20

40

60

80

100

120

Time (min)

Fig. 10. End results of the freezing curve. The right chamber temperature profile will prevent latent heat damage and reduce the fluctuation of the crystal size with temperature variations.

Y.-H. Park et d.

604

~m: Fig. 11. After freezing of the saphenous vein with a cardiac homograft freezing curve, the programme found a new ideal freezing curve, and this graph shows actual chamber and sample temperature curves by new program. (The black line is the chamber temperature and the gray line is the sample temperature.)

as combinations of several kinds of curves. After the sample is frozen, the freezing rate of l°C/minute is applied until the sample temperature reaches -80°C. Figure 11 shows the new ideal freezing curve for a saphenous vein. By this method, we found the new ideal freezing curve specific to a sapheous vein (Fig. 11). We found the new curves for various tissue engineered products (Fig. 12). (a) PLGA 75:25 with hMCS (PLGA 75:25 scaffolds cultured with human mesenchymal stem cells). (b) hBone Chip with OBC (human bone chips cultured with osteoblast cells derived from mesenchymal stem cells). (c) FLGA with Rb Chondrocyte (PLGA scaffolds cultured with rabbit chondrocyte).

Finding the Ideal Freezing Curve for Tissues

%%$fi&:

,.. , -. ,.

...•.--..,.,•••...•

s r •*.**-

Specimen I

Specimen II

PLGA 75:25 with hMCS

hBone Chip with OBC

PLGA with Rb Chondrocyte

CAp-AtCol+PLLA with RCO

H

U

r

. . . , .

Specimen III

.,,

-,

.

**-,...

-.,..

^ i . ^ , -

•-••

Specimen IV

Specimen I:

PLGA 75:25 with hMCS (PLGA 75:25 scaffolds cultured with human mesenchymal stem cells) Specimen II: hBone Chip with OBC (human bone chips cultured with osteoblast cell derived from MSC) Specimen III: PLGA with Rb Chondrocyte (PLGA scaffolds cultured with rabbit chondrocytes) Specimen IV: Cap__AtCol + PLLA with RCO (Carbonate apatite-atelocollagen + PLLA composites cultured with rat calvarial osteoblasts) Fig. 12. New ideal freezing curve according to various tissue-engineering products.

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(d) Cap_AtCol + PLLA with RCO (Carbonate apatite-atelocollagen + PLLA composites cultured with rat calvarial osteoblasts). These four tissues were frozen by the simple freezing method as well as by the ideal freezing method, and compared with a no-freezing group. The viability using rate-controlled freezing is better than that using mechanical freezing; however, there were no significant differences between the simple freezing group

120

O

u. >.

• PLGA 75:25 with hMCS

3 hBone Chip with OBC

• PLGA with Rb Chondrocyte

I Cap-AtCol+PLLA with RCO

100

80

m

60

i*B

n

.55 40 > 0)

O

20

non-treated control

mechanical freezing

rate-controlled freezing

Freezing method •

PLGA 75:25 with hMCS: PLGA 75:25 scaffolds cultured with human mesenchymal stem cells H hBone Chip with OBC: human bone chips cultured with osteoblast cell derived from mesenchymal stem cells • PLGA with Rb Chondrocyte: PLGA scaffolds cultured with rabbit chondrocytes • Cap_AtCol + PLLA with RCO: Carbonate apatite-atelocollagen + PLLA composites cultured with rat calvarial osteoblasts Fig. 13. Viability of cells according to freezing methods. Mechanical freezing means freezing at -70°C in a deep freezer. In rate-controlled freezing, each sample is frozen by individual ideal freezing curves.

Finding the Ideal Freezing Curve for Tissues

607

and the ideal freezing group. The rate-controlled freezing means that the specimens are frozen by the new, ideal freezing curve respectively. (Fig. 13) 6. Discussion Cryopreservation methods are well known as an ideal preservation method for cardiac homograft valves. The survival of fibroblast in the extracellular matrix is important for improvement of durability. Each process of harvesting, processing, freezing, storing and thawing is important for cellular survival. Lessening the warm and cold ischemic time, and dissecting the heart on the ice, are essential for cellular survival. During soaking in the antibiotic solution, the temperature and the duration will affect cellular survival. During thawing, the rapid increase of temperature will damage the tissues less than a slow increase. Every process will affect the cellular survival, and should be strictly managed in the tissue bank. The reason for freezing is to achieve a longer preservation period. In the frozen state, the metabolic rate decreases rapidly, and the cell can survive without much of an energy supply. But between -15°C and -80°C, ice crystals will grow and shrink inside the tissue depending on the temperature. Below -130°C, liquid water that is still not frozen, changes into a glassy state. Its viscosity is infinite, and the water cannot be moved. Below -196°C, there is insufficient thermal energy for chemical reactions. Therefore the cardiac valve should ideally be stored in liquid nitrogen. In this study, we want to find an ideal freezing curve that may be applied to various kinds of tissues. Mazur emphasised that intracellular ice crystals are the most harmful factor during freezing and thawing (Mazur, 1984). If the freezing is too fast, the tissue will be super-cooled because intracellular water does not go out; and if the freezing is too slow, the tissue will be damaged because intracellular water is lost due to high oncotic pressure outside the cell, and the cell

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will shrink. For this purpose, the cryoprotectants (DMSO; DimethylSulfoxide) 1M are useful to prevent cellular shrinkage by maintaining the cellular oncotic pressure. Around the temperature of latent heat, the computer-controlled programme is necessary to ensure that the tissue temperature will not increase during freezing. Mazur reported that the freezing rate of 1°C/ minute is ideal to minimise intracellular ice crystallisation after freezing (Mazur, 1984). Takamatsu observed the compression damage between forming ice crystals during rapid freezing, by using a cryomicroscope (Takamatsu, 1999). Most damage will occur around -1.8°C. Brockbank feels that intercellular ice formation would damage the extracellular matrix, and the debris will evoke calcification (Brockbank, 2000). In the carotid artery, Song reported that the vascular function was maintained well when adequate DMSO concentration was below 15%, and the freezing rate was about 0.69°C/minute (Song, 1995). He also developed the vitrification method (socalled ice-free freezing method) which was applied to vascular freezing. He observed that the smooth muscle cells and endothelial cells were functioning well and the structures were well maintained after thawing (Song, 2000). During thawing, Pegg suggested that from -196°C to -100°C the tissue should be thawed slowly so that the fracture of tissue might be prevented (Pegg, 1997). In a tissue bank, several kinds of tissues will be treated, and various ideal freezing curves will be necessary for various tissues. Products of tissue engineering techniques should be frozen by the ideal freezing curve. So, this indirect calculation method can be an alternative method to finding ideal freezing curve. 7. Conclusion Because we cannot know the thermophysical properties of all tissues and solutions, the direct calculation approach is almost

Finding the Ideal Freezing Curve for Tissues

609

impossible. In the indirect calculation approach, data taken from experiments are approximately fitted with the polynomial curve. By the results of experiments with new ideal freezing curves, we found that there are no significant differences among four samples; however, we think that there is a significant difference if the sample size is larger. This method will lower the cost and time, and can be applied to all kinds of tissues via computer simulation. 8. Summary Liquid nitrogen freezing techniques have already met with widespread success in biology and medicine as a means of long-term storage for cells and tissues. The use of cryoprotectants such as glycerol and dimethylsulphoxide to prevent ice crystal formation, with carefully controlled rates of freezing and thawing, allows both structure and viability to be retained almost indefinitely. Cryopreservation of various tissues has various controlled rates of freezing. To find the optimal freezing curve, we investigated two methods of thermodynamic calculation of tissues freezing curves. One is the direct calculation method. We should know the thermophysical characteristics of all components: latent heat of fusion, area, density and volume, etc. This kind of calculation is highly sophisticated, and some variables cannot be determined. The other is the indirect calculation method. We performed the tissue freezing with already used freezing curves and observed the actual freezing curve of that tissue. We also modified the freezing curve by several steps of calculation, polynomial regression analysis, time constant calculation, thermal response calculation and inverse calculation of chamber temperature. We then applied that freezing programme on mesenchymal stem cells, chondrocytes and osteoblasts, but could not find any differences in tissue survival. The reason could be that the freezing material is small and contains cellular components. We expect a significant difference of cellular viability, if the freezing curve is applied to tissues on a large scale.

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We conclude that this programme would be helpful for determining the ideal freezing curve easily. 9. A c k n o w l e d g e m e n t This study was supported by a grant (HMP-98-G-2-052) of the HAN (Highly Advanced National) Project, Ministry of Health & Welfare, Republic of Korea. 10. References ANGEL, J.D., CHRISTOPHER, B.S., HAWTREY, O. and ANGEL, W.M. (1976). Fresh, viable human heart valve bank: Sterilisation sterility test and cryogenic preservation, Transplant Proc. 8 (Suppl. 1), 139 BROCKBANK, K.G., LIGHTFOOT, F.G., SONG, Y.C. and TAYLOR, M.J. (2000). Interstitial ice formation in cryopreserved homografts: A possible cause of tissue deterioration and calcification in vivo, }. Heart. Valve Dis. 9, 200-206. TAKAMATSU, H. and RUBINSKY, B. (1999). Viability of deformed cells, Cryobiology 39, 243-251. MAZUR, P. (1984). Freezing of living cells: Mechanisms and implications, Am. J. Physiol. 247, C125-142. PEGG, D.E., WUSTEMAN, M.C. and BOYLAN, S. (1997). Fractures in cryopreserved elastic arteries, Cryobiology 34, 183-192. SONG, Y.C., PEGG, D.E. and HUNT, C.J. (1995). Cryopreservation of the common carotid artery of the rabbit: Optimisation of dimethy sulfoxide concentration and cooling rate, Cryobiology 32, 405-421. SONG, Y.C, HAGEN, P.O., LIGHTFOOT, F.G., TAYLOR, M.J., SMITH, A.C. and BROCKBANK, K.G. (2000). In vivo evaluation of the effects of a new ice-free cryopreservation process on autologous vascular grafts, /. Invest. Surg. 13, 279-288.

Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

27 THE SAFE AND EFFECTIVE USE OF ALLOGRAFT TISSUE: AN UPDATE

SCOTT A. BARBOUR a n d W A R R E N KING Palo Alto Medical F o u n d a t i o n Palo Alto, California

1. Introduction The use of allograft tissue in orthopaedic surgery has been a tremendous advance in the management of a variety of orthopaedic problems. Today, allograft tissues are commonly used for a variety of procedures, including limb-salvage, ligament reconstructions, cartilage resurfacing, and as osteoconductive and inductive substrates, to name a few. The role for allograft tissue is constantly expanding, and becoming more common. In 1999 alone, US tissue banks distributed over 750,000 allografts (Gadzag and Lane, 1995). The use of allograft tissue has many advantages over autografts including unlimited size, lack of donor site morbidity and availability for revision surgery. However, allograft is not without some disadvantages. It has decreased osteoinductive and osteoconductive characteristics, as well as increased incorporation times when compared to autograft tissues (Gadzag and Lane, 1995). But the most significant disadvantage is the risk of disease transmission.

611

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Currently, the risk of viral transmission through allograft tissue transplantation is extremely low. Proper donor screening and tissue processing help prevent the transmission of viral disease. However, window periods of infection where detection is missed through serological tests, and human error, make transmission still possible (Tomford, 1983). As of 1995, two documented case reports of HIV, one case of Hepatitis B and three cases of Hepatitis C had described infected donors being involved in transplantation of musculoskeletal allografts, resulting in several cases of disease transmission (Tomford, 1983). It is important to note that with steadfast adherence to the screening methods available today, most if not all of these infections would have been avoided (Tomford, 1983). The risk of bacterial infection from allograft tissue is unknown (CDC, 2001). This is in part due to several confounding factors. The lack of a standardised procurement protocol for tissue harvesting; failure of surgeons to recognise or confirm the allograft as the source of infection; and the lack reporting when this complication does occur. We present four cases of Clostridium septicum infection, which occurred following Anterior Cruciate Ligament (ACL) reconstruction with contaminated allografts from the tissue bank. 2. Case 1 A 50-year-old male sustained a torn right anterior cruciate ligament while playing baseball, and received a hemipatellar tendon allograft reconstruction in March, 1998. The fresh frozen graft was removed from the package on the back table, and a sample of the bone and tendon was sent for aerobic and anaerobic culture prior to implantation. The patient was seen in follow up four days later, and was doing well; physical therapy was prescribed. Seven days later the patient was evaluated in the clinic and found to have an erythematous, warm right knee. The patient complained of a significant increase in pain, and reported feeling

The Safe and Effective Use of Allograft Tissue: An Update

613

febrile. Culture results of purulent fluid aspirated from his right knee identified the gram-positive rod, Clostridium septicum. The patient was immediately taken to the operating room where an arthroscopic debridement and removal of the graft and bioabsorbable screws was performed. He was admitted to the hospital and placed on intravenous (IV) antibiotics and his right knee was subsequently debrided multiple times in the operating room. Several intraoperative cultures confirmed Clostridium septicum infection. The patient completed a four-week course of IV antibiotics followed by a two-week course of oral antibiotics as well as physical therapy. His case was further complicated by arthrofibrosis with a range of motion of 15-90 degrees. Three months following the initial operation he was again taken to the operating room for an arthroscopic lysis of adhesions and manipulation under anaesthesia, improving his range of motion to 5-100 degrees. The primary surgeon routinely sends allograft tissue for culture prior to implantation, to determine if any subsequent postoperative infections were from contaminated tissue. In this case, the preoperative tissue culture results correlated with the rare organism cultured from the patient's knee aspirate, indicating that the tissue came from the tissue bank already contaminated. For this reason, the tissue bank was contacted and informed of the incident. They responded that their testing revealed no such contamination of the allograft tissue, and held the position that the infection was the result of contamination at the time of surgery. The tissue bank was also unwilling to share any information regarding methods used to harvest the tissue, or the circumstances of the cadaver donor's death and medical history. Our patient, however, filed a lawsuit against the tissue bank, allowing us access to the cadaver donor's autopsy report and harvesting methods of the tissue bank. From these documents we were able to track other tissues from the same donor to other recipients around the country. It was learned that a patient in another part of the country received the other half of the

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hemipatellar tendon allograft from this donor, and contracted a post-operative infection with the same rare organism, Clostridium septicum. Despite the findings, the tissue bank still held the position that the infections were separate complications, resulting from perioperative tissue contamination. The tissue bank ultimately reached an out-of-court settlement with the patient that included a confidentiality clause prohibiting disclosure of the facts of this case. 3. Case 2 A 50-year-old male was taken to the operating room by a surgeon on April 1, 1998, where a bone patella tendon bone allograft right ACL reconstruction was performed without complication. The patient did well initially; however, approximately 10 days post operatively the patient began to experience increasing pain and swelling of his right knee, as well as high fevers. He was re-admitted to the hospital and taken to the operating room for an arthroscopic debridement of his right knee. Cultures taken intraoperatively eventually grew out Clostridium septicum. On May 5, 1998 the patient was again taken to the operating room where he had a debridement and removal of the interference screws and graft. Intraoperative findings included lysis of the graft and separation of both the tibial and femoral bone plugs from the tendon. On May 11, 1998, the patient had a colonoscopy to rule out a colonic lesion. The findings of that study were negative. The patient was treated with six weeks of appropriate antibiotics, and the infection eventually resolved. Medical records were not available to know the total number of debridements required, or the patient's final condition. The senior author, and the surgeon of this patient, were later discussing the unusual findings of the first two cases at an orthopaedic meeting. Subsequent investigation lead to the realisation that this patient received his graft from the same cadaver donor as the patient in Case 1.

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The tissue bank was informed of these findings, and following investigation, the tissue bank concluded that the two cases were unrelated and no further action was taken. 4. Case 3 On April 27, 1998 a 16-year-old female had her right ACL reconstructed with a bone patella tendon bone allograft by a surgeon. On May 3, 1998 the patient began to experience increasing pain and swelling of her right knee, as well as fevers to 103°F. Her knee was aspirated and sent for culture, and she was admitted to the hospital for intravenous antibiotic treatment. Cultures were negative at five days; however, the patient's condition failed to improve, and she was ultimately taken to the operating room for debridement and removal of the graft on May 8, 1998. Six days later, culture results confirmed infection with the Clostridium septicum species. She was treated with a six-week course of appropriate antibiotics, and a repeat debridement on June 17, 1998, and her condition eventually resolved. She regained full range of motion of her right knee and eventually had a revision autograft bone patella tendon bone ACL reconstruction that proceeded without complication. The patient eventually filed a lawsuit against the tissue bank that was settled out of court with a monetary award to the patient, with an associated gag order. 5. Case 4 A 51-year-old male, an avid runner was treated with a left bone patella tendon bone allograft ACL reconstruction on April 20, 2001 by a surgeon. He had an unremarkable post operative course initially; however, he later began to experience increasing pain and swelling of his left knee, with associated fever to 102°F, and was readmitted to the hospital on May 2, 2001. He was taken to the operating room where an irrigation and

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debridement was performed. The patient had culture results that were positive for Clostridium septicum infection, and he was placed on oral antibiotics that alleviated his symptoms. He was discharged from the hospital on oral antibiotics. Following his discharge the patient suffered a loss of knee motion and was taken to the operating room again on May 25, 2001 for a manipulation under anaesthesia. The patient seemed to improve following the manipulation; however, he again began to experience high fevers to 102°F and was readmitted to the hospital on June 15, 2001. X-ray evaluation of his left knee revealed a distal thigh abscess consistent with a gas-producing organism. He was taken to the operating room on the same day for debridement of his left distal thigh, and cultures again grew out Clostridium septicum. He was subsequently treated with appropriate antibiotics, and his clinical course improved. Unfortunately, he developed a flexion contracture of his left knee, and when he was seen at our institution, he had a left knee range of motion from 40° to 45°. The senior author treated the patient with an arthroscopic lysis of adhesions and manipulation under anaesthesia, ultimately improving his range of motion to 5° to 80°. His legal case is currently in litigation.

6. Discussion Clostridia species are gram-positive rods found in soil, decaying vegetation, marine sediment, and in the intestinal tract of humans, insects and other vertebrates, and most of the species possess the ability to sporulate when exposed to unfavourable environmental conditions (Bennett, 2000). Clostridium septicum has been identified in 2% of stool cultures in previous normal flora studies of humans, and carriage rates of 10-63%, most commonly in the appendix, have been reported in the literature (Godette and Kapta, 1996). Clostridium septicum is most often encountered clinically as bacteraemia associated with intestinal malignancy, or a relapse of leukaemia (Larson and Bubrick, 1995).

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However, careful review of the literature identifies agonal bacteraemia in potential cadaver donors as a significant finding, as it pertains to the procurement of allograft tissues (Isenberg and D'Amato, 1995). MacEwen reported the first use of musculoskeletal allograft in 1880 (Dolano and Kapta, 1991). Lexer reported on 23 cases of articular cartilage transplantation between 1908 and 1925. These reports included phalangeal, elbow and knee joints, and he claimed a 50% success rate (Lexer, 1925). Noyes et ah, reported allograft ligament reconstructions in 1981 (Noyes and Barber, 1990) and Milachowski reported on transplanted menisci in 1984 (Milachowaki and Wermeier, 1989). Today the use of allograft tissues is extremely common, leading to an increasing demand for tissue, and requiring surgeons to keep informed of the risks and benefits of this ever-expanding technology. Musculoskeletal allografts are processed using a variety of methods depending on the tissue being transplanted, and on the requirements of the surgeon. Fresh allograft refers to tissue that is harvested under sterile conditions, that is transplanted directly from the host to the recipient. The tissue is normally maintained in lactated Ringer's solution at 2°C-4°C for a period up to seven days. Studies show that viable cartilage cells diminish substantially in culture media after 24 hours, and are virtually nonexistent after seven days (Shelton and Treacy, 1998). This type of tissue is necessary for successful articular cartilage and meniscal cartilage transplants to preserve viable cartilage cells. Although these tissues are thoroughly washed prior to transplantation, they still contain some marrow elements and viable cells from the donor; thus, an immune response does occur (Langer and Czitron, 1975). Currently, no standard of processing these tissues with regard to washing, antibiotic treatments, or sterilisation treatments (gamma radiation, ethylene oxide, etc.) exists (Oversight Committee on Tissue Banking, 2001). Cryopreservation is a process of controlled-rate freezing using DMSO and glycerol to remove water during the freezing process in an attempt to preserve viable cells. The process works by

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altering water crystallisation during the freezing process, which preserves up to 80% of cells (Shelton and Treacy, 1998). Grafts are cooled to 0°C and processed within 48 hours. They are then incubated in an antibiotic solution for 24 hours at 37°C, and frozen to -135°C and packed in a cryoprotectant solution for up to 10 years (Shelton and Treacy, 1998). This technique works well for meniscal cartilage; however, the process damages articular cartilage. The increased cost prohibits its use for other types of tissue, as other cheaper techniques work just as well (Shelton and Treacy, 1998). Fresh frozen tissue is the most common technique for processing ligament allografts. After tissue harvest under sterile conditions, the tissue is usually cultured and frozen while serologic test results are carried out in a process that takes two to four weeks. It is then soaked in an antibiotic solution at room temperature for one hour, and then packaged and frozen without solution for up to five years (Shelton and Treacy, 1998). No viable cells reliably survive this process (Shelton and Treacy, 1998), which has several important clinical ramifications. Loss of cells decreases the likelihood of immune reactions or disease transmission; however, the effectiveness of this treatment is controversial. Finally, freeze-dried allograft tissues are also commonly used in ligament reconstruction. After harvest under sterile conditions, the tissue is frozen pending serologic and bacterial culture results. The tissues are then subjected to a one-hour bacterial solution soak at room temperature. A process of refreezing and lyophilisation to residual moisture of less than 5%, followed by packaging and storage for up to five years is then carried out. The colour and strength of the tissues are altered; however, studies have shown no deleterious effects on clinical outcomes of ligament reconstruction (Shelton and Treacy, 1998). Allogeneic tissues function as a scaffold that is eventually incorporated into the host. Incorporation generally occurs through three stages. First, death of cells from the donor tissue occurs. This stage is usually considered to be a characteristic of fresh, or

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cryopreserved tissues, because other processing methods (fresh frozen or freeze-dried) are considered to have already caused cell death in donor tissues. The second stage includes revascularisation of the donor tissue, with repopulation of the donor tissue by host cells (Shelton and Treacy, 1998). These first two stages occur relatively rapidly compared to the final stage — remodelling — that occurs more slowly. Jackson et al., demonstrated complete replacement of donor cells by host cells in goat ACL's in four weeks (Backson and Simon, 1963). It has been shown that complete remodelling of allograft tissue may take one-and-a-half times as long as autograft tissue to regain comparable strength (Cordrey and McKorkle, 1963). Prolongation of the remodelling phase may be the result of tissue-antigen mismatch, resulting in a subclinical immune response (Shelton and Treacy, 1998). Several studies in dogs and goats have noted similar gross, and histologic patterns of tissue, between native and allograft ligaments at six months to 1-year post transplantation (Garrett, 1996; Mankin and Doppelt, 1983; Zukor and Paitich, 1989). In addition to proper donor screening, adherence to sterile techniques during procurement and processing, and appropriate serologic and bacteriologic testing and post harvest sterilisation, can improve the safety of allograft tissue. Currently, the two most common methods of sterilisation are ethylene oxide treatment and gamma irradiation. Unfortunately, both of these treatments can have deleterious side effects, and are therefore not universally employed. Ethylene oxide leaves behind a chemical residue that may cause chronic synovitis and graft failure (Jackson and Windier, 1990), and gamma irradiation greater than 3.0 Mrad weakens collagen tissue (Pelker and Friedlaender, 1987). Tissues are weakened by irradiation due to the destruction of collagen chains, probably mediated by oxygen free radicals (Hamer and Stockley, 1999). Recent literature suggests that gamma radiation can be efficaciously employed to sterilise allograft material without a deleterious clinical effect. In 1951 Meeker and Gross reported that 1.5 Mrad destroyed 95% of bacterial organisms, but that 3.0 Mrad

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caused significant tissue damage. Fideler et al, showed that 30 kGy was necessary to eradicate HIV in bone-patella tendonbone allograft at -70°C (Fideler and Vangsness, 1995). In 1956 Turner et al, reported that bone grafts sterilised at 2.0 Mrad underwent abnormal absorption and fragmentation, but reported only two infections in 100 patients receiving 189 allografts, demonstrating the effective sterilising properties of gamma radiation. In 1959, Bassett and Packard reported a less than 1% infection rate in 1,037 patients receiving bone graft sterilised with 2.0 Mrad of gamma radiation. Fideler showed in 1995, that the initial biomechanical strength of fresh frozen bone-patellar tendon bone allograft was reduced 15% after irradiation with 2.0 Mrad of gamma radiation, but that stiffness, elongation, and strain were not reduced with statistical significance. Goertzen reported in JBJS (Br) 1995, that canine BPT-B allograft treated with 2.0 Mrad gamma radiation protected with Argon gas, compared favourably with non-irradiated controls with regard to maximum load to failure at 12 months. The irradiated group failed at 718.3 N (63.8% of normal ACL's), and the control group failed at 780.1N (69.1% of normal). In addition, histologic studies showed no difference between the groups with regard to collagen structure. Silver staining showed the presence of Golgi tendon organs and free nerve endings in both groups, and only a slight hypervascularity was noted in the controls compared to the irradiated group. The literature on sterilisation demonstrates that no technique is 100% effective at rendering allogeneic tissues sterile, but that improved sterility with certain treatment protocols can be employed to improve the quality of these tissues without adversely effecting clinical outcomes. Torisuka et al., studied the effect of 25 kGy of gamma irradiation, and freeze-drying on patella tendon graft remodelling after transplantation in the rat model (Toritsuka and Shino, 1997). They studied four groups, including fresh-frozen, freezedried, fresh-frozen gamma-irradiated, and freeze-dried gamma irradiated. They discovered that freeze drying and gamma irradiation temporarily accelerated graft remodelling in the early

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phase (first 12 weeks), accompanied by an increase in newly synthesised collagen, and a decrease in donor collagen (Toritsuka and Shino, 1997). Histologically, no difference in the progression of cellular repopulation was observed between the four groups. It was thus assumed that the chemotactic properties of the graft materials were unaffected, and that accelerated graft remodelling was potentiated. Although it can be assumed that the rapid decrease in donor collagen would initially weaken the grafts treated in this manner, further studies would be required to determine if this phenomenon has any clinical significance requiring a modification of rehabilitation protocols. One can conclude that grafts treated in this manner would be safer, less likely to promote immunologic reactions, and accelerate incorporation of the grafts. Our current knowledge of the safe procurement of allogeneic tissues was originally adapted from procedures developed by the United States Navy Tissue Bank, and has been modified through clinical experience over many years. As our understanding of the risks and means by which disease is transmitted is further delineated, efforts have been made to modify techniques in order to insure the highest possible safety of allograft tissue. Current understanding of safe tissue banking procedures has lead to the development of a comprehensive screening process for potential donors. These include a detailed medical and social history; serologic tests for HIV I/II antibodies, HIV antigen, PCR HIV, hepatitis B surface antigen, hepatitis B surface antibodies, hepatitis B core antibodies, hepatitis C virus antibodies, and the RPR test for syphilis. In addition, an autopsy of potential donors, with a separate study of lymph nodes, completes the screening process. Currently, only a medical history, social history, and serologic testing are required by the FDA. It is also pointed out by Malinin et ah, in 1985, that procurement of tissue is a surgical skill requiring strict adherence to sterile technique. Procurement is also a clinical service, which carries with it the responsibilities incumbent on any physician providing medical care. As such, it

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would seem logical that personnel who procure tissue be held to the same standard as other medical professionals. Buck and Malinin reported on their experience at the University of Miami Tissue Bank in 1994. They discussed the reasons for exclusion of 187 of 1,000 consecutive donors over a six-andone-half-year period. Eighty-five were excluded on the basis of bacteriologic criteria alone. Seventeen had bacteriological exclusionary criteria as well as serologic a n d / o r morphologic exclusionary criteria. Sixty-eight cases were excluded for hepatitis detected using serologic markers, or morphologic changes, and one case based on histologic changes alone (Buck and Malinin, 1994). Documented or suspected HIV accounted for 10 exclusions. In one case a donor was excluded based on classic nonspecific changes of HIV noted in the lymph nodes (Murchadha and Wolfe, 1987; Racz and Tenner-Racz, 1986). Another donor was excluded based on birefringent material consistent with drug abuse, noted in granulomas located in the liver and lungs (Tomashefski and Hirsch, 1989). Seven donors were rejected for granulomatous disease; another four for unsuspected malignancies, and two for myocarditis. The experience of Buck and Malinin clearly demonstrates the need for screening beyond serologic tests to insure the safety of allograft tissue. In order to understand the potential risks of bacterial infection with the use of allograft tissue, one must first identify the manner in which these tissues become contaminated. A review of the literature reports a 5% to 44% rate of bacterial contamination of allograft tissue. Due to variability of the culture techniques employed it is not possible to compare the contamination rates. Diejkers et al., analysed the incidence and predisposing factors of bacterial contamination of allograft tissue in JBJS [Br] 1996. They evaluated 1,999 bone and soft tissue grafts from 200 cadaver donors under sterile operating conditions. After removal of the grafts from the cadaver donors, they were rinsed with an antibiotic solution in the first 150 donors, and with saline only in the last 50 donors. Swabs were then taken from the entire

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graft surface and placed in a transport medium. The swabs were subsequently inoculated onto blood agar and chocolate agar plates, and cultured under aerobic and anaerobic conditions. The entire swab sticks were then placed in a brain heart infusion broth and cultured for another 72 hours under both aerobic and anaerobic conditions. Blood samples from the cadaver donors were cultured aerobically and anaerobically for seven days. Microbial load was considered low if the microorganisms grew only in the broth, and high if growth occurred directly on the plates. They found that 50% of the grafts cultured positive for organisms of low pathogenicity, and 69% were of a low microbial load. Three percent of the grafts grew out organisms of high pathogenicity. The authors determined that organisms of low pathogenicity (for example, coagulase-negative staphylococci, Corynebacterium, Propionibacterium acnes) were likely to have contaminated the grafts exogenously during procurement. Organisms of high pathogenicity originated endogenously from the donor, and were usually contaminants from the gastrointestinal tract or the upper respiratory tract, and were more likely to cause a clinically significant infection in the recipient. They found that contamination with organisms of high pathogenicity was 3.4 times higher in donors with a traumatic cause of death, and 5.2 times higher in those with positive blood cultures. It was also noted that while washing the grafts with antibiotic solution reduced the organisms of low pathogenicity by a factor of two, organisms of high pathogenicity were not reduced with antibiotic soaks. The authors concluded that exogenous contamination was most affected by the procurement team, and that endogenous contamination was best controlled through careful donor selection (Diejkers and Bloem, 1997). Martinez et al., reported on the microbiologic cultures of blood and bone, of 239 cadaver donors, and 58 "beating heart donors" who had been aseptic prior to tissue harvest. The incidence of positive blood cultures was significantly lower in

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the "beating heart donors" (8.6%) compared to the cadaver donors (38%) (Martinez and Malinin, 1985). Agonal bacteraemia is a well-described process whereby endogenous bacteria, such as normal intestinal flora, are disseminated throughout the body after death in cadaver donors (Isenberg and D'Amato, 1995). This process is thought to be facilitated by the loss of barrier function of gut capillaries, and may also result from trauma or manipulation procedures during resuscitation attempts around the time of death (Diejkers and Bloem, 1997; Veen, 1994). The difference in blood culture results between the two groups of this study was attributed to the persistence of anatomic barriers to microbial invasion, and competent microbial clearing mechanisms in the "beating heart donor" group. Microorganisms were isolated from the tissues of 55.4% of the cadaver donors, and 67.9% of the "beating heart donors" that had negative blood cultures. Clostridium species were the second most commonly isolated group behind coagulase negative staphylococcus. It was also noted that 60% of donors with positive blood cultures for Clostridium species also cultured positive from tissue samples (Martinez and Malinin, 1985). They found the predictive value of positive blood cultures to be 83.5%; however, the predictive value of negative blood cultures was only 44.5% in the two groups. These results demonstrate that blood cultures are ineffective in confirming the sterility of allograft tissue, and that the Clostridium species seem to be disseminated at the time of death in the cadaver donors, and often seed tissues prior to harvest. The tremendous increase in allograft tissue applications, and thus, demand, has resulted in a failure to adequately supervise the safety of this industry. Generally, allograft tissues are extremely safe, and complications resulting from disease transmission are extremely small. However, when they do occur, the consequences are often catastrophic, and potentially avoidable with strict adherence to known protocols designed to insure safety.

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In January 2001, the Office of the Inspector General, Department of Health and Human Services, convened an Oversight committee to profile the current state of tissue banking in this country. It was noted that oversight of tissue banking practices occurs at three levels; the Food and Drug Administration (FDA), the American Association of Tissue Banks, and at the state level. The Food and Drug Administration requires donor screening and testing to prevent the transmission of communicable diseases. The FDA had conducted 188 inspections of 118 tissue banks known to the government at that time, since 1993. The American Association of Tissue Banks (AATB) conducts a voluntary accreditation program that evaluates the procurement practices of each tissue bank. As of January 2001, 58 tissue banks had been accredited, and another 90 identified banks were not. According to the oversight committee's report, only New York and Florida require licensing and inspection of tissue banks, although authorities in California report that AATB certification is required in that state. Furthermore, the Oversight Committee points out that many tissue banks do not seek AATB accreditation because there is no incentive to do so. The committee reported that no cases of disease transmission have been identified since the FDA's regulation regarding donor screening and testing in 1993. However, it is our contention that bacterial infections have occurred as the result of contaminated allograft tissue as demonstrated by our current Case 1 report. We are treating two other patients who had ACL allograft transplants by other surgeons, complicated by postoperative Clostridia infections. In addition, we are aware of a fourth patient who received the other half of the hemi-patellar tendon graft that caused our patient's infection, and contracted the same rare Clostridium septicum infection. In these particular cases, no preoperative graft cultures were taken to confirm the graft was the source of infection, but three patients received a legal settlement from the tissue bank with a nondisclosure clause, and a fourth is currently pending a legal action. Despite the lack preoperative culture results from three of these cases, Clostridium is an

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extremely rare organism in post operative wound infections, and it seems likely that the graft material was the source of infection. We believe that the rarity of contamination, failure of surgeons to recognise allografts as a source of infection, and the tissue bank's failure to properly investigate these complications, has resulted subsequent infections that may have been otherwise avoided. The oversight committee found 36 tissue banks that have never been inspected, out of 154 identified tissue banks, and notes that the actual number and location of all tissue banks is unknown. While the FDA does regulate donor screening and testing, no regulations exist for quality and handling of tissue. Of the 118 tissue banks inspected by the FDA since 1993, 26 notices of official action requiring the banks to take corrective action were issued. In 72 others, notices were issued that suggested changes to improve quality (Department of Health and Human Services, 2001). The FDA reported a list of examples of safety and qualities problems found in those tissue banks inspected. These included "lack of adequate controls to assure product sterility, lack of standard operating procedures to prevent cross contamination of human tissue during manufacture, and distribution and implantation of soft tissue grafts from a single donor with possible bacterial contamination," just to name a few. Clearly, the current supervision and accountability of the tissue banking industry is woefully inadequate and poses an unnecessary risk to recipients of allograft tissue. 7. Summary The use of allogeneic tissue in orthopaedic surgery is generally safe and efficacious. The indications for these tissues continue to expand, and represent a marked advance in the implementation of medical care. Recently, the risks of bacterial infection from contaminated grafts, and the devastating consequences, have become recognised, prompting the CDC and other government agencies to scrutinise the tissue banking industry.

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Several changes in the practice of tissue banking, and the use of allograft tissue, will be required to improve the quality of allogeneic tissue. First it is imperative that tissue banks be closely regulated to insure that proper practices for safe tissue banking are being followed. Furthermore, research needs to be conducted to develop safer methods for tissue procurement. A review of the literature demonstrates that blood and swab cultures, and antibiotic washes, are ineffective in confirming the sterility of donor tissue. In addition, current literature on known methods of sterilisation such as gamma irradiation, demonstrate efficacious employment, demanding that standard protocols be implemented for all tissue banks. An argument can also be made to require that only trained professionals, held to the same standards as other medical professionals, be allowed to harvest tissue. It is also necessary to make donor medical records available to surgeons using their graft material, to aid in clinical decision-making. Finally, reporting of bacterial infections to a central agency to monitor future outbreaks, should be mandatory. When complications do occur, these cases need to be investigated, rather than buried by legal sanctions, to improve tissue-banking techniques. Based on these case reports, we would recommend that the implanting surgeon send cultures of allograft tissue, so that appropriate action may be taken should high pathogenic bacteria such as Clostridia be encountered. These changes are likely to increase the cost of tissue banking, and may create difficulty in attaining graft materials. However, even though the complication rate associated with the use of allograft tissue is extremely low, the devastating impact on morbidity and mortality under the current system is unacceptable. Additionally, it is incumbent on the orthopaedic surgeon to keep informed on the various tissue types and indicated uses. Understanding these factors will allow orthopaedic surgeons to inform their patients more knowledgeably on the potential risks, and to use the various graft types available in the most efficacious manner.

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8. References BARRIOS, R.H., LEYES, M , AMILLO, S. and OTEIZA, C. (1994). Bacterial contamination of allografts, Acta Orthopaedica Begica 60-2, 152-154. BECHTOLD, J.E., EASTLUND, T.D., BUTTS, M.K., LAGERBORG, D.F. and KYLE, R.F. (1994). The effects of freeze-drying and ethylene oxide sterilisation on the mechanical properties of human patellar tendon, Am. }. Sports Med. 22, 562-566. BENNETT, L. (2000). Gas gangrene and other clostridium-associated diseases. In: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Disease, 5th Ed. Churchill Livingstone, Philadelphia, PA, pp. 2549-2561. BETTEN, D., DETHLOFF, M., STEINBECK, J. and POLSTER, J. (1994). Organisation of a bone and tissue bank, Z. Orhtop. Ihre. Grengeb. 132, 453-458. BOLANO, L. and KOPTA, J.A. (1991). The immunology of bone and cartilage transplantation, Orthopaedics 14, 987-996. BRIGHT, R.W., FRIEDLAENDER, G.E. and SELL, K.W. (1977). Current concepts: Tissue banking: The United States Navy tissue bank, Milit. Med. 142, 503. BUCK, B.E. and MALININ, T.I. (1994). Human bone and tissue allografts, preparation and safety, Clin. Orthop. 303, 8-17. BUCK, B.E., MALININ, T.I. and BROWN, M.D. (1989). Bone transplantation and human immunodeficiency virus: An estimate of risk of acquired immunodeficiency syndrome (AIDS), Clin. Orthop. 240, 129-136. CENTER FOR DISEASE CONTROL (CDC) (2001). Septic arthritis following anterior cruciate ligament reconstruction using tendon allografts, MMWR 50, 1081-1083.

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CENTER FOR DISEASE CONTROL (CDC) (2001). Unexplained deaths following knee surgery, Minnesota, MMWR 50, 10351036. CORDREY, L.I., MCCORKLE, H. and HILTON, E. (1963). A comparative study of fresh autogenous and preserved homogenous tendon grafts in rabbits, /. Bone Joint Surg. [Am.] 45, 182-195. DEPARTMENT OF HEALTH AND HUMAN SERVICES, OFFICE OF INSPECTOR GENERAL, OVERSIGHT OF TISSUE BANKING (2001), January. DIEJKERS, R.L.M., BLOEM, R.M., PETIT, P.L.C., BRAND, R., VEHMEYER, S.B.W. and VEEN, M.R. (1997). Contamination of bone allografts, analysis of incidence and predisposing factors, /. Bone Joint Surg. [Br.] 79-B, 161-166. FARRINGTON, M., MATTHEWS, I., FOREMAN, J., RICHARDSON, K.M. and CAFFREY, E. (1998). Microbial monitoring of bone grafts: Two years' experience at a tissue bank, /. Hosp. Infect. 38, 261-271. FIDELER, B.M., VANGSNESS, C.T. Jr., MOORE, T., LI, Z. and RASHEED, S. (1994). Effects of gamma irradiation on the human immunodeficiency virus: A study in frozen human bonepatellar tendon-bone grafts obtained from infected cadavera, /. Bone Joint Surg. [Am.] 76-A, 1032-1035. FIDELER, B.M., VANGSNESS, C.T. Jr., LU, B., ORLANDO, C. and MOORE, T. (1995). Gamma irradiation: Effects on biomechanical properties of human bone-patellar tendon-bone allografts, Am. J. Sports Med. 23, 643-646. FIDELER, B.M., VANGSNESS, C.T. Jr, LU, B., ORLANDO, C , MOORE, T., FINEGOLD, S.M., ATTEBERY, H.R. and SUTTER, V.L. (1974). Effect of diet on human fecal flora: Comparison of Japanese and American diets, Am. J. Clin. Nutr. 27,1456-1469.

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GARRETT, J.C. (1996). Osteochondral allografts for reconstruction of articular defects. In: Operative Arthroscopy, 2nd Ed. R.B. Caspari, R.W. Jackson, J.B. McGinty, and G.G. Poehling, eds., Lippincott-Raven, Philadelphia, pp. 395-403. GAZDAG, A.R., LANE, J.M., GLASER, D. and FORSTER, R.A. (1995). Alternatives to autogenous bone graft: Efficacy and indications, /. Am. Acad. Orthop. Surg. 3, 1-8. GEORGE, W.L. and FINEGOLD, S.M. (1985). Clostridia in the human gastrointestinal flora. In: Clostridia in Gastrointestinal Disease, S.P. Boriello, ed., CRC, Boca Raton, Fla., pp. 1-37. GODETTE, G.A., KOPTA, J.A., and EGLE, D.M. (1996). Biomechanical effects of gamma irradiation on fresh frozen allografts in vivo, Orthopaedics 19(8), 649-653. GOERTZEN, M.J., CLAHSEN, H., BURRIG, K.F. and SCHULITZ, K.P. (1995). Sterilisation of canine anterior cruciate allografts by gamma irradiation in argon, /. Bone Joint Surg. [Br.] 77-B, 205-212. HAMER, A.J., STOCKLEY, I. and ELSON, R.A. (1999). Changes in allograft bone irradiated at different temperatures, /. Bone Joint Surg. [Br.] 81-B, 342-344. HIRN, M.Y., SALMELA, M. and VUENTO, R.E. (2001). Highpressure saline washing of allografts reduces bacterial contamination, Acta Orthop. Scand. 72, 83-85. ISENBERG, H.D. and D'AMATO, R.F. (1995). Indigenous and pathogenic microorganisms in humans. In: Manual of Clinical Microbiology, 6th Ed. E.J. Baron, P.R. Murray, M.A. Pfaller, F.C. Tenover and R.H. Yolken, eds., ASM Press, Washington, D.C., pp. 5-18. IVORY, J.P. and THOMAS, I.H. (1993). Audit of a bone bank, /. Bone Joint Surg. [Br.J 75-B, 355-357.

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Rduances in

Tissue Banking Vol.7

This is the most comprehensive volume dealing with tissue banking presently available, with 27 contributions from the most distinguished and experienced practitioners in the field: surgeons, microbiologists and tissue bankers. Safety of allografts is now a major concern due to possible microbial and viral contamination of tissues, even in the most sophisticated centres. Thus, publication here of the International Atomic Energy Agency's Code of Practice for the Radiation Sterilisation of Tissues is important, as is their guidance on Standards and Public Awareness regarding this often misunderstood technology. The volume spans all the methodologies used in the field and covers a spectrum of tissues: bone, skin, cardiovascular grafts, corneal grafts and sperm banking. Of particular interest in these days of gigantic disasters is the evaluation of the value of an effective tissue bank during the Volendam burns disaster in the Netherlands and the horrific disaster in "Messa Redonda" Peru. Orthopaedics, as usual, has the premier usage of tissues and this volume is graced by a landmark contribution from that doyen of massive allograft surgery, Henry Mankln. Balancing out the US experience is a contribution from Russia, which outlines new approaches to using allograft and autograft bone. The motivation for such a comprehensive volume came at the congress held In Boston, which drew together all the international associations of tissue banking: American, AsiaPacific, Latin American and European.The whole world has been harnessed to construct this outstanding and historic volume. ISBN 981-238-723-4

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