Transfusion Immunology and
Medicine
This Page Intentionally Left Blank
Transfusion Immunology Medicine edited by
Care1 J. van Oss
State University of New York at Buffalo Buffalo, New York
Marcel Dekker, Inc.
New York. Basel Hong Kong
Library of Congress Cataloging-in-PublicationData Transfusion immunology and medicine / edited by Carel J. van Oss. cm. p. "Proceedings of the Twelfth International Convocation on Immunology" "Previouslypublished in Immunologicalinvestigations, vol. 24, no. 1, 1995""T.p. verso. Includes bibliographical references and index. ISBN 0-8247-9640-3 (alk. paper) 1. Blood-Transfusion-Congresses. I.van Oss, Carel J. 11. International Convocation on Immunology (12th : 1994 : State University of New York at Buffalo) WB 356 T77265 19951 [DNLM: 1. BloodTransfusion-congresses. RM171.T66 1995 615'.39-d~20 DNLM/DLC for Library of Congress 94-23955 CIP
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PREFACE
Transfusion Immunology ana' Medicine addresses the immunological andmicrobiological safety of bloodtransfusion in all its ramifications, as well as new findings on the red cell surface, novel aspects of the use of blood components and alternatives, and advances in transfusion strategies. It is intended to serve as a reference for hematologists; immunologists; transfusionists; surgeons; infectious disease specialists; epidemiologists; pathologists; cell biologists; medical, scientific, and technical personnel in blood centers, blood transfusion services, and hospital blood banks; and graduate and medical school students in these disciplines. The work may thus well be considered a general treatise on the state of the art of modem blood transfusion. Bacteriological and virological safety, and its possible further improvement, is considered, from both the point of view of the removal of infectious agents from the blood supply (Dodd; Friedman et al. ; Mohr et al.; Steneker et al.; Dzik; Bowden) and the aspect of improved testing (Sazama; Busch; Bianco; Blajchman). Then the increasing awareness of the importance and function of red cell, leukocyte and platelet allotypes is treated (Tippett; Anstee et al.; Daniels; Garratty; Balakrishnan and Adams; von dem Borne et al.; Reed). The immunological effects of blood transfusion are discussed by Tartter; Perkins; MincheffandMeryman;BlajchmanandBordin;Davenport;and Snyder. Blood components and possible alternatives are treated by Strauss; Sweeney et al.; Heaton; Broxmeyer; and Klein. Blood substitutes are discussed by Dracker, who also addresses the use and collection of hematopoietic stem cells. Transfusion strategies are discussed by Menitove, and immunological hazards of blood transfusion, such as graftversus-host disease and possible remedies, are treated by Davey. Autogeneic bloodtransfusion is addressed by Qutaishat, and two very different treatments for immune hematologic disorders are discussed by Bussel and Szatrowski. Finally, at the beginning of the book, Dr. Greenwalt, who gave the plenary Ernest Witebsky Memorial Lecture at the iii
iv
PREFACE
Convocation on which this book is based, treats in detail the modem knowledge of the molecular make-up of the erythrocyte, in his paper “Red but Not Dead: Not a Hapless Sac of Hemoglobin”. I gratefully acknowledge the help of a large number of collaborators, colleagues and institutions, who all aided in makingthis work a success: my co-members of the Convocation Committee, Drs. R. K. Cunningham, R. A. Dracker, J. Hay, R. J. Kratzel, R. M. Lambert, J. F. Mohn, F. R. Orsini and S. Qutaishat; the institutional, industrial and commercial contributors: Abbott Diagnostics, the American Associationof Blood Banks (AABB), Armour Pharmaceutical Company, Baxter Biotech, Conferences in the Disciplines, the State University of New York at Buffalo, The Research Foundation of the State University of New York, Haemonetics Corporation, Immunex Corporation, IMRB Corporation, Jewett Refrigerator Co. Inc., Miles, Inc.-Biological Business Unit, Olympus America Inc., Ortho Diagnostic, Pall Biomedical Products Company, Vital Systems, Inc., and W. B. Saunders; the invited speakers, more than 90% of whom contributed one, and in some cases, two manuscripts to this volume; my former Chief and very good friend Dr. Tibor J. Greenwalt, for agreeing to give the Ernest Witebsky Memorial Lecture and for helping tremendously with the early organization of this work; Dr. Leonard Friedman, whose suggestions for authors were also veryuseful; Mrs. J. Dalkey and Mrs. S. Wilson of the Center for Immunology; and the registered attendees, who came from four continents to partake in all the functions. Finally, a special word of thanks to Dr. Richard Bettigole, of the American Red Cross Regional Blood Center ofBuffalo, for his help with and interest in these prefatory comments, and to Dr. Edward Leonard of the Department of Chemical Engineering, ColumbiaUniversity, who brought the microbiological problems of blood transfusion, as well as many of their possible solutions into sharp focus by organizing andchairing the American Red Cross Advisory Committee on Reducing the Infectious Potential of Transfused Red Blood Cells, a Committee which regularly convened at the American Red Cross Holland Laboratory, at Rockville, Maryland, during 1991 and 1992.
Carel J. van Oss
CONTENTS
Preface
.............................................
Contributo............................................
iii
ix
THE ERNEST WITEBSKY MEMORIAL LECTURE Red but Not Dead: Not a Hapless Sac of Hemoglobin
T. J . Greenwalt
...............
3
PART I: INFECTIOUS AGENTS AND THEIR REMOVAL FROM BLOOD AND BLOOD COMPONENTS Viral Contamination of Blood Components and Approaches forReduction of Infectivity R. Y. Dodd
................................
Reducing the Infectivity of Blood Components-What We HaveLearned.
........................................ L. I. Friedman, R. R. Stromberg, and S. J . Wagner Photodynamic Virus Inactivation of Blood Components ..............
25
49 73
H.Mohr, B. Lambrecht, and A . Selz Leukocyte Filtration Mechanisms: Factors Intluencing the Removal of Infectious Agents from Red Cell Concentrates..........................................
87
I . Stenekr, R. N. I. Pietersz, and H. W. Reesink Use of Leukodepletion Filters for the Removal of Bacteria W. Dzik Transfusion-TransmittedCytomegalovirusInfection
R. A. Bowden V
............
95
................ 117
CONTENTS
Vi
PART II: TESTING FOR INFECTIOUS AGENTS
.............. 131 Testing Blood Donors for €€W Current : Controversies .............. 147 M . P. Busch HepatitisTesting ....................................... 155
Existing Problems in the Testing for Infectious Diseases
K. S a z m
C. Bianco
Bacterial Contamination of Blood Products and the Value of Re-transfusionTesting
...................................
163
M. A . Blajchman PART IIk ALLOTYPES What
IS Important on the Red Blood Cell Surface
................. 173
P. Zppett Functional Factors in the Red Cell Membrane: Interactions Between the Membrane and Its Underlying Skeleton D.J . Anstee, N. J . Hemming, and M . J . A. Tanner
................ 187
Hot Spots in the Red Cell Membrane: Molecular Aspects of Some RedCellAntigens
..................................
199
G.Daniels Blood Group Antigens as Tumor Markers, ParasiticlBacteriall Viral Receptors, and Their Association with Immunologically Important Proteins
......................................
G. Garratty The Role of the Lymphocyte in an ImmuneResponse
213
............... 233
K. Balakrishnan and L. E. Adams Neutrophil Antigens, from Bench to Bedside
.....................
A . E. G. Kr. von dern Bonre, M. de Haas, D.Roos, C. H. E. Homburg, and C. E. van der Schoot Anti-idiotypes to H L A and Their Role in Transplantation
245
............ 273
E. Reed PART TV: IMMUNOLOGICAL EFFECTS OF BLOOD TRANSFUSION Immunologic Effects of Blood Transfusion
P. I. Tamer TransfusionReactions:TheChangingPriorities
......................
277
.................. 289
H. Perkins Blood Transfusion, Blood Storage, and Immunomodulation
M. S. Minchefland H . T. Meryman
........... 303
CONTENTS
vii
The Tumor Growth-Promoting Effectof Allogeneic Blood Transfusio............................................ 311 M. A. Blajchman and J . 0. Bordin The Role of Cytokines in Hemolytic Transfusion Reactions 319
............
R. D.Davenport The Role of Cytokines and Adhesive Molecules in Febrile Non-hemolytic Transfusion Reactions
.......................... 333 E. L. Snyder Neonatal Anemia: Pathophysiology and Treatment ................. 341 R. G. S t r a w Quality of Platelet Concentrates ............................. 353 J . D. Sweeney, S. Holme, and A . Heaton
.............................
The Quality of Red Blood Cells W. A . L. Heaton Growth Factors and Cord Blood Stem and Progenitor Cells
371
........... 391
H.E. Broxmeyer The Development and Use of Oxygen-Carrying Blood Substitutes R. A. Dracker Novel Cellular Therapies
...........................................
403
..................................
411
H . G. Klein PART V: TRANSFUSION STRATEGIES TransfusionStrategies:OpportunitiesforImprovement
............. 423
J . E. Menitove Transfusion-Associated Graft-Versus-Host Disease and the Irradiation of Blood Components
............................
431
R. J . Davey Autologous Blood Transfusion: Evaluation of an Alternative Strategy in Reducing Exposure to Allogeneic Blood Transfusion
.......................................... 435 Hematopoietic Stem Cells: "Form .Method .Characteristics" ......... 443 S. Qutaishat
R. A . Dracker
Uses of Intravenous Gammaglobulin in Immune Hematologic Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 1 J . B. Bussel and T. P. Szatrowski AuthorIndex
457
SubjectIndex
459
......................................... .........................................
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Louis E. Adams Cincinnati, Ohio D. J. Anstee
HLA Laboratory, Hoxworth Blood Center, University of Cincinnati,
International BloodGroupReferenceLaboratory,
KamalaBalakrishnan HLALaboratory,HoxworthBlood Cincinnati, Cincinnati, Ohio Celso Bianco
Bristol, England Center, University of
New York Blood Center, New York, New York
M. A. Blajchman Departments of Medicine and Pathology, McMasterUniversity, and the Canadian Red Cross Society, Hamilton, Ontario, Canada J. 0. Bordin Departments of Medicine and Pathology, McMaster University, and the Canadian Red Cross Society, Hamilton, Ontario, Canada Raleigh A. Bowden Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, andDepartmentof Pediatrics, University of Washington, Seattle, Washington Hal E. Broxmeyer Departments of Medicine and Microbiology and Immunology, and the WaltherOncology Center, IndianaUniversitySchool of Medicine, Indianapolis, Indiana M. P.Busch Department of Laboratory Medicine, University of California, and Irwin Memorial Blood Centers, San Francisco, California James B. Bussel New York Geoff Daniels
New York Hospital-Cornel1 University Medical Center, New York, MRCBloodGroup Unit, WolfsonHouse,London,England
R. D. Davenport
Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
ix
X
CONTRIBUTORS
Richard J. Davey
National Institutes of Health, Bethesda,Maryland
MasjadeHaas DepartmentofHematologyandCentre for Blood Cell Research, Academic MedicalCentre, and Department of Experimental Immunological Hematology, Central Laboratory ofthe Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands Roger Y.Dodd AmericanRed Cross, Holland Laboratory, Rockville, Maryland Robert A. Dracker State University of New York-Health Science Center at Syracuse, Syracuse, New York
W. Dzik Departments of PathologyandMedicine,DeaconessHospitalandHarvard Medical School, Boston, Massachusetts Leonard I. Friedman American Red Cross, Holland Laboratory, Rockville, Maryland G. Garratty ResearchDepartment,AmericanRedCrossBlood California Region, Los Angeles, California
Services, Southern
Tibor J. Greenwalt, M.D. Emeritus Professor ofTransfusionMedicine,Hoxworth Blood Center, University of Cincinnati Medical Center, Cincinnati, Ohio AndrewHeaton
IrwinMemorialBlood Centers, San Francisco, California
W. A. L. Heaton IrwinMemorialBlood Centers, andDepartment of Laboratory Medicine, University of California, San Francisco, California
N. J. Hemming International Blood Group Reference Laboratory, SteinHolme
Bristol, England
AmericanRed Cross, Norfolk, Virginia
ChristaH. E. Homburg Department of HematologyandCentre for BloodCell Research, Academic Medical Centre, and Department of Experimental Imunological Hematology, Central Laboratoryof the NetherlandsRedCrossBloodTransfusion Service, Amsterdam, The Netherlands Harvey G. Klein Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland
B.Lambrecht Blood TransfusionServiceof Lower Saxony, SpringeInstitute, Springe, Germany J. E. Menitove HoxworthBloodCenterandDepartmentof University of Cincinnati College of Medicine, Cincinnati, Ohio
Internal Medicine,
H. T. Meryman Transplantation Department,Holland Laboratories, AmericanRed Cross, Rockville, Maryland M. S. Mincheff Transplantation Department,Holland Laboratories, AmericanRed Cross, Rockville, Maryland H. Mohr Germany
BloodTransfusion Service ofLowerSaxony,Springe
Institute, Springe,
H.Perkins IrwinMemorialBlood Centers, San Francisco, California
CONTRIBUTORS R. N. I. pietersz
xi Red Cross Blood Bank Amsterdam, Amsterdam,
The Netherlands
S. Qutaishat The Ernest Witebsky Center for Immunology, Department of Microbiology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York
E. Reed Department of Pathology, College of University, New York, New York H. W. Reesink
Physicians and Surgeons of Columbia
Red Cross Blood Bank Amsterdam, Amsterdam, The Netherlands
Dirk Roos Department of Hematology and Centre for Blood Cell Research, Academic Medical Centre, and Department of Experimental Immunological Hematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands Kathleen Sazama Department of Pathology and Laboratory Medicine, Medical College of Pennsylvania, Philadelphia, Pennsylvania A. Selz BloodTransfusion Germany
Service ofLowerSaxony,Springe
Institute, Springe,
Edward L. Snyder DepartmentofLaboratoryMedicine,Yale University School of Medicine,andBloodTransfusion Service, Yale-NewHaven Hospital, New Haven, Connecticut
I. Steneker Red Cross Blood Bank Amsterdam, Amsterdam,
The Netherlands
Ronald G . Strauss DepartmentsofPathologyand Pediatrics, University ofIowa College of Medicine, DeGowin Blood Center, University of Iowa Hospitals and Clinics, Iowa City, Iowa Robert R.Stromberg Joseph D. Sweeney Ted P.Szatrowski New York
American Red Cross, Holland Laboratory, Rockville, Maryland BloodBank, The Miriam Hospital, Providence, Rhode Island
New York Hospital-Come11University Medical Center, New York,
M. J. A. Tanner Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, England Paul I. Tartter York
Department of Surgery, Mount Sinai Medical Center, New York, New
PatriciaTippett MedicalResearchCouncilBloodGroup University College London, London, England
Unit, WolfsonHouse,
C. EllenvanderSchoot DepartmentofHematologyandCentre for BloodCell Research, Academic Medical Centre, and Department of Experimental Immunological Hematology, Central Laboratory of the NetherlandsRedCrossBloodTransfusion Service, Amsterdam, The Netherlands
Care1 J. van Oss State University of New York at Buffalo, Buffalo, New York
xii
CONTRIBUTORS
Albert E. G. Kr. von dem Borne Department of Hematology and Centre for Blood Cell Research, Academic Medical Centre, and Department of Experimental Immunological Hematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands Stephen J. Wagner
American Red Cross, Holland Laboratory, Rockville, Maryland
THE ERNEST WITEBSKY MEMORIAL LECTURE
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RED
BUT
NOT DEAD: NOT A HAPLESS
SAC OF HEMOGLOBIN
Tibor J. Greenwalt, M.D. Emeritus Professor of Transfusion Medicine Hoxworth Blood Center University of Cincinnati Medical Center PO BOX 670055 3130 Highland Avenue Cincinnati, OH 45267-0055
ABSTRACT
The purpose of this presentation was to demonstrate that the red blood cell is not a hapless sac of hemoglobin and that much researchisstillneededforbetterunderstandingofits complexities. After a brief historical introduction the following subjectsarepresented: 1). Phosphofructokinaseistherate limiting step in the anaerobic glycolytic pathway. Ribose-5phosphate, a metabolite of the oxidative pentose phosphate pathway is essential for ,the generation of phosphoribosylpyrophosphate whichinturn 1s neededforthesynthesisofadenosine monophosphate from adenine by the action of adenine phosphoribosyl transferase. 2). There are at least 17 blood group systems with more than400 epitopes expressed on the red cell membrane. Rh The null and the McLeod phenotypes associated with abnormally shaped red cells and hemolytic anemia are briefly describedas is the present understanding of the nature of the Rh complex. 3). The structure of the cytoskeleton and the composition and behavior of the lipid bilayer are presented with some discussion of the MN a Ss sialoglycoproteins and the Leach phenotype. 4). Touched upon is the role of phosphoinositides with some emphasis on recent discoveries relating to the glycophosphoinositide protein anchor. 5 ) . The intricacies of the many faceted transport mechanisms are introduced. Briefly mentioned are the mechanisms activated when regulatory volume adjustments occur in fine tuning red cell volum after exposure respectively to hypotonic or hypertonic stress. Sufficient evidence is presented to convince that a cell doesn't have to have a nucleus to be respected even though it is just a corpuscle.
In 1984 I read a stimulating editorial entitled "More red than dead"whichgavemetheinspirationforthetitle of my presentation (1). The author stated that "The opinion has been too often voiced that the mammalian red cell, having shed its nucleu ribosomes and cytoplasmic cytoskeleton, is no more than a floati corpse,fitfortheattention of nothingbetterthana pathologist.1v I will in the time allotted do my best to dispel t 3
4
GREENWALT
misconception I was taught as a student that the red cell is nothing but a sac of hemoglobin. I tlsacll picked rather than "sack" because Webster's definition **a soft-walled cavity or pouch, usually having a narrow opening or none at all, and in many cases containing some special fluid" seemed better. HISTORY
To my knowledge, before World WarI1 there were only two blood banks in theU.S. , one founded in 1937 by Bernard Fantus at the Cook County Hospital in Chicago and the other started by Paul Hoxworth at the Cincinnati General Hospital in 1938. Sodium citrate had been found to be a suitable anticoagulant but blood could not be stored for more than five days because very little known about the metabolism of the red blood cell and its 1916 Rous and Turner (2) had reported nutritional requirements. In that red cells could be kept foras asa long month in glucose plus citrate. But this important observation appears to have passed relatively unnoticed. In 1925 Greenwald (3) documented that red cells contained a surprising amount of 2,3-diphosphoglycerate (DPG) 4 0 years. The impetus but its function was not understood for over for learning more about the metabolism of the red cell may have been the serendipitous observation by Loutit and Mollison ( 4 ) that blood could be stored effectively for 21 days with an acidified citrate dextrose solution (ACD) Dextrose and citrate could not be autoclaved together because at alkaline pH the dextrose carmelize It was necessary to lower the pH to 5 about to prevent this. Thus ACD was born. It was learned that low pH favored the maintenance the easily hydrolyzable organic phosphates (adenine nucleotides) in the red cells (5) It was also shown that the difficultly hydrolyzable organic phosphate (DPG) was better maintained at higherpHduringstorage (5). The maintenance of adenosine triphosphate (ATP) is generally accepted as closely correlated with the in vivo survival of stored red cells (6) The effort therefore has been focused on supporting the level of ATP during No storage. practical way by which both ATP DPGand can be maintained has been found because low pH is not favorable for the desired actions of the enzymes in the Rapoport-Luebering cycle to generate DPG. It would be desirable to sustain both of these organic phosphate molecules because it has been demonstrated that DPG interacts specifically with hemoglobin to keep the oxygen dissociation curve ideal for oxygenation in the lungs and deoxygenation in the venou Capillaries (8,9). During the first week of storage DPG drops drastically causing shift of the hemoglobin P50 to the left where
.
.
.
5
RED BUT NOT DEAD
oxygen is bound more tightly. This is called the Valtis-Kennedy effect (10) described in 1954 but not explained as due to decreased DPG until 1967 (8,9). Normally the oxygen tension at which hemoglobin is 50 percent saturated (P50) is 26 mm Hg. During storage of RBCs the P50 drops 1 8 - 2to 0 mm Hg at which it holds on to oxygen more tightly. GLYCOLYSIS
Before discussing a few areas of the carbohydrate metabolism of red cell which have been of particular interest to our laboratory, I must refresh memories by presenting the Embden-Meyerhof anaerobic glycolytic pathway (FIGURE 1) which handles 90 percent of the glucose utilized by the red cell. We have explored the remarkable ability of a combination of ammonium (NH4+) and inorganic phosphate (Pi) ions, when added to the usual formulation of a preservativeadditivesolution,tomaintainATPlevelsandredcell survivability for extended periods (11-14). Phosphofructokinase (PFK) is the most important rate limiting enzymatic step in this scheme. ATP is known to have an allosteric inhibitory action on which is released in the presence of ammonium ions (15,16). On the other hand Pi has a stimulatory action on PFK. This combined action is postulated to stimulate glucose utilization by anaerobic glycolysis, thus supporting the generation of more ATP from its substrates (17,18)
.
NH,' and Pi also act on the oxidative pentose phosphate pathway through which approximately 10 percent of the glucose used by the red cell is metabolized. Their combined action is postulated to increase the generation of ribose-5-phosphate (17,19-21). In FIGURE 2 it is indicated how R-5-P04 can be used to generate phosphoribosylpyrophosphate (PRPP) by the action of PRPP synthetase (also known as ribose-5-phosphate pyrophosphokinase) in the presence of ATP.
Glyceraldehyde phosphate dehydrogenase (GAPD) can conceivably become a rate limiting step in the metabolism of stored red cell if sufficient lactate should accumulate. The metabolic steps are outlinedinFIGURE 3. Ithasbeenreportedthatwhenthe 1actate:pyruvateratioexceeds 25 theoxidationofreduced nicotinamide adenine dinucleotide (NADH) to NAD decreases (22). NAD is required as a co-enzyme for GAPD to synthesize 1,3diphosphoglycerate.Blockageatthislevelwouldslowthe utilization of the trioses required for the generation of ATP fro
Glucose hk
J.
Glu ose - 6 - P 0 4 wi
Fructose 6 - P 0 4
J.
PR
Fructose 1,6 - Di - P 0 4 4 aldolase DihJ.droxyacctone P 0 4
EMBDEN-MEYERHOF ANAEROBIC GLYCOLYTIC PATHWAY
enol.se
Phosphoenolpyruvate pk
Pyruvate 4-, NADH
Idh
+
+-+ NAD+
Lactate
FIGURE 1 EMBDEN-MEYERHOF ANAEROBIC
GLYCOLYTIC
PATHWAY
Abbreviations: hk, hexokinase; gpf, glucosephosphate isomerase; pfk, phosphofructokinase; tpi, trlosephosphate isomerase; gapd, glyceraldehydephosphatedehydrogenase; pgp, diphosphoglycerate phosphatase; pgk, phosphoglyceromutase;pk, pyruvate kinase; ldh, lactate dehydrogenase.
PENTOSE PHOSPHATE PATHWAY
I
R-S-P
ATP
PRPP Synthetase
GMP AMP
Adenine I
+ ATP
ak "-t
2ADP
adok
Adenosine FIGURE 2 PENTOSE
PHOSPHATE
PATHWAY
The role of phophoribosylpyrophosphate (PRPP) in the synthesis of adenine nucleotides. Abbreviations: R-5-P, ribose-5-phosphate; aprt, adenine phosphoribosyl transferase; ak, adenylate kinase; adok, adenosine kinase.
7
RED BUT NOT DEAD Role of LactatelPyruvate and NADRVADH Ratios
Glucose Glyceraldehyde - 3 - P j
+ NAD
GAPD
v
-
Pyruvate NADH
Lactate
LDH
NAD
FIGURE 3 ROLE OF LACTATE/PYRWATE AND
NAD/NADH
RATIOS
Abbreviations: GAPD, glyceraldehydephosphate dehydrogenase; NAD, nicotinamide adenine dinucleotide; LDH, lactate dehydrogenase.
ADP by the
kinasesactive in the lower end of the Embden-Meyerhof
pathway. The
mostsignificantrecentpracticaladvance
in
redcell
preservation was the discovery that exogenous adenine could be utilized by red cells for maintaining ATP levels (23). The mechanism for incorporating adenine into nucleotides is depicted in FIGURE 2. Adenine phosphoribosyl transferase synthesizes adenosine monophosphate(AMP)fromadenine andPRPP. Adenylatekinase converts one mole of AMP plus one mole of ATP to two moles ADP which can than be used in the Embden-Meyerhof pathway.
Early
BLOOD GROUPS inmy career in transfusion medicineI had a
serious
interest
in alloimmunity but for almost three decades have focused more the biochemistry ofthe red bloodcell and its membrane. The title of this Convocation is Transfusion Immunology and Medicine which givesconsiderablelatitude to the subjectmatter included. Nevertheless, I decided to address some subjects of special interest to immunologists. I noted that Dr. Tippett would address llWhatfsimportant on the RBC Surface?11 and that Drs. Anstee's,
on
8
GREEWALT
Daniels, and Garratty's titles address matters related to the corpuscular surface. I trust that my eclectic review will not overlap their presentations.
It seems natural that blood bankers for decades focused attention on the outer surface of the sac. Alloimmunization from the led to the increased use of blood transfusions after WorldI1 war reporting of more than 600 blood group epitopes. There was considerable duplication because lack of standardized nomenclature. 400 (personal The correct number is probably closer to communication, Dr. Peter Issitt) classified into at 17least blood group systems. There are very few answers as to why they are th certainly not solely to harass the crossmatch staff. It has been established that persons with the Duffy phenotype, Fy (a-b-) are protected from infestation by Plasmodium v i v a x and also Plasmodium knowlesi (24). This appears to be the result of evolutionary selection because this phenotype occurs 68 percent in of African Americans and is also present in Blacks of Equatorial Africa.
The Rh,,,,,, phenotype is associated with stomatocytosis of red blood cells and mild to moderately severe hemolytic anemia (25-28). Clearly the Rh substance must have some role in membrane integrity The early attempts to identify its nature gave inconclusive results (29-30). E.A. Steane and I were convinced that a peptide structure was involved because we were able to store glutaraldehyde treated ghosts for months without loss of their ability to adsorb anti-D antibodies (31). We now know much more, but all the riddles have not been resolved. The subject is too complex to present in detail. The recognition of peptides 28 toof 32 Kd in the membranes with presumed genotypes D/D or D/d was the initiating event in the beginning of the understanding of the Rh locus (32,33). Another important contributing element was the development of monoclonal antibodies which could be used to precipitate the D polypeptide from the solubilized membranes Dofpositive RBCs which had been labeled with'"I. The polypeptide was purified to homogeneity by SDS-PAGE. A very similar polypeptide was obtained'"I from labeled D positive RBCs by hydroxylapatite chromatography and preparative electrophoresis of SDS-solubilized membranes. Classical amino acid sequencing of the isolated peptides made it to isolate possible the cDNAwhichwaslocalizedtochromosomelp34.3-p36.1which corresponds with the locus previously deduced by segregation
RED BUT NOT DEAD
9
analysis. The cDNA was found to encode a 416 amino acid chain which bears no glycosylation sites. Most of the amino acid sequence is believed to lie within the lipid bilayer. The model suggested consists of 13 bilayer spanning domains with the C-terminus on the outside and the N-terminus extending from the cytoplasmic of face the membrane as shown in 4FIGURE . The sequences of Dthe , the C/c and E/e peptides are similar. The reader is referred to the cited review f o r details ( 3 4 ) . It is likely that there are two closely linked loci for D and Cc/Ee. It now seems that D positive individuals have two Rh polypeptide genes whereas negatives have only one. Unfortunately all this has not revealed the nature of the Rh antigens as they are expressed on the erythrocyte membran A heterogeneous collection of glycosylated proteins with related amino acid sequences were coprecipitated by the anti-D, anti-c and anti-E reagents. Their exact function is unclear but it has been postulated that the actual Rh antigen may be a complex Rhof the polypeptides,theRh-relatedglycoproteins,andsomeofthe adjacent phospholipids ( 3 4 ) . We still do not know the exact structure of the Rh antigen nor all the details missing LW from the and RhnulL mysteries.
The Kell blood group system is worthy of some attention in this brief overview.It has also become cluttered with many serological complexities which will not be discussedOf here. special clinical interest is the McLeod phenotype (35). Briefly McLeod individuals have RBCs which appear on routine to testing lack the Kell antigens like PP cells but actually have very weak expression of k and Kp(b+) which may be demonstrable only by adsorption-elution studies. Of special interest is that such individuals lackX-an chromosome linked gene, Xk, responsible for the expression of Kx o red cells and/or granulocytes. Some McLeod phenotypes also have sex linked chronic granulomatous disease (CGD) and others do not. Thus it appears that the genes f o r the McLeod phenotype and granulomatous disease are linked but not identical. An interesting feature of people with the McLeod phenotype is the presence of acanthocytic red cells which are irregular in size and shape with numerous blunt protrusions. Such cells do not survive normally and the usual manifestations of hemolytic anemia may be present which is reported to increase in severity after 40. McLeod age phenotype individuals who do not have CGD have to been have shown high levels of serum creatine phosphokinase of the MM type of cardiac and skeletal muscle derivation. With progressing age these people have been found to develop a neurological disorder associated with
10
GREENWALT
Palmitate
FIGURE 4
Suggested topography of the Rh peptide 13 showing bilayer spanning domains with C-terminus on the outside and N-terminus on the cytoplasmic side of the membrane. o=cysteine residue. Adapted from: P. Agre, J.P. Carton, Blood, 7 8 , 551 (1991)
dystonic and choreiform movements and wasting of limb muscles and some also developed cardiac failure with cardiomyopathy (36). It has been hypothesized that these associations can be explained by closeness on theX chromosome of the Xk locus to loci for genes responsible for the clinical conditions mentioned. THE
MEMBRANE
Recently investigators have directed most attention to the membr as of the red cell. is Itreadily available in quantity and serves a suitable model from which to launch studies of other cells. In the following a few of the major developments relating to the cytoskeleton, the lipid bilayer, the protein glycolipid anchor and the transport mechanisms will be presented. The revolution of our understanding of the red cell membrane was started when Singer and Nicolson proposed the fluid mosaic structure of membranes (37) followed closely by the bilayer couple theory concept introduced by Sheetz and Singer (38). Singer's basicconceptwasthatintegralproteins(band 3 andthe glycophorins) are embedded in and float in the sea of the lipid
11
RED BUT NOT DEAD Glycophorin pprotein 4.1 interaction
Band 3
Spectrin dimer-dimer interaction
I
'Actin Spectrin-protein 4.1 AdducinP
p "
actin interaction FIGDRE 5
A
schematic
diagram of erythrocyte
membrane
organization.
Reprinted with permission from: J . G . Conboy, N. Mohandas. In Red Blood Cell Membranes, P. Agre and J.G. , eds. Parker , Marcel Dekker , Inc. New York ( 1 9 8 9 ) .
bilayer first suggested by Gorter and (39) Grendel and confirmed by DavsonandDanielli (40). Theperipheralproteinsonthe cytoplasmic surface of the membrane form the cytoskeleton (FIGURE 5)
-
Cytoskeleton
The major cytoskeletal protein is spectrin, so named because it was isolated from erythrocyte ghosts ( 4 1 ) . There are approximately 2 0 0 , 0 0 0 copies of this rod shaped protein per red cell. It is a heterodimer made up of an alpha subunit of 2 6 0 , 0 0 0 daltons and a beta subunit of 220,000 daltons which probably is present as tetramers and a smaller percentage of oligomers (FIGURE 5 ) . In sodiumdodecylsulfatepolyacrylamidegel(SDS-PAGE)patterns stained by Coomassie Brilliant Blue it is seen as bands 1 and 2. The protein bands in SDS-PAGE gels stained with Coomassie Blue a numbered on the basis of their relative electrophoretic mobility according to the scheme first introduced by Steck (42). The spectrin mesh is stabilized by interaction with other cytoskeletal proteins and linked by some proteins to integral membrane proteins floating in the lipid bilayer. Ankyrin (anchor) furnishes the major high affinity link between the cytoskeleton and the lipid
12
bilayer lying external to it.
GREENWALT
There are about 100,000 copies of
the 215,000 dalton globular ankyrin molecule. It has binding sites for spectrin and the 92,000 dalton Cl-/HCO,- anion transporter (band 3). The binding site on spectrin is on itsbeta subunit. Each spectrin heterodimer has a binding site for actin filaments. molecular weight, 43,000 daltons, in the red cell membrane exists as oligomeric filaments made up of 12-20 monomers. It has been estimated that there are25,000 to 30,000 such oligomers per red cell. This implies that each should be associated with an average of six spectrin molecules. These interactions areweak. They are enhanced by the action of protein 4.1, another cytoskeletal protein. Protein 4.1 has a molecular weight of 78,000 daltons and approximately 200,000 copies are present per cell. Protein 4.1 also interacts with a class of integral sialoglycoproteins referred to as the glycophorins. The 4.1-glycophorin interaction is greatly enhanced by the phosphoinositides
which
are
largely
located in the
inner lipidbilayer. Phosphatidylinositol 4,5- biphosphate appears to have the greatest effect (43). Tropomyosin has more recently identified in the erythrocyte membrane. It is a heterodimer consisting of 27,000 and 29,000 dalton peptides. One function appears to be the stabilization of the membrane portion of actin (43)* The glycophorins A, B, C, and D of which glycophorinA is the most abundant, are of special interest to transfusionists as carriers blood group antigens. Thereareabout 1,000,000 copies of
of
glycophorin A (MN sialoglycoprotein) per cell. This 31,000 dalton glycoprotein hasthe MN antigens on chains of 70 amino acids on its external amino terminal end. Thirty-five amino acids form the cytoplasmic extension which interacts with protein 4.1. Less is known about glycophorinB (Ss sialoglycoprotein) which is similar in structure to theMN sialoglycoprotein and bears the S,s and the related U antigens (43). The serologic aspects of the Gerbich-Leach phenotypes have become so cluttered with time that they would be comprehensible only to a dedicated blood group philatelist (44). They are mentioned here because red cells the of Leach phenotypelack glycophorins Cand D and also donot have thebinding site for protein 4.1. The missing binding site must reside on either glycophorin C or D (45). Of interest is that such cells are elliptocytic. Presumably the altered red cell shape is secondary to the abnormal protein
Actin,
13
RED BUT NOT DEAD
structure. The other Gerbich phenotypes have normal appearing biconcave red cells.
The roles of some other proteins found in the red cell membran less clear. Protein 4 . 2 is a7 2 , 0 0 0 dalton polypeptide with some 2 0 0 , 0 0 0 copies per cell. It binds to 3.band It is phosphorylated in the presence of cadmium and mercury ions and has been identif as the major binding site for mercurial compounds such as pchloromercuribenzoate. There are 30,000 copies of adducin per cell. It is a heterodimer 103 with ,000 and 9 7 ,000 dalton subunits. It plays some function with the calcium regulatory protein, calmodulin, and with protein 4.1 promotes the association of spectrin and actin. Two other proteins identified in the red cell membrane are myosin and protein 4 . 9 . Their roles are not well understood. Undoubtedly other components will continue to be identified. Further research will be necessary to understand the complex interrelationships and importance of the components of the red cell cytoskeleton. Lipid
Bilayer
The phospholipid components of the membrane are given in TABLE I. The lipid asymmetry of the bilayer of the erythrocyte membrane was first recognized by Bretscher ( 4 6 ) . Using trinitrobenzene sulfonic acid (TNBS),as a non-penetrating label for the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), Gordesky, Marinetti, and Love ( 4 7 ) demonstrated their asymmetric distribution. Other techniques using phospholipase A2, spin-labeled and fluorescent analogues of the phospholipids and the activation of the prothrombinase complex by the presence PS on ofthe surface of the membrane have established the normal asymmetric distribution of the phosholipids in the red cell membrane and their dynamic behavior ( 4 8 , 4 9 ) . Inhumanredcells 82 percentofthe sphingomyelin and 7 6 percent of the phosphatidylcholine are in the outer lipid layer whereas 7 0 percent of the PE and virtually all of the PS are in the inner leaflet. The asymmetry was at first believed to be due to the selective binding of the aminophospholipids to elements of the cytoskeleton, most likely spectrin (50,51) Seigneuret and Devaux( 5 2 ) first reported that protein-mediated translocationof spin-labeled PE and PS occurred (50,51). This has now been shown to be dependent on the of action a translocase (flippase) requiring Mg2+ATP to rapidly transport added PE and PS to the inner lipid layer (flip). The mechanism to
.
14
GREENWALT
TABLE
I.
Phospholipid
Composition RBC of Membrane
PHOSPHOLIPID
%
Phosphatidyl choline
32
Sphingomyelin Phosphatidyl ethanolamine
22
Phosphatidyl serine
11
25
Phosphatidyl inositol
2.5
Phosphatidic
1.5
acid
transport PS and PE in the opposite direction (flop) has not been clarified. At first it was suggested that the translocase protein was the 32,000 dalton (band 7 ) Rh peptide. The evidence now supports that it 115 is Kda Mg'+ATPase which may be associated with the Rh complex. It still may be that the slow flop of internalized PS and PE isduetointeractionwithcomponents of the cytoskeleton. It has been hypothesized that red cells with exposed PS are sequestered from the circulation. PS is thrombogenic and may enhance adherence to the endothelium and recognition by the reticuloendothelial system for phagocytosis. This may beso for sickle cells but the evidence that this may be the mechanism €or the shortened survival of older red cells and the red cells of neonates is not convincing. Our own data using the TNBS (53) label have not shown any flop of PS in red cells stored for prolonged periods (unpublished observations).
Phosphatidylinositides constitute but a small percentage of the total phospholipids of the red cell membrane. They are largely localized in the inner lipid bilayer. Recently evidence has been published that a small fraction of phosphatidylinositol 4,5biphosphate, phosphatidylinositol and phosphatidic acid but no phosphatidylinositol 4-phosphate is detectable on the surface of the red cell membrane(54). Ferrell and Huestis(55) demonstrated that loss of a small percentage phosphoinositides from the inner lipid layer is sufficient to produce marked shape changes. Phosphoinositides may also represent the pathway for an important cellular signaling system. When acted upon by a phosphoinositide specific phospholipase C, inositol triphosphate (IP,) and diacylglycerol (DAG) are released. IP, mobilizes calcium from intracellular organelle and plasma membrane sites and DAG and CaZ+ activate phosphorylation of proteins by protein kinase.
RED BUT NOT DEAD
15
Glycosyl-phosphatidylinositol (GPI) protein anchors Phosphoinositides play another important role in cell membrane structure and function. They are an integral part of a glycolipid anchor for attaching some proteins covalently to cell membranes. It was first described in trypanosomes (56), and has been much studied in the formed elements of the blood in patients with paroxysmal nocturnal hemoglobinuria (PNH). Here the discussion will be restricted to a brief synopsis of what has been reported for the red cells in PNH (57,58). The general structure of the glycolipid anchor is depicted in FIGURE 6. The diacylglycerol end of the phosphoinositide (PI) molecule is inserted into the lipids of the membrane. The PI is attached to four hexoses, one of which is aminated (hexosamine). The protein is attached by an amide bond formed between its terminal carboxyl group and ethanolamine as shown. Most of the proteins known to be lacking completely or almost completely from the surface of the most severely affected type I11 erythrocytes in PNH are listed in TABLE 11. The first one identified was acetylcholinesterase which has also been shown to bear the Cartwright (Yt") blood group antigen (59). Decay accelerating factor (DAF or CD55) functions to disrupt the convertase complexes of complement (C4b2a of the classical pathway and the C3bBb complexof the alternative pathway). The membrane inhibitor of reactive lysis (MIRL or CD59) controls the formation of C5b-9, the membrane attack complex of complement. The function of another missing protein on type I11 PNH red cells, the C8 binding protein, also called the homologous restriction factor (HRF), has not been defined. TRANSPORT
My interest in the preservation of red cells required me to devel some familiarity with the transport mechanisms of their membranes. Delving into the literature on this subject quickly convinced me that it would be necessary to one's devote scientific career to the study of membrane transport to speak with authority about it. I therefore will take the liberty of quoting from (60) Tosteson who has done so. "It is remarkable that the very existence of cell membranes was problematic when I and most of the other authors of this volume began our work.I1 "...it was not until the emergence of the electron microscope, and particularly, adequate techniques for the isolation, purification, and chemical characterization of
16
GREENWALT
PROTEIN _____)
1
Asp
I
ETHANOLAMINE
4? O O=Po 0
INOSITOL
DIACYLGLYCEROL
\
\
W
Z
m W
FIGURE 6 GLYCOSYL-PHOSPHATIDYLINOSITOL (GP11 ANCHOR The general structure of the GP1 molecule which anchors many proteins to cell membranes is shown. The diacylglycerolis inserted into the lipid bilayer. The carboxy-terminus forms an amidebond with the protein. Adapted from: W.F Rosse, Blood, 75, 1595 (1990)
RED BUT NOT DEAD
TABLE 11.
17
SomeGlycophosphoinositide(GPI)AnchoredMembrane Proteins
Acetylcholinesterase (Yt') Decay accelerating factor (DAF, CDSS) CDS9) Membrane inhibitor of reactive lysis (MI-, C8 binding protein or homologous restriction factor (BRF) Lymphocyte functional antigen-3 (LFA-8, CDSS) Fc-y-receptor IIIa (CD16) Endotoxin binding protein (CD14) Urokinase-type plasminogen activator receptor Blood arour,factors: Yt', JMH, Inab-phenotype Inab RBCs lack all blood antigens belonging to the Cromer complex, including Cr., Tc"'~ , ES', Dr., WESn.b and
IFC.
membranes, that these structures were recognized components of cells and organelles". (60)
as essential
Fifty or sixty years ago the movements of ions were explained on the basis of how they were expected to behave to satisfy the concept of the Donnan equilibrium. The red cell membrane was conceived to be an inert semipermeable sac containing hemoglobin which passively influenced the concentration of the ion content. Modern techniques have revealed that many mechanisms exist for th transport of ions, sugars, amino acids, and other compounds in and out through the red cell membrane (see 111). TABLE I will touch upon two phenomena which present intriguing implications relating to the changes in erythrocyte the collection and storage of red cells.
volume
during
Red cells possess mechanisms that enable them to regulate their volume precisely. Placing them in a hypoosmotic environment producing an increase in mean corpuscular volume, has been observe to improve their in vitro characteristics during prolonged storage (11,61). Hemolysis and the shedding of microvesicles are markedly reduced and their posttransfusion recovery is improved (unpublished observations). The physiologic reasons are poorly understood. It has been suggested that the increase in cell volume prevents the vesiculation and the resulting loss of membrane by elevating membrane tension (11). Perhaps more important is the observation that during refrigerated storage the red cells gradually pump down to normal volume. This phenomenon has been described as the
GREENWALT
18
TABLE 111. Major
Transport
Mechanismsof the Red Blood Cell
Na+-K+ Pump Na*-K+-2Cl' cotransporter K+-Cl-cotransporter Na+-H+ exchanger CaZ+ transport Ca2+ activated K+ channel (Gardos effect) cr/nco; Glucose transport Nucleotide transport Amino acid transport: L system prefers large neutral amino acids Lyt system specific for dibasic amino acids ASC system is Na-dependent and selective for neutral amino acids of intermediate size
regulatory volume decrease (RVD) of cells placed in a hypoosmotic medium. It has been attributed to the activation of the K-Cl cotransport (61-64) which results in the loss of water, potassium and chloride. It is not understood what else occurs to make this possible and to improve the preservation qualities of RBCs.
Remarkably, when red cells are placed in a hypertonic medium the respond by regulatory volume increase(RVI) which is mediated by inward ion fluxes through the activation of the Na-K-C1 cotran and water movement into the cell.
Space and time have precluded including all the areas of interest being studied about the red cell. Apologies to those whose speci areas of interest have been bypassed. I trust that sufficient evidence has been presented to convince that a cell doesn't hav have a nucleus to be respected with awe even though is just it a corpuscle. There is yet another reason for the pursuing research of the red cell which is very personal. Sir John Dacie titled an editorial in the American Journal of Medicine, "Research on the Red Cell- A Recipe fora Long Life" (65). In 1909 Sir Rickard Christophers- who died in1978 at the age of 104 - with C.A. Bentley first designated as spherocytes small dark corpuscles whose "appearance is due mainly, if not altogether, to changes in their elasticity which prevent them being flattened as are normal corpuscles.1* Two other nonagenarians
fro
RED BUT NOT DEAD
19
of red cell research mentioned by Sir John were Winifred Peyton Rous. And now Sir John has joined the at ranks age82.
Ashby
In concluding I am reminded about another anecdote about age and productivity. Someone asked Bruce Bliven, the editor of theNew Republic, then in his seventies, what it felt like to be an old man. Bliven retorted "1 don't feel like an old I man. feel like a young man with something the matter with (66) him." I am motivatedto continue research relating to the red cell. ACKNOWLEDGEMENTS
Appreciation is expressed to Angelica Washam for her help in preparing this manuscript and Umakant J. Dumaswala, Ph.D. for reading the draft. Supported in part by a grant from the NIH HL44897.
W,
REFERENCES 1. Gratzer. Nature, 2 , 368-369 (1984). P. Rous and J.R. Turner. J Exp Med,2 3 , 239-248 (1916). 2. I. Greenwald. J Biol Chem, Q, 339-349 (1925). 3. J.F. Loutit and P.L. Mollison. Brit Med J, 2 , 744-745 4.
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9. 10. 11.
an
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Rapoport, eds., University Park Press, Baltimore (1974) pp. 55-92. 19.
A. Hershko, A. Razin, and J. Mager. Biochim Biophys Acta,
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R.B. Dawson. Transfusion, 16, 450-454 (1976). R.B. Dawson, L.D. Sisk, D.R. Meyer, R.T. Hershey, and C.S. Meyers-Hilbert. Transfusion, Zl, 215-218 (1981). W.M. Tilton, C. Seaman, D. Carriero, and S . Piomelli. J Lab Clin Med,118, 146-152 (1991). E.R. Simon, R.G. Chapman, and C.A. Finch. J Clin Invest,41,
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L.H. Miller, S.J. Mason, J.A. Dvorak, M.H. McGuinniss, and I.K. Rothman. Science, 189, 561-563 (1975). G.H. Vos, D. Vos, R.L. Kirk, and R. Sanger. Lancet,1, 14-15 (1961).
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P. Levine, M.J. Celano, F. Falkowski, J. Chambers,O . B . Hunter, and C.T. English. Nature, 2 0 4 , 892-893 (1964). P.J. Schmidt, M.M. Lostumbo, C.T. English, and O.B. Hunter, Jr. Transfusion, Z, 33-34 (1967). T. Ishimori and H. Hasekura. Transfusion, 1, 84-87 (1967). F.A. Green. J Biol Chem,2 4 7 , 881-887 (1972). F.V. Plapp, M.M. Kowalski, L. Tilzer, P.J. Brown, J. Evans, and M. Chiga. Proc Nat Acad Sci, 76, 2964-2968. T.J. Greenwalt, E.A. Steane, and E. McFaul. Immunol Comm, 2,
32.
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37. 38.
C.G. Gahmberg. FEBS Lett, 1 4 0 , 93-97 (1982). P. Agre and J.P. Cartron. Blood,2 8 , 551-563 (1991). P.D. Issitt, In ARDlied Blood GrOUR Serolosy, Montgomery Scientific Publications, Miami, F1(1985) pp. 297-301. L.L. Tang, C.M. Redman, D. Williams, and W.L. Marsh.Vox Sang, 40, 17-26 (1981). S.J. Singer and G.L. Nicolson. Science, 175, 720-731 (1972). M.P. Sheetz and S.J. Singer. Proc Nat Acad Sci, U , 44574461 (1974).
39. 40. 41. 42. 43.
E. Gorter and F. Grendel. J Exp Med, Q, 439-443, (1925). J.F. Danielli and H. Davson.J Cell Comp Physiol,5 , 495 V.T. Marchesi, E. Steers. Science, 159, 203-204 (1968). T.L. Steck. J Cell Biol,6 2 , 1-19 (1974). K. Gardner and G.V. Bennett. in Red Blood Cell Membranes, P. P. Agre and J.C. Parker, eds, Marcel Dekker, Inc., New York, (1989) pp. 1-29.
44. 45.
P.D. Issitt, Amlied Blood GrOUR Serolosy, Montgomery Scientific Publications, Miami,(1985) pp.397-399. J. Smythe, B. Gardner, and D . J . Anstee. Blood, 8 3 , 1668-1672 (1994)
46. 47.
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M.S. Bretscher. Nature, 2 3 6 , 11-12 (1972). S . E . Gordeski, G.V. Marinetti. and R. Love. J. Membr Biol,
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This Page Intentionally Left Blank
PART I:
INFECTIOUS AGENTS AND THEIR REMOVAL FROM BLOOD AND BLOOD COlMPONENTS
This Page Intentionally Left Blank
VIRAL CONTAMINATION OF BLOOD COMPONENTS AND APPROACHES FOR REDUCTION OF INFECTIVITY Roger Y. Dodd American Red Cross Holland Laboratory 15601 Crabbs Branch Way Rockville M D 20855 USA
ABSTRACT
Currently, the United States blood supply offers a relatively low risk of viralinfection.Thisis a resultofcarefulselectionofdonors and extensive laboratory testing using sensitive procedures. Epidemiologic data show that there is some room for improvementin donor selection, but such improvements cannot be expectedt o entirely eliminate the collection of blood from infectious donors. Similarly, increased numbers of tests, along with improvements in the analytic sensitivity of these tests, may further reduce risk, but again, complete safety cannot be assured.Consequently,there is continuing interest in the development of safeand effective procedures for viral inactivation of single donor blood components. In order t o establish appropriate expectations for such inactivation procedures, it is necessaryt o understand the titersand distributions of viral contaminants in blood components. Viruses may variously occur freein the plasma, as replicative forms in actively infected leukocytes, as integrated proviral DNA and perhaps, nonspecifically associated with cellular surfaces.
INTRODUCTION
Although the past ten
years havebeencharacterized
manage the risk of transfusion transmitted infection
by the need t o with the Human
Immunodeficiency Virus (HIV), the period has, nevertheless brought a profound increase in the overall safety of the blood supply. Continued concern over the 25
DODD risks of transfusion has resulted in ongoing attention to the issue and there is public expectation that additional
measures will be taken. In this paper,
I will
reviewthecurrentestimatesoftheriskofinfectionbykeytransfusiontransmitted viruses in the United Statesand will review some of the approaches which may further reduce this risk. Major concern focusses on the risk of HIV infection,eventhough transmitted.
it istheagentwhichisperhapsleastlikely
to be
HTLV 1 and HTLV II also offer some continued risk of infection,
while infection with the hepatitis B virus is nowan almost vanishingly infrequent outcome of transfusion. However, the hepatitis C virus continuesto be the agent whichismostfrequentlytransmitted.Anumberofothervirusesmaybe transmitted by transfusion, but do not appear to offera growing risk. A complex of measures has been introduced to establish blood safety, including judicious use of blood, donor selection and screening, and laboratory testing. A t least t w o methodsfortheinactivationofviruses
in freshfrozenplasmahavebeen
developed to the point ofuse or clinicaltrial; research on inactivation of cellular products continues. RESIDUAL RISK OF TRANSFUSION TRANSMITTED VIRAL INFECTION A recent editorial suggests
that the risk of viral infection from the fully
screened blood supply is extremely low (1 1. Indeed, fewer than one recipient in 3000 is now thought to be at risk of clinically apparent disease as a result of viral infection transmitted by transfusion of single donor products. As a result of the use of effective viral inactivation
procedures, products prepared from
pooled plasmaare now thought to offer less riskthan that associated with single donorproducts.
Per unitestimatesoftheriskoftransfusiontransmitted
infection are summarized in Table I. The Human Immunodeficiency Virus (HIV)
HIV can be transmitted to, recipients by blood which is collected during the so-called window period, which represents some fraction of the time between
27
VIRAL CONTAMINATION OF BLOOD COMPONENTS TABLE I:
Transfusion-transmittedinfections in theUnitedStates
Risk of transmission
Agent
Test
in donors (%)
(per unit)
HBV
:200,000 1
HCV
Detection rate
1:3,300
HBsAg
0.04
anti-HBc
1.27
anti-HCV
0.25
ALT
1.64
HIV
1:225,000
anti-HIV
0.006
HTLV 1/11
1:70.000
anti-HTLV I
0.006
infection with the virus and the development of detectable levels of antibodies t o HIV. There have been
a wide variety of estimates of the frequency of this
occurrence, ranging from l in 40,000 t o 1 in 225,000component units. Direct measures of the frequency of posttransfusion infection have been made upon a
in Baltimore and Houston, suggesting large population of cardiac surgery patients an overall risk of 1 in 60,000, based upon two infections ( 2 ) . A similar figure of 1 in 61,000 wasdevelopedin lymphocytes from blood units
San Francisco byculturingpoolsof
(3). Estimates of 1 in 40,000 t o 1 in 153,000
were developed by estimating the chance of collecting blood during the window period (4,5).Such estimates were based uponthe assumption that the window
period was about eight weeks long and that the frequency of new HIV infecti amongpreviouslytesteddonorswas
areasonable
predictorofincidence.
Kleinman and Secord, working in Los Angeles, developed an estimate of
68,000,baseduponobservationsof
1 in
theproportionofinfectionsamong
recipients of prior donations from repeat blood donors newly found to be positi
28
DODD
for HIV (6). Petersen and colleagues recently extended this lookback method t o a study on seroconverting donors from 40 blood centers in the United States. They wereable to establish that 20 percent recipients of of the last seronegative donation from a seroconverting donor became infected. As might beexpected, the frequency of such infection was proportional to the time interval between the anti-HIV positive donation and the prior seronegative
one.By
comparing this
relationship with a mathematical model, they wereable to show that the average infectious window period was 45
days.Further,
distribution and probably is less than about
this window hasa narrow
150 days in 90% of cases ( 7 ) . By
using this windowperiod, Petersen was able to show that,based upon measures of HIV incidence in the donor population, the risk of infection in 1990 was about
1 in 225,000 (1 1. Subsequently, the HIV antibody tests
in use have become
more sensitive andare able to detect seroconversion 5t o 1 5days earlier that the test in use in the late 1980s ( 8 ) . Since the frequency of positive tests among donors continues t o decline, this suggests that the actual risk of HIV infection is n o w significantly less than the 1990 estimate.
The Human T-lymphotropic Retroviruses (HTLV) Recognition, in Japan, ofthe
transmissibility of HTL.V I byblood
transfusion led to concern that this virus may States. Epidemiologic studies donors was about 0.025% (9). t o HTLVwasintroduced Interestingly,thisprocedure
showed that the prevalence of infection among In 1989, uniform donor testing for antibodies
in theUnited
procedures,baseduponalysate
also offer a risk in the United
States.The
testsusedwere
ELISA
of purified HTLV 1 as thecapturereagent. also detectsantibodies
tothe
closelyrelated
HTLV II. It was found that more than 50% of donors confirmed positive in tests for HTLV I were in fact, infected with HTLV II. Risk factors for HTLV I infection among donors were largely geographic, whereas risk for HTLV II infection was primarily associated with injecting drug use (10). It is not clear whether or not there is significant window period risk for recipient infection with HTLVI or -11. However, Nelson has shown that there was one infection among a population
VIRAL CONTAMINATION OF BLOOD COMPONENTS
29
of cardiac surgery patients who received a total of 69,272 units collectedafter the initiation of donor testing.
Thus, the current risk of posttransfusion HTLV
infection appears to beapproximately 1 in 70,000 per unit (2). In this study, the post-test infection was caused by HTLV II, suggesting that the infection may have resulted from the known limitations of sensitivity for anti-HTLV-ll of tests based upon an HTLV I lysate (1 1). Data from Japan and from the United States confirm that infectivity is confined to cellular transfusion products (10,12). Hepatitis B virus (HBV). There are no definitive
measures of
the contemporary risk of
posttransfusion infection with HBV. However, Alter has developed estimates based upon thesensitivities of current tests for HBsAg and for anti-HBc and has concluded that the risk is of the order of 1 in 200,000 per unit ( 1 1. Dodd has reportedthatthefrequencyofreportedposttransfusionhepatitis distinguishable from the background rate in
B is not
a non-transfused population (13).
Nevertheless, there are documented cases of posttransfusion hepatitis B which havebeentracedtodonorswhosubsequentlydevelopedmarkers infection. However, tests for HBsAg
of HBV
overall, it seems likely that the combination of sensitive
(i.e. those capable of detecting less than 1 ng/mL) and the
routine use of tests for anti-HBc, along with donor screening measures, have effectively eliminated the risk of hepatitis B infection for recipients. In contrast to Japanese practice, there is
no attempt to discriminate among donors with
anti-HBc by accepting those with low titers, and/or the presence of anti-HBs. However, as shown in Table I, the overall frequency of donations reactive for anti-HBc is only l .2%. Hepatitis C Virus (HCV). HCVis
nowknownto
betheagent
responsible formost
cases of
posttransfusion non-A, non-B hepatitis (NANBH) and particularly of chronic forms of the disease. NANBH was first recognized
as a result of the availability of
30
DODD
tests for infection with the hepatitis B and A viruses. Residual
posttransfusion
hepatitis after the implementation of testing for HBsAg was found to be devoid of serologicevidence
of infection with these two known viruses.For
many
years, the causative agent could not be identified, despite careful analysis of a number of prospective studies on posttransfusion hepatitis. However, certain of these studies did reveal that recipients of blood from donors with elevated levels of ALT, or with antibodies to theHBV core antigen, were themselves more likely t o develop NANBH ( 1 4,151. As a result of these observations,
by early
1987, all donor blood was screened for elevated ALT levels and for anti-HBc in theUnited
States.Isolation
ofthe genome of HCVandexpression
ofviral
proteins in yeast systems led to the availability of ELISA tests for antibodies to theC100-3
protein,expressed
by a non-structuralcomponentoftheviral
genome (16,171. This test came into general use in the United States in1990, but has subsequently been superseded by tests based upon multiple expressed antigens from HCV. Donahue and colleagues have reported on the of riskposttransfusion HCV infection,baseduponstudiesontransfused
cardiacsurgerypatients.These
studies were conducted over a period which started prior to the introduction of ALT and anti-HBc testing, and continued until after the C100-3 antibody tests had been introduced. As a result, this group was able to show that the per-unit riskof
HCVinfectionwas0.45%prior
introductionof
to anytesting,
so-calledsurrogatetests,and
0.1 9 % afterthe
0.03%aftertheadditional
implementation of anti-HCV tests (1 8). When the patients were retested using second-generationtests
of improvedsensitivity,thefrequency
of recipient
infection was found to be somewhat higher, although it must berecognized that thesedatarepresentresidualinfectionafterdonorscreeningbythefirstgeneration assay (19). Thus, the risk of anti-HCV infection has, in all likelihood, beensignificantlyreduced
as a result of the implementation of the secondnow as low as 1 in 6,000 (20).
generation, multi-antigen test
and is perhaps
Thedevelopment
versions of thesetests
offurther
expressed antigens or synthetic peptides continues.
based uponadditional
31
VIRAL CONTAMINATION OF BLOOD COMPONENTS Other viruses.
A number of other viruses are known to be transmitted by transfusion. Cytomegalovirus is perhapsof greatest concern, since its transmission can lead t o seriousdisease
with
ordeathamongprematureinfantsandindividuals
seriously compromised or ablated immune systems. Currently, such patients are generallysupported
with bloodcomponentsfromdonorswhohavebeen
serologicallytestedandshown
t o beseronegative.However,sincethe
prevalence rate for anti-CMV is usually at least 50% amongdonors, and may be as high as 90%, thisapproachdoespresentlogisticaldifficulties.Emerging evidence suggests that even relatively modest levels of leukodepletion may be effective in preventing transmission of CMV by transfusion (21-25). The B1 9 parvovirus is known to products, at leastprior
be transmissible by blood and blood
totheintroduction
ofadvancedheattreatment
procedures for labile plasma derivatives. In the majority ofcases, B1 9 infection is of little consequence to the recipient. However, maternal infection is known
to cause serious problems for the fetus, and patients with hemolytic anemias or HIV infection may suffer aplastic crises as a result of B1 9 infection. Unlike most other transfusion transmissible viruses, B1 9 is an essentially epidemic infection and is transmitted essentially only during the preacute phase. Additionally, the virusisnot
lipid-enveloped, so is not susceptible tomanyinactivation
procedures. Infection risk is o f 1 in 10,000 t o 50,000,
generally thought to be low, perhaps of the order although a muchhigherfrequencyhasbeen
suggested by studies based upon PCR (26,271. Interestingly, the hepatitis A virus, another nonenveloped agent causing acute infection, may also be transmitted in the presymptomatic phase (28-30). Such occurrences
are very rare and
donot
appear towarrant
special
precautionary measures, although blood should certainly not be collected from individuals who may havebeen exposedto thesource of an outbreak. However, there have recently been puzzling reports of an outbreak of HAV infection which
32
DODD
appear to have resulted from the use of pooled plasma products inactivated by a solvent-detergent method (31,321.The source of the virus has not yet been definitively established, but it is possible that the product contaminationdid not originate from the plasma donors. Viral distribution in the blood Transfusion transmissible viruses may be found
in a number of
compartments within the blood. Morespecifically, viruses may be found freein the plasma, or associated with cells, or in both compartments. Table II outlines the distribution of key viruses. The hepatitis B and C viruses, which are presumed to replicate in the liver, are found primarily as freely circulating virionsin the plasma. The infectivity titer of HBV has been reported to be as high as 10’ infectious doses or more permL, but these levels are confined to situations in which HBsAg is readily detectable.
It has been estimated, on thebasis of the sensitivity of tests for HBsAg, that the titers of HBV could not exceed 1O5 particles per mL in a screened product and, in fact, would be likely to be very much less (33).Similarly, HCV is most likely
presentonlyinthe
plasma ininfectiousform,withtiterswhichhavenot
normally exceeded IO2 infectious dosespermL.Interestingly, HBV nucleotide sequenceshavebeen
both HCV and
detected in leukocytesfrominfected
individuals - perhaps of evenmoreinterestisthefindingthatreplicative intermediates were also detected, indicating the potential for viral multiplication within the leukocytes. However,it is not clear whether such apparently infected leukocytes can transmit the virus recipients. to HAV and the B19 parvovirus also appear to be present onlyas free virions, although as noted below, all of these viruses might also be associated with cell surfaces. Conversely, infectious forms of CMV and HTLV
1 (and presumably, by
extension, HTLV-Ill appear to be exclusively cell-associated, as indicated by the absence ofinfectionsfrom
plasma, single donorcryoprecipitate,orpooled
33
VIRAL CONTAMINATION OF BLOOD COMPONENTS Table II: Distribution of Infectious Agents in Blood
Agent
Leukocytes
Plasma
Proportionof PBMC
Infectedcells per:
1nfected:Uninfected RBC
CMV HBV HCV
HIV (Acute) (Asympt) HIV
+ + + +
HTLV
plasma fractions.
PC
x io3
x io3
Present
I
Present
NA
NA
Present
NA
NA
1:lOO
6 X IO6
1 X IO6
1:50,000
Ix
2 x io3
1:5,000
I
io4
x lo5
I
2x
lo3
For CMV, this is substantiated by the apparent efficacy
leukodepletion in preventing transmission. In the
of
case of HTLV, infectivity is
reduced upon storage, presumably representing loss of viability of the infected lymphocytes. HIV may bepresent, in infectious from, both freein the plasma and within leukocytes. Indeed, it is possible that HIV-infectedleukocytes are the major source of infection. Nevertheless, plasma and non-sterilized plasmacomponents readilytransmit
HIV.
The infectioustiterofHIV
in the plasmais
characterized, although there have been estimates of from
notwell
1O2 to about lo5
virions per mL, depending upon the stage of disease and the technique used to measure the titer. Table I I outlines the potential titer of infectious agents and
34
DODD
virallyinfected
cells in blood components. An
added complication with
retroviruses (HIV and HTLV) is that the lymphocyte can be provirally infected. That is, the cell is not actively replicating
virus, but a DNA copy
of the viral
genome is integrated into thecell's own genomic DNA. Such provirally infected cells can be activated by
a variety of stimuli
(perhaps including exposure to
allogenic cells) and then express the virus. A final complication in defining the locus of viral agentswithin the blood is the possibility that naked virions may also associate with the cell surface. A variety of studies have suggested that laboratory cultures HIV and viruses, other used as models, do not wash out ofred cell suspensions in a manner consistent with simple dilution.
Rather, it appears that there is a nonspecific association
with the cellular compartment.Suchassociationisdifferentfromthat where there is a known viral receptor on the erythrocyte surface. Busch has
shown remarkable a association
presumably mediated
seen In addition,
between HIV and platelets,
by nonspecific adhesion
remembered that virus-antibodycomplexes
(34).
It should also be
are quitelikely
to beboundto
erythrocyte or platelet surfaces. As a consequence of all of these findings, it is clear that an effective viral removal or inactivation procedurewill have to have the capabilityof dealing with viruses in each of these compartments. Perhaps most difficult tomanage is the proviral form of the retroviruses.
DONOR SELECTION The population of active blood donors ahas lower prevalenceof infectious disease markers than does the overall population of the United States. This is particularly apparent for antibodies to HIV, where the current prevalence is about one fiftieth of that for which would be
seen in a random population sample,
assuming that there are indeed one million HIV-infected persons in the country. Similarly, population-based studiesof thefrequency of infectionwith HBV clearly
35
VIRAL CONTAMINATION OF BLOOD COMPONENTS
demonstratelowerratesthanthose
seen among donors.
Clearly, these
differences reflect the complex of activities which contribute to blood safety. At the population level, specific and deliberate measures have been taken
to
eliminatepopulationsofpotentialdonorswho
to
clearly offeredexcessrisk
recipients - namelypaiddonorsandinstitutionalized
individuals, including
prisoners. In addition, it is evident that the donor population is self-selected from those who are motivated to support society by donatingblood. More subtly, at least some of the criteria for donor acceptability
are widely known, and many
individuals with unacceptable medical or risk histories do not attempt
t o give
blood. Alldonors
are routinelyinterviewedabouttheirmedicalhistoryand
aspects of their behavioral background. The fact that this resultsin the deferral of a significant number of individuals, for a variety of causes, clearly indicates that this process contributes to blood safety.
The donor interview includes a
number of questions designedto elicita known history ofdisease, including viral age of lo), leishmaniasis,Chagas
hepatitis(occurringafterthe
disease,
babesiosis, syphilis and gonorrhea. In addition the interview includes questions reflecting a history of actualor potential exposure to viral agents, such as blood transfusion, close contact
with a hepatitis patient, tattooing etc.
specific, direct questions
are asked to elicit a history of risk behavior which
In addition,
might haveexposed the donor toHIV or HBV infection. This includes questions about direct risk, such as injecting drug use and sexual activity between males, plus those about indirect risk such as a history ofsexual contact withindividuals who are themselves at risk of HIV infection. As pointed out above, these selection
measures, along with laboratory
testing and deferral policies, lead t o a high degree of selection against infected individuals in the donor population. However, such measures are not perfect,as shown by the continued finding of HIV-infected donors (currently about
6 in
every 100,000 donation episodes). Interview studies on these infected donors showthatmorethan
25% ofHIVinfected
male, andalmost
40% ofHIV-
36
DODD
infected female donorsare unable to identify a specific risk factor(35). It should be noted that only about
4% of AIDS cases fail to report risk factors, clearly
indicating that donor selection procedures
are rather effective in eliminating
those with known risk for infection. However, these same interview studies do show thata surprisinglyhigh proportion of HIV-infected donors did actually know that they were at
risk. A variety of reasons for donating are given by such
individuals, includingpeer pressurein the donation environment, a sensethat the individual was, in some way, different from those considered risk, at and a desire to obtain a test result (36).This latter finding is somewhat hard to understand, since confidential or anonymous test sites have been availablefrom the initiation of HIV testing. It is not entirely clear what measures could be taken to improve the efficacyof donor interviews. However, further education, including advising the donor about the risk of window period infection, may be appropriate.
In
addition, it is generally recognizedthat the writtenmaterials presentedt o donors may not be readily understood,
so there have been a number of attempts to
simplify, and improve the impactof, these materials (37).Finally, there has been some apparent success in the use of an automated questionnaire, rather
than
relying upon interactionwith a human interviewer(38).Ultimately, however, the majorlimitations
of the donor interview
will continue to be the difficulty of
dealing with donors who are not able t o identify their risk status. It is clear that interview questions of adequate specificity couldnot bedevised t o deal with this issue (39).
A procedure which is in general use is that of confidential unit exclusion (CUE), or the related callbackmechanism. In bothcases, these procedures were conceived t o deal with an individual who recognizes that he or she may be at risk of infection,but for one reason or another, feels compelled to continue with the donation process. The CUE procedure requires that each donor specifically define, by a confidential mechanism, whether or not the blood unit should be used for transfusion to
a patient. This occurs at the donation
blood has been given. The callback procedure provides
site, after the
a donor with a means
to contact the bloodcenter after he orshe has left: a telephone number and the
VIRAL CONTAMINATION OF BLOOD COMPONENTS wholebloodnumber
37
are provided; thedonorisencouragedtomake
a
confidential call if heor she recognizes a problem. Although thereare some data t o suggestthatthere
are increasedprevalencerates
ofinfectious
disease
markers among blood units which have been defined as unsuitable by the donor, remarkably few HIVinfecteddonorsusethe
CUE option,even
subsequently acknowledge that they understood the situation
ifthey
(36).Although
there have been suggestions to eliminate the CUE process, it seems more likely that attempts will be made to improve the procedures in some way, so as to make them more effective. Another aspect of the donor selection process is the use of deferral files.
If a donor is found
to be unsuitableon
the basis of information relating to
infectious disease risk, or apositive testresult, then this finding is entered in the donor's record. Most often, this is
a computerized record.
the current findings must be compared with the previous
A t each donation, record. Thus, if the
donation has been givenby a donor who was previously regarded as unsuitable, then this check will reveal that fact. The donation can be located and withheld. The American Red Cross has chosen to undertake this procedure on a national basis, so that anydonorwhois deferred at onelocation
regarded as unsuitableandispermanently
will berecognized
if heor
she givesatanyother
location. It is important to recognize that, at this time, the process does not normally extend to recognition of the donor's status prior t o donation, although efforts are being madeto develop procedures to achieve this. Careful review of the concept of donor deferral suggests that, inthe absence of laboratoryerrors, theprocessactuallymakes
arelativelyinsignificantdirectcontributionto
transfusion safety. Rather, it provides an additional layer of safety which acts as a backuptoother
procedures.
Care needs to betaken
toavoidmaking
permanent deferral records for factors which have no impact on recipient safety.
LABORATORY TESTING Laboratory testing plays a major role in assuring blood safety. However, as pointed out above, it cannot be relied upon t o assure complete safety, as a
38
DODD
result of the occurrence of the infectious window period. Thus, donor selection continues to be of major importance. Current tests are extremely sensitive and specific; it is of interest to ask whether significant improvements could be made
in this aspect. For
example, the analytic sensitivity
of existing tests could be
improved, or additional test methods could be introduced.
With the exception of the tests for HBsAg tests for transfusion safety
and elevated ALT levels, all
are designed to detect antibodies to the relevant
viruses. In general, in the United States, ELISA technology is used. The capture reagent is viral lysate and/or recombinant or synthetic viral proteins and the probe reagent is an enzyme labelled antiglobulin. However,a recently introduced test for antibodies to HIV-1 and HIV-2 uses labeled antigenic peptides
as the
probe, thus relying upon the bi- or poly-valent nature of the antibody molecule. A key question relatingto the sensitivity of these antibody tests is the extent to which increases in their sensitivity are expressed in a reduction in the length of the window period. In the case of the test for antibodies toHIV, it appears that the mostsensitive currently available test has reduced the window period by say
12-13 days, relative to previous procedures( 8 ) . This may be a reflection of the abilityoftheantigen-antigensandwichformattodetectIgMantibodies effectively and without loss of specificity. Similarly, tests for antibodies t o HCV are becoming more sensitive. However, in this case, this is due t o a progressive increase in theabilityto
epitopes, or more appropriate Ultimately,however,
of HCV, so thatmore
expressantigenicproteins
epitopes, are included in the capture reagent.
it seems likely that thereis
a limittotheimpactof
increased sensitivity in all o f these tests, since theremust always bea finite time between infection and the development of antibodies. Arethereotherapproacheswhichmaybeused duringthewindow
period?Interestingly,this
to identify infectivity appears to have beenlargely
achieved for hepatitisB, where donorsare screened for HBsAg and for anti-HBc. In this case, however, the primary test has always been for the viral antigen; the use of anti-HBc is thought to identifyindividuals in a brief window period which
VIRAL CONTAMINATION OF BLOOD COMPONENTS mayoccurduringtheresolution
39
phase of anacuteinfection,priortothe
appearance of protective anti-HBs, or perhaps carriers with undetectable levels of HBsAg. HBV infection is unusual inasmuch
as copious excess of the viral
protein issynthesized, and this antigen isa very sensitive indicatorof infectivity. In contrast, during theearly stages of HIV infection, althougha viral antigen may bedetected,
it ispresent,
ifat
all, atverylow
concentration,andonly
transiently. In fact, the most sensitive of current tests for anti-HIV almosteliminateanypotentialbenefitforHIVantigentesting.
appear t o Prior to the
availability ofthecurrentversionoftheHIVantibodytest,therewas considerable interest in the potential benefit of HIV antigen testing.
Alarge,
prospective study on more than 500,000 routine donors was performed; in no case was a donorfoundwhowas
HIVantigenpositiveinthe
absence of
detectable anti-HIV (40). Another study was performed upon a subset of donors selected on the basis of high risk for HIV infection.
In this study, a population
judged to beequivalent, in terms of HIV risk, t o about one million contemporary donors, was tested and no antigen positive donor was found (411. There have, however, been cases of HIV antigen-positive, antibody negative donors in the United States (42).These were not located as a result of prospective studies. There has been no move towards widespread implementation of antigen testing. IntheUnited relatively mature,
States, the epidemic of HIVinfectionisjudgedtobe with a stable incidence
rate, which appears t o balance the
death rate for AIDS. In contrast, in Thailand, there is an explosive epidemic of infection. As a consequence, incidence
rates are high. In this environment,
has been shown that the donor population includes an
it
appreciable number of
individuals who are HIV antigen positive in the absence of detectable antibody. Thus, at least until the epidemic is under control,it may be justifiablet o use this test in Thailand,or
countries with similar problems.
Finally, it doesappear
possible that use of gene amplification techniques, suchas the polymerase chain reaction, might be able to detect infectivity somewhat days) earlier thanantibodytests
(i.e. a matter of a f e w
(8). It is not clear, however,whetherthe
potential benefit of this technique would offset the very considerable difficulties and costs of implementing it.
DODD
40
One furtheraspectoftestimprovement
relates to the occurrence of
additional types, strains or variants of viruses. The best example is that of HIV2, a viruswhichisdistinctfrom
HIV-1, but which also causes
AIDS. Viral
lysate-based tests for anti-HIV-l were able to detect the majority of individuals infected with HIV-2, but there was considerable concern that introduction of HIV-2 into the United States might eventually result recipients could be infected.
in situations where blood
At the time of greatest concern, only
thirty cases ofHIV-2infectionhad
twenty to
been identifiedintheunitedStates.
Extensive surveillance activities were initiated in order to provide early warning of HIV-2 infection, particularly among donors. This was done
by relying upon
the fact that the HIV-1 antibody tests in use were expected to be able to identify 60 t o 95% of all HIV-2 infections.
Consequently,alarge
number of samples
foundrepeatedlyreactiveinHIV-1testswereevaluatedinHIV-2
ELISA and
Western blot tests. Although about 25,000 samples, representing well over 2 0 million donations, were tested, no HIV-2 infections were identified (43).In the meantime, prospective donors from areas where HIV-2 infection was known to be endemic were excluded. This practice continued until it was possible t o use combination tests, permitting detection of anti-HIV-l and anti-HIV-2 in the same reaction. Somewhat
similarly,
crossreactivities for anti-HTLV-ll.
tests anti-HTLV-l for have significant Given the fact that more than half of HTLV-
infected donors in the United States are actually infected with HTLV-II, tests are being modified to increase their sensitivity for this virus.
VIRAL INACTIVATION The final approacht o increasing the safety of the blood supply is to accept that there are always likelyto besome residual viral agents in a small proportion ofcomponentsandtodevelop
means t o eliminatetheseagents
by some
inactivation procedure (33).Clearly, this has been accomplished in the case of clotting factor concentrates and other proteins prepared from pooled plasma. Not unexpectedly,freshfrozenplasmahasproven inactivationprocedures.IntheUnitedStates
most amenable t o
and Europe, proceduresbased
VIRAL CONTAMINATION OF BLOOD COMPONENTS
41
upon the use of organic solvents and detergents have been developed and the products have been entered into clinical
trials,or
are in use (44,451.
Also in
in the use of a methylene
Europe, considerableprogresshasbeenmade
blue/visible light photoinactivation process and plasmatreated in this fashion has beeninuse
sinceFebruary
of 1992 (46). In addition, filters capable of
withholdingtransfusiontransmitted
viruses, whilepassingthemajorityof
therapeutically important plasma proteins, have been developed in Japan (47). In fact, these methods reflect the three major
approaches to viral inactivation:
physical, chemical and photochemical. Although no virally inactivated platelet concentrates are available, there hasbeenconsiderableprogress
in the laboratory.Anumber
of groups have
demonstrated that theuse of various psoralens and long wavelength ultraviolet
light can inactivate modelviruses, such as VSV, and target viruses, such as HIV, in platelet concentrates, while preserving the in vitro properties of the platelets
(48-51). Additionally, it hasbeen
shownthatthisprocedurecaninactivate
intracellularvirusesandintegrated
gene sequences
(52).
Finally, treated
platelets have been shown to retain their hemostatic potential in an animal model (53). Published work from several years ago has also shown that the psoralen approach inactivates both hepatitis B and C viruses, as shown by chimpanzee infection studies (54). The major concern about the useof psoralens isthat this class of compounds is generally regarded t o be mutagenic and, in at least one study, residual levels of psoralens in treated platelet concentrates were clearly shown to be mutagenicin a bacterial test system (55). Thus, it may be difficult t o balance the risks and benefits of this approach in circumstances where the residual risk of infection is so low.
It has notproven quite so easy to developasuccessfulmethodfor inactivatingviruses
in redcellproducts.
The mostpromisingapproachis
photodynamicinactivation,usingdyeswhich preferably in the range whichisnot
are activatedbyvisiblelight,
absorbed b y hemoglobin.Themajor
problems have been that many compounds have adverse effects upon the red
DODD
42
cells when used inconditionsknowntoinactivatevirusesandthatthese methods are generally ineffective on cell-associated viruses, including proviral forms (33). To some extent, this problem can be resolved by the use
of high
efficiency leukocyte filters, which are now available, at least on an experimental basis. The photochemical currently showing most promise for red cells is again methylene blue (56).
COMMENT The blood supply in the United States is currently safer than been, with extremely low risk for infectivity for HIV and decreased risk for HCV infection.
it has ever
HBV, andavastly
Recent data suggest that the frequency of
seriousclinicaloutcomesfromHCVinfection,atleastinthe18-20years following transfusion, may not be
as great as had previously been supposed
(57,581. It islikelythatcontinuedimprovementsindonorselectionand questioning will further reduce the risk of infection. However, this approach cannot be expected to
deal with individuals who do not
recognize, or who
deliberatelysuppress,theirriskbehaviors.Serologictestingcontinues
to
improve, but again, may not be able to identify all early infections. considerableprogress
in the development of viralinactivation
There is
procedures;
however, it remains to be seen whether the balance of risksand benefits favors this approach, and whether or not societywill support the added costs. Finally, although this isa review devoted to viral aspects of transfusion safety, it should berecognized
that parasitic disease continues to bearisk,
particularly as
international population movementsincrease. Also, the risk of serious bacterial sepsis from contaminated platelet concentrates appears to exceed that of viral disease.
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19.
K.E.
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and
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CONTAMINATION VIRAL OF
BLOOD COMPONENTS
45
22. Y.C.E. De Graan-Hentzen, J.W. Gratama, G.C. Mudde, L.F. Verdonck, J.G.A. Houbiers, A. Brand, F.W. Sebens, A.M. Van Loon, T.H.The,
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25. K.L. Smith, T. Cobain, and R.A. Dunstan, Er. J. Haematol.,83,,640-642 (1 993). 26. B.J. &hen,
A.M. Field, S. Gudnadottir, S. Beard, and J.A.J. Barbara, J.
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(1 990).
27. F. McOmish, P.L. Yap, A. Jordan, H. Hart, B.J. Cohen, and P.J. Simmonds, J Clin Microbio1,=,,323-328
(1 993).
28. F.B. Hollinger, N.C. Khan, P.E. Oefinger, D.H. Yawn, A.C. Schmulen, G.R. Dreesman, and J.L. Melnick, JAMA,Z,,2313-2317 (1 983). 29.
R.C.
Noble,
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M.A.
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S.A.
Reeves,
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I.
Roeckel,
(1 984).
30. P.H. Azimi, R.R. Roberto, J. Guralnick, T. Livermore, S. Hoag, S. Hagens, and N. Lugo, Amer J Dis Children,=,,23-27
(1986).
31. P.M. Mannucci, Lancet,E$2,,819 (l 992). 32. A. Gerritzen, K.E. Schneweis, H.-H. Brackmann, J. Oldenburg, P. Hanfland, W.H. Gerlich, and G. Caspari, Lancet,=,,1231-1232
(1992).
46
DODD
33. S.J. Wagner, L.I. Friedman, and R.Y. Dodd, Transfus Med Rev,!5,,18-32 (1991).
34. T.-H. Lee, R.R. Stromberg, D. Henrard, and M.P. Busch, Science,=,,
1585
(1 993). 35.
L.R.
Petersen,
L.S.
Doll,
and
HIV
Blood
Donor Study
Group,
Transfusion,a,,698-703 (1 991 1. 36. L.S. Doll, L.R. Petersen, C.R. White, J.W. Ward, and HIV Blood Donor Study Group, Transfusion,a,,704-709 (l 991). 37. D.J. Mayo, A.M. Rose, S.E. Matchett, P.A. Hoppe, J.M. Solomon, and K.K. McCurdy, Transfusion,31,,466-474
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38. S.E. Locke, H.B. Kowaloff, R.G. Hoff, C. Safran, M.A.Popovsky, Cotton, D.M. Finkelstein, P.L.Page,
D.J.
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(1 992). 39. L.R. Petersen, L.S. Doll, C.R. White, E. Johnson, A. Williams, and HIV Blood Donor Study Group, Transfusion,33,,552-557 (1993). 40. H.J. Alter, J.S. Epstein, S.G. Swenson, M.J. VanRaden, J.W. Ward, R.A.
Kaslow, J.E. Menitove, H.G. Klein, S.G. Sandler, M.H. Sayers, I.K. Hewlett, A.I. Chernoff, and HIV-AntigenStudy
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and
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42. R.O. Gilcher, J. Smith, S. Thompson, L. Chandler, J. Epstein, and F. Axelrod, ISBT/AABB 1990 Joint Congress,Abstracts,,60 (1 990).(Abstract)
47
VIRAL CONTAMINATION OF BLOOD COMPONENTS 43. CDC, MMWR.,s,,829-831 (1 990).
44. B. Horowitz, R. Bonorno, A.M. Prince, S.N.Chin, B. Brotrnan, and R.W. Shulrnan, Blood,B,,826-831
(1 992).
45. Y. Piquet, G. Janvier, P. Selosse, C. Doutrernepuich, J. Jouneau, G. Nicolle, D. Platel, and G. Vezon, Vox Sang.,=,,251-256 46.
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Larnbrecht, Mohr, H.
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Schrnitt,
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Sang.,m,,207-213 (1991 1. 47. T. Yuasa, G. Ishikawa, S. Manabe, S. Sekiguchi, K. Takeuchi, and T. Miyamura, J. Gen. Viro1.,72,,2021-2024
(1 991 1.
48. L. Lin, G.P. Wiesehahn, P.A. Morel, and L. Corash, Blood,74,,517-525 (1 989). 49. R.Y. Dodd, G. Moroff, S. Wagner, M.H. Dabay,
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Ribeiro, J. Shurnaker, and L.E. Benade, Transfusion,31,,483-490 50. L. Corash, L. Lin, and G. Wiesehahn, Blood Cells,j&,57-74
(1 991). (1992).
51. H. Margolis-Nunno, B. Williams, S. Rywkin, N. Geacintov, and B. Horowitz, Transfusion,32,,541-547
(1992).
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48
Corash, G.C. Smith, H. Popper,and
DODD J.W. Eichberg, Lancet,~,,l446-1450
(1988).
55. S.J. Wagner, R. White, L. Wolf, J. Chapman, D. Robinette, T.E. Lawlor, and
R.Y. Dodd, Photochem. Photobio1.,57,,819-824 (1 993). 56. S.J. Wagner, J.R. Storry, D.A. Mallory, R.R. Stromberg, L.E. Benade, and
L.I. Friedman, Transfusion,S,,30-36
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F.B. Hollinger, G. Gitnick, R.G. Knodell, R.P. Perrillo, C.E. Stevens, C.G. Hollingsworth, and NHLBl Study Group, N. Engl. J. Med.,327,,1906-1911 (1992). 58. R.L. Koretz, H. Abbey, E. Coleman, and G. Gitnick, Ann. Intern.
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REDUCINGTHE INFECTIVITY OF BLOOD COMPONENTS - WHAT WE HAVE LEARNED Leonard I . Friedman,Robert R. Stromberg,Stephen J. Wagner ProductDevelopmentDepartment Jerome H. HollandLaboratory American Red Cross Rockville, MD 20855 ABSTRACT The s a f e t y o f the nation’s blood supply has improved over the last several years as a r e s u l t o f more intensivedonorscreening and v i r a l t e s t i n g . C o n c u r r e n t l y , t h e r e has beenmore j u d i c i o u s use o fb l o o d components. A l t h o u g ht h er i s ki s small,transmissionofbloodborneviruses,bacteria and p a r a s i t e s canoccur. I n v e s t i g a t o r s have studied a myriad o f processes f o r pathogendepletionand/or i n a c t i v a t i o n i,n c l u d i n gt h e use o f chemicals, extended storage, filtration, heating, irradiation, photochemicals and washing. Pasteurization, methylene blue and solvent-detergentprocesses have been i n t r o d u c e di np a r t so f Europe f o r improvingthesafetyof plasma used f o rt r a n s f u s i o n . The FDA i sr e v i e w i n g a 1 i c e n s ea p p l i c a t i o nf o rt h es o l v e n t - d e t e r g e n tp r o c e s s .F o rr e dc e l l s , use o f h i g h l y e f f i c i e n t l e u k o d e p l e t i o n f i l t e r s i s b e l i e v e d t obe e q u i v a l e n t t o a n t i b o d y t e s t i n g f o r t h e p r e v e n t i o n o f CMV disease transmission. Otherwise, no successful treatments have yet been i d e n t i f i e fdorre cd e l l ospr l a t e l e t sS. e v e r a l photochemicals,which may be u s e f u lf o rt r e a t i n gt h e s e components, arebeing studied. However, t h e r e appear t o be t r a d e - o f f s between theextentofpathogen i n a c t i v a t i o n ,p l a t e l e to rr e dc e l l damage, and g e n o t o x i c i t y . These as w e l l as w i l need t o be f u r t h e r otherbiologicalparameters and operationalissues evaluatedbeforeimplementationcan be considered.
INTRODUCTION Carefuldonorselection and increasedlaboratorytesting have g r e a t l y improved thesafetyofthebloodsupply.Nevertheless, a small r i s k of v i r u s transmission by transfusion s t i el l x i s t s . The most r e c e ne ts t i m a tf eo r HIV-1, 1 i n 225,000; HBV, 1 i n 200,000; HCV, 1 i n t r a n s m i s s i o no fv i r u s e si s : and HTLV 1/11, 1 i n 50,000.(1)Otherbloodbornevirusesinclude CMV, HIV-2, HAV, non-A,B,C h e p a t i t i s and parvovirus B19.(2) If 20 m i l l i o nb l o o d components a r e t r a n s f u s e d a n n u a l l y i n t h e U . S . , and assuming t h a t H I V - 1 i s 100% f a t a l , HBV 0.1% f a t a l , ( 3 ) HCV 1.5% f a t a l ( 4 ) and HTLV 1/11 (combined) 1%f a t a l , thenthe maximum number o f r e l a t e d deaths i s about 185. Since many o f these
3,300;
l i d i eo ft h e i rp r i m a r yi l l n e s so rf o ru n r e l a t e d reasons,the individuals w number o f f a t a l i t i e s as a d i r e c t r e s u l t o f v i r a l l y t r a n s m i t t e d d i s e a s e i s much lower. There are also increasing concerns about transmission of bacteria.(5,6) I n 1990, t h e FDA r e c e i v e d r e p o r t s o f 6 f a t a l i t i e s r e l a t e d t o b a c t e r i a l s e p s i s ;
49
50
FRIEDMAN, STROMBERG, AND WAGNER
5 in platelets and 1 in red cells. Six fatalities due to bacterial sepsis, all in platelet products, were reported during1991.(7) The incidence of non-fatal sepsismaybeashighas 1 per1700transfusionsofpooledplatelet concentrates.(8) Finally, parasitic diseases, such as Chagas, are also being followed with interest.(9) Infectiousdiseasesarenottheonlycauseoftransfusionrelated fatalities. In 1990 and 1991, 29 deaths were due to "administrative errors", such as the incorrect unit being transfused or testing the wrong crossmatch sample; 58 deaths were attributed to serologic incompatibility and other causes.(7) Although the number of deaths attributable to transfusion are relatively small in comparisontothenumberofpatientstransfused, implementation of infectious agent removal and/or inactivation processes should provide an added level o f safety. This could possibly limit the need for additional donor screening or testing, and may reduce or eliminate disease transmission due to current or future blood borne pathogens. PRODUCT DEFINITION It is useful to think of plasma, red cells and platelets as "impure"blood components. P1 asma contains residualp1 atelets, 1 eukocytes and red cell s. Red cells contain residual leukocytes, platelets and plasma. Platelets contain residual red cells, leukocytes and plasma. Depending on the infectious agent, it may be "freely" suspended in the plasma, extracellular but "associated" with red cell, leukocyte or platelet membranes,or intracellular. Table I shows the location of various viruses in these compartments; there are many unknowns. Similarly, bacteria can be found extracellularly and intracellularly, and parasites may also be locatedin different compartments. In order to define an infectious disease removal or inactivation process, the maximum bioburden expected to be present mustbe estimated. Assuming that blood components will be derived from fully tested donations, it is likely that a 106-fold reduction (99.9999%) in virus titer will need to be achieved in extracellular, intracellular and cell-associated compartments.(lO,ll) Table I1 shows the dataused to define the6 log,,, objective. For many of the bacteriaassociatedwithbloodtransfusion,their concentration at the time ofblood collection is
OF BLOOD COMPONENTS
INFECTIVITY REDUCING
51
Table I . Location of Viruses in Blood Components
P1Leukocytes asma Cells Red
Virus
P1 ate1 ets
HIV-1
Yes
Yes
?
Yes
HIV-2
? Yes
?
? ?
?
HBV HCV HTLV-I HTLV-I I CMV
No
B19
HAV
?
?
? ?
No
?
?
yes ?
?
?
No
Yes
No
No
Yes
?
?
?
Yes
?
?
?
Yes
?
Table 11. Estimate of Window Period Virus Load ~~
Virus Infectious Particles Infectious Leukocytes
P1 asma (per mL)
In
HIV-l HCV HBV HTLV-I CMV
(Per mL)
102-104 [Virus isolates]
105-103 [DNA hybrid]
10' [Chimp studies]
?
t5X105 particles [EIA test]
?
0
lo4 [DNA hybrid]
?
?
OVERVIEW OF VIRUS REMOVAL AND INACTIVATION PROCESSES Over
the
past
10
years
a
number
of
virus
removal
Bear
in
mind
that
some
processes
inactivation
proces
I 1 1 for each blood
have been investigated. They are categorized in Table component.
and
may
not
be
suitable
alone,
but
might
be acceptable if combined with another (eg. leukodepletion combined with photochemical treatment). It is also important to recognize that "removal" and "inactivation" are equivalent in terms of final product infectivity, as long as the
process
removes
(eg.
filtration)
or
inactivates
(eg.
chemical)
same extent. PLASMA PROCESSING Although the indications for fresh frozen plasma for transfusion are limited,(l5-17) the clinical demand for this product has not decreased. Approaches for virus removal and inactivation are presented below.
viruses
to
th
52
FRIEDMAN, STROMBERG, AND WAGNER Table 111. Overview of Virus Removal/Inactivation Processes Studied
Process
Cells P1Red asma
column Affinity
X X
Chemical storage
P1 ate1 ets
X X
Extended Filtration size
excl
X on
usi
1 eukodepl Heating
et
i
pasteurization mild heat
X
high T/shortt
X
X
X
on
X
Irradiation ul
travi
ol
et
X X
gamma Photochemical
X X
Washing
Affinity column Affinity adsorption columns have been used successfully for protein purification. In these cases, specific proteins adsorb to selected solid phase matrices; contaminating viruses are washed away. For plasma, however, the approach to
the
has
to
matrix.
removal matrices
was
For
reversed;
about 1 log,,.
would
MiCroDorous
be When need
these
viruses
experiments
(18) Another to
be
made
and
infected
were
disadvantage selective
cells
attempted, for
of
this
each
have
the
to
selectively
extent
approach type
of
of is
"free" that
virus
or
the
infecte
membrane 1 trat f ion i virus
been studied.(l9)
reduction
in
plasma,
size
exclusion
membrane
based
filtration
In this process, plasma is passed through a microporous
membrane in which the mean pore diameter is larger than most proteins, yet smaller than freely suspended viruses and cells containing viruses. Extracellular HIV-l, with a size of about 100 nm, did not pass through nm a 105 pore diameter membrane.
However, due to the small size of B19
hepatitis delta (28-39 nm), HCV (30-60 nm) and diameter
bin
viru
membrane
is
required.
(18-26 nm),
HBV (42 nm),(20) a smaller pore
When p1 asma is filtered
through
a
40
nm
membrane,
there is considerable loss of high molecular weight clotting factors.(21)
ha
OF BLOOD COMPONENTS
INFECTIVITY REDUCING
53
Gamma irradiation High
energy
gamma
irradiation
has
been
used
to
inactivate
viruses
in
fresh
frozen plasma. At 40 kGy, 10e.5 infectious dose(ID)/mL of HIV-1 was inactivated in plasma at -4O"C.(22) However, there was concurrent clotting factor activity
VIII:C, loss of14.6, 38.8 and 23.3% for factors
V1II:vWF and IX, respectively.
In an unrelated study, using similar irradiation conditions, more significant protein
damage
occurred
.
and
the
process
was
found
to
be
unacceptable
for
further
development (23) Iodine Two iodine based methods for virus inactivation have been investigated. Iodine, when bound to cross-linked polyvinylpyrrolidone of VSV in
inactivate >5 log,,
cryoprecipitate-poor
(XLPVP), was able to
plasma
while
decreasing
factor
IX activity by <10%.(24) However, since XLPVP was not able to bind iodine as tightly as was desired, another carrier was considered. Iodine bound to crosslinked and
starch
B19).(25)
>75% factor
inactivated lo>9 g,, Under
these
of
VSV and >7log,, EMC (a model virus for HAV
conditions
there
was
retention
70% VI11 factor and
of
IX activity. The disadvantages to such a process are the need to
remove the starch-iodine following incubation, the potential release of iodine from the matrix and a reduction in coagulation factor activity with increasing starch-iodine concentration or incubation time. Pasteurization Pasteurization (10 hrs @ 60°C) of thawed, pooled and stabilized plasma has been implemented in France.(26,27) Studies indicate preservation of 75-95% clotting factor activity and no protein denaturation or coagulation factor activation. In vitro studies demonstrated that more 5than log,, log,,
HIV-1 and >5.7
Sindbis (a model virus forHCV) were inactivated. However, no chimpanzee
studies, using
HIV-1, HBV and HCV spiked and processed plasma, have been
reported. These data are needed in order to evaluate the overall effectiveness of this specific pasteurization process.(28) Preliminary
virus
inactivation
studies
in
plasma
were
also
conducted
using
a high-temperature short-time heating method.(29) The problem is to determine the
temperature
and
time
conditions
where
the
rate
of
virus
inactivation
exceeds
the rate of protein damage. For example, with a process duration of S0.25 and
HIV-1 was inactivated with a 15-20% V loss of factor VI11 and IX activity; there was a greater loss of factor activity. Three log,, of HIV-1 (74"C), 25.8 log,, EMC (172°C) and 24.4 log,, VSV a
hold
time 0.006 of
S
at 77"C, 24.4 log,,
of
(175°C) could also be inactivated. Solvent
Deterqent
Virus inactivation in plasma, using a solvent detergent process, has been implemented in parts of Europe. In
it, the thawed and blood group specific
FRIEDMAN, STROMBERG, AND WAGNER
54
pooled plasma is incubated with 1% tri(n-buty1)phosphate l%Triton and X-l00(a non-ionic detergent)
at30°C
for 4 hours.(30) Subsequently, the chemical
additives are removed by chromatography and the resulting plasma is sterile filtered and refrozen. The final product contains approximately 200 mL and has a shelf-life of >2 years when stored at -30°C.
Protein recovery is good and
coagulation factors are not activated. In vitro inactivation studies
VSV kill of 27.2,
demonstrated HIV-l, Sindbis and
and27.5
16.9
log,,,
respectively. Chimpanzee studies resulted in virus inactivation of r l 0 ' CIDSO for HBV and r105 CID,, underway
for HCV.(31)
3 years over
for
and
show
Extensive clinical studies have been
comparability
between
untreated
fresh
frozen
plasma and solvent-detergent treated plasma. Solvent/detergent
processes
have
been
used
for
virus
inactivation p1 asma
of
derivatives for many years. One drawback is its inability to inactivate nonlipid
enveloped
suchHAVasand
viruses,
B19. HAY transmission
has
recently
been
reported in Europe following treatment of plasma derivatives.(32-34) Another potential issue may relate to residual solvent and/or detergent in transfusions to neonates. Also, as with pasteurization or any process based on treatment of pooled components, a breach in manufacturing or a process inadequacy will have an affect on many patients.
B1 ue ene
Methvl
A photodynamic method, using methylene blue (MB), has been introduced in partsofGermanyandSwitzerlandforvirusinactivationofplasmafor transfusion.(35) In this process,MB is metered into individual thawed units of previously
frozen
plasma
by
sterile
connection,
1 p M concentration. to make a
The
units are then incubated in the dark with agitation at4°C for 1 hour before irradiation by visible light(50,000 lux) for 1 hour. Refreezing is the final stepinthisprocess.Mostplasmaproteinsarenotinfluencedbythe process(36); there is usually less than a 20% impact on those that are. For example, under slightly different irradiation conditions there is a loss of approximately 13% of factor studies
indicated
VI11 and 17% of factor IX activity.(37) These extracellular HIV-1, VSV, HSV
that 22.5, 25.7, r 3 . 0 , 25.5 log,,
and influenza, respectively, were inactivated. However, non-lipid enveloped viruses were not greatly impacted; poliovirus was not inactivated and only1 log,,
adenovirus was inactivated. Of additional concern is the fact that
treatment withM8 does not inactivate intracellular virus. The initial freezethaw
step
is
intended
to
lyse
residual
leukocytes
or
make
the
intracellular
vi
more accessible to the M8 treatment. However, the freeze-thaw treatment is not 100%effective.(38) microporous
membrane
It isreasonabletoexpectthatleukodepletionor filtration
would
be
more
effective
in
removing
intracellu
viruses. In terms of toxicity, methylene blue has been used clinically for many years
and
at
significantly
higher
doses.
There
have
been
only
occasional
reports
ONENTS BLOOD
OF INFECTIVITY REDUCING
55
of adverse effects. Over 100,000 units of MB treated FFP have been transfused in Germany. In comparison with transfusionsof untreatedFFP, there has been no increase in adverse outcomes. A more controlled clinical study of methylene blue treated FFP is now being conducted in Germany and a review comparing MB and solvent detergent methods for virus inactivation in FFP has recently been pub1 i shed. (39) Mi scell aneous aDDroaches A number of other virus inactivation processes for plasma have been studied.Forexample,beta-propiolactone in combinationwithultraviolet irradiation has beenused in Germany for cold-sterilization of pooled serum and several types of plasma derivatives for many years.(40) Psoralen compounds and UVA light have also been studied, but usually as part of investigations involving platelets and/or red cells. In one study,aminomethyltrimethylpsoralen (AMT) was found to inactivatez6 log,, ID,, of VSV; factor VI11 recovery averaged 57%.(41) Use of glutathione, as a quenching agent, increased the recovery to 87% without affecting virus kill. A second studyused a newly developed brominated psoralen derivative; S log,, inactivation of two non-enveloped bacteriophages was obtained while retaining 90"/0 factor VI11 activity.(42) Ozone, administered through a microporous membrane system was demonstrated to inactivate HIV-Iin plasma and factor VI11 concentrate.(43) Medium-chain saturated and long-chain unsaturated fatty acids and monoglycerides have also been shown to inactivate significant levels of HIV-l, HIV-2 and HSV in plasma and serum.(44) Summarv of Dlasma Drocesses Three processes have been implemented outsidetheofU.S. for inactivating viruses in plasma: chemical (solvent detergent); heat (pasteurization); and photochemical(methylene blue) . Table IV showssomeoftheprocess considerations and indicates data which have not yet been reported. Whether as stand-alone processes orin combination with one or more additional steps, it is 1 i kely that they are able to reduce the virus transmission risk of plasma for transfusion. The solvent detergentand pasteurization processes should also be able to inactivate contaminating bacteria and parasites; it is not known whether the methylene blue process is effective. This may, however, be a moot point since most plasma products are frozen during storage and proliferation of pathogens or generation of significant endotoxin is unlikely. RED CELL PROCESSING Manyresearchgroupshavestudiedvirusdepletion and inactivation processes in red cells using various approaches. In addition, since several species of bacteria can grow in red cell suspensions during storage and some parasites are associated with red cells, the "ideal" process would be one in
56
FRIEDMAN. STROMBERG, AND WAGNER
IV. Virus Inactivation of Plasma
Table Consideration
Pasteurization SolventMethylene
B1 ue
Detergent Extracell
ul rus ar
vi
Intracellular virus Virus limitations
Yes
Yes
Yes
Yes
Yes
?
Lipid
Lipid
Chimp studies
Yes
Protein loss
Yes
? ? Yes
Potential toxicity
Low
No
Low
Control1 ed
Yes
?
?
Central manufacturing
Yes
Yes
Possible
Pooled p1 asma
Yes
Yes
Uniform
Yes
Yes
Product QC
Yes
Yes
No No No
Process QC
Yes
Yes
Yes
?
Yes
?
No
No
Minor
tri a1 S
product
Suitable for all patients Hospital practice change
which
pathogens
below
reviews
would some
of
be
removed
the
work
or
inactivated
which
has
been
? Yes
in
all
compartments.
The
section
reported.
Leukodepletion Leukodepletion has been investigated as a means of removing leukocyte associated viruses. Early clinical studies in which approximately 1-2 log,, leukocytes were removed by washing
of
or filtration resulted in significant
CMV transmission.(45-50) As leukocyte reduction filters became more efficient, the data indicating CMV elimination became more
reduction or elimination of
compelling.Finally,in a recentmulti-centercontrolledclinicalstudy involving bone marrow transplant patients, the use of filtered red cells was
CMV infection. compared to the use of seronegative red cells for prevention of There was no significant difference in the incidence CMV of between the tested and filtered red cells.(51) Thus, the use of filtered red cells should be comparable to the use of seronegative red cells for prevention of CMV disease.
HTLV-I, Leukodepletion may have an effect on other pathogens. In the case at of least a 3 log,,
reduction in
infectivity
was
reported
after
leukocyte
removal
filtration of blood spiked with cultured HTLV-I-transformed cells.(52) With
HTLV-I infected individuals, the number of infected cells was reduced by 1-3 . , g o l
Although filtration does reduce infectivity, there are
no data to
by
REDUCING INFECTIVITY OF BLOOD COMPONENTS
57
demonstrate that filtration will prevent its transmission. With HIV-l, both "free" and cell-associated virus is present. Therefore, it is not feasible for transmission to be prevented by filtration alone. However, using PCRand/or coculture methods, it has been demonstrated that filtration reduced the number of infected leukocytes in HIV-l spiked red cells(53-55) and in blood obtained from seropositive donors (54,55) Thus, leukocyte removal may reduce the infectious di sease bi oburden. Additional studies have indicated that Yersinia and other bacteria in red cel 1 S can be reduced by plasma proteins and filtration.(56-60) P1 ate1 et DeDl et ion Platelets are a major "contaminant" in red cells. A number of viruses such asinfluenza,(61) vaccinia(62) and herpes(63) havebeenassociatedwith platelets. More recently, a strong association between platelets and free HIV-1 has beenreported.(64,65) Leukodepleted and washed platelets obtained from HIV-1 seropositive donors were found to be positive for HIV-1 and PCR-RNA negative for PCR-DNA, showing that the detected virus was not incorporated in leukocytes. Extensive washing did not remove significant quantities of virus, indicating binding between the platelets and HIV-l. The presence of HIV within platelets has also been shownby electron microscopy.(66) Thus, although the infectivity of HIV-1 associated with platelets has not been demonstrated, residual platelets in red cells may need to be considered as a vector for virus transmission. Washinq In the late 1970's it was believed that red cell freezing, deglycerolization and washing would eliminate HBV. Initial reports based on retrospectiveanalysessuggestingasubstantialreduction in hepatitis infectivity(67)werenotconfirmed. In aprospectivestudy,noclinical hepatitis was contracted with frozen, deglycerol ized and washed red cells compared toseveral cases in the control arm. However, the number of experiments were not sufficient to be statistically significant.(68) On the other hand, frozen, deglycerol ized and washed cells have been reported to have no effect on reducing the incidence of post-transfusion hepatitis. (69) Further evidence that suchtreatmentdoesnotpreventtransmissionofhepatitiswasshown by transfusing 4 chimpanzeeswithhumanredcellsthat had been frozen, deglycerolized and washed after being spiked with hepatitis virus that were CEP negative but RIA positive.(70) All chimpanzees demonstrated unequivocal evidence of infection. More recently, a series of leukodepletion and washing experiments were conducted.(71,72) Prior to processing, the red cells were spiked with VSV, Sindbis, pseudorabies, or the bacteriophage Experimental filters were used to remove 6 log,,, of leukocytes(73-75); about 2t log,, of platelets were concurrentlyremoved.Based on dyeexperiments, it wasestimatedthat
$6.
58
STROMBERG, FRIEDMAN,
AND WAGNER
commerci a1 ly avai 1 ab1 e cell washers would remove about1 og,,5t of p1 asma. Under conditions which should have resulted in approximately 6 log,, virus washout, the maximum reduction observed was typically 4-5 log,, from the supernatant, but <2 log,, from the red cells. (71) Extensive test tube experiments were also conducted in which the wash solution to red cell volume was considerably increased from thatin the cell washers. Although virus reduction was improved, residual model virus was always present in the red cell portion. These results suggested virus association with red cells or with contaminating platelets. Virus reduction was not improved by modifying the wash conditions. This was doneby slightly elevating the temperature or changing the ionic strength or protein level of the wash solution. A series of experiments was conducted in which different detergents were added to VSV spiked red cells to determine whether they could augment the release of virus from red thecell membrane and thus improve washout efficiency. The detergents included nonionic and anionic, short and long chain aliphatic and aromatic compounds. No improved virus depletion was observed unless accompanied by significant red cell damage.(72) Mild temperature elevation Although a slight temperature elevation will not inactivate most blood borne pathogens, HIV inactivation in culture media was reported to occurat a rate of about 1 log,,/hr at 45"C.(76) Thus, if inactivation occurred linearly, a simple6 hr incubation would be able to reduce or eliminate HIV from cells.red Unfortunately, when the studies were conducted using red cell suspensions, only about 0.25 log,,/hr was inactivated at 45"C.(77) In addition, thered cells were irreversibly damaged. These data indicate that heat treatment will not be effective for HIV inactivation. Extended storaqe In a retrospective study of individuals who were later found been to have HIV-I positive at the time of donation, the infectivity of transfused red cells was significantly reduced during storage. There anwas 89% transmission rate for products stored from 8-14 days, compared to a50% transmission rate after 22+ days of storage.(78)In an unrelatedstudyusingco-cultureassays,the infectivityofredcellsspikedwithHIV-1infectedT-cellsdeclined exponentially during storage, although the rate appeared to be dependent on the suspension medium.(79) Thus, while this is a simple approach, most infectivity of intracellular virus is retained during storage. Chemical aooroaches Chemical approaches have alsobeen explored for virus inactivationin red cells. The literature indicates that ozone is capable of inactivating HIV1(80,81) and other pathogens.(82-84) In a series of test tube experiments using
INFECTIVITY REDUCING
COMPONENTS OF BLOOD
59
VSV and @, it was demonstrated that 5-6log,, virus kill was only achieved at low (-5%) hematocrit and thatextensivehemolysis (-30%) occurred.(85) Unexpectedly, this level of hemolysis could not be visually observed in the supernatant because of ozone-induced destruction (bleaching) o f extracellular hemoglobin. The use of sodium chlorite and lactic acidto generate chlorous acid and chlorine dioxide for HIV inactivation has been suggested.(86,87) Recent studies were unable to achieve effective virus kill without significant damage to red cells, using C102 generated from sodium chlorite by addition of lactic acid and chloride ions.(88) One report in the literature also indicated that decreasing the pHin cell-free media inactivatedHIV-1.(89) We have been unable to achieve HIV-I inactivationat pH levels as low as 4.9 in the absence o f red cells. Medium chain saturated and long chain unsaturated fatty acids have been reported to inactivate enveloped viruses. Monoglycerides of the acids were also found to have antiviral activity, frequently at considerably lower concentrations. (90) However, wefoundthatmonoglycerideetherscaused unacceptably high hemolysis. Photochemical amroaches Most published work on the inactivation of viruses in red cells have utilized photochemicals such as merocyanine 540 and its derivatives,(91,92) benzophorphyrin derivative,(93-95) aluminum phthalocyanine and related compounds, (96,97) and methylene blue. (98,99) Evidence suggests that the hydrophobic dyes merocyanine 540 and benzophorphyrin derivative target virus envelopes for damage. Thus, these photochemicals are dependent on subtle differences between virus and red cell membranes to inactivate virus without significantly altering the structure or function of red cells. Although they inactivate lipid enveloped extracellular (and in some cases, intracellular) viruses, it has been difficult to maintain red cell properties, especially during subsequent storage.' Less information is available about the likely mechanism for virus inactivationusingaluminumphthalocyanine and itsderivatives.However, phototreatment of red cells with aluminum phthalocyanine tetrasulfonate under virucidal conditions resultedin some alteration ofred cell surface properties and reduction of circulatory half-life.(IOO) More recent studies using new phthalocyaninederivativesshow model virusandHIV-linactivationwith maintenance of selectedin vitro red cell properties.(lOl) Methylene blue, a phenothiazine dye which can intercalate between nucleic acid bases(l02), has also been studied as a virucidal photochemical. Upon exposure to visible light of wavelengths that hemoglobin does not appreciably absorb, a varietyof nucleic acid lesions have been observed. More than log,, 5 of some model viruses becaninactivated in 30% hematocrit suspension with modest alterations o f in vitro red cell properties, and stability over 42 days of
60
FRIEDMAN, STROMBERG, AND WAGNER
storage.(98) Although methylene blue can inactivate extracellular HIV-1 under conditions which do not compromisein vitro red cell properties, other results have been discouraging. Different viruses have very different sensitivitiesto methylene blue photoinactivation.(103) The conditions required to inactivate M6 resistant viruses, such as VSV, result in large changes in red cell ion permeability and the binding o f plasma proteins to red the cell surface(98); nonlipid enveloped viruses are not usually inactivated.(99) Furthermore, methylene blue treatment does not inactivate HIV-1 infected H9 cells or Sindbis infected Vero cells under conditions that readily inactivate extracellular virus.(99) Other photoactive chemicals are currently under investigation. Hypericin, a drug currently being evaluated in clinical trials in AIDS patients, has recently been reported to inactivate 6 0 4 TCID ofHIV-I in whole blood.(l04) No red cell damage was observed during 21 days of post-treatment storage. There have been limited studies using anovel brominated psoralen derivative.(42) In this case, 6.6 log,,, o f was reported to be inactivated in a 60% hematocrit suspension with no significant alterations to selected in vitro red cell properties. For UVA activated photochemicals, however, light absorption by hemoglobin strongly inhibits virucidal activity.(l05) Summarv of red cell Drocesses In spite of the large number of studies of both physical and chemical processes, there are no near term approaches which appear to be suitable for "generic" inactivation of viruses and/or bacteria in red cells. High efficiency leukocyte depletion is expected to eliminate the transmission of CMV disease. With other approaches, significant virus inactivation is usually accompanied by red cell damage. Thus, a combination of processes may be needed. For example, leukodepletion by filtration could remove most white cell associated viruses, and to a lesser extent, those which may be associated with residual platelets. In addition,thisstepmayalsopotentiallyreducebacterialbioburden. A subsequent photochemical step would inactivate "free" and membrane-associated viruses. The photochemical should be one which is activated at a wavelength which is largely unaffected by the presence of hemoglobin and plasma proteins. PLATELETPROCESSING
For platelet products, a process to inactivate bacteria as well as viruses might permit extension of the shelf-life of platelets, thus reducing a portion of the outdating which now occurs. Miscellaneous apDroaches While leukodepletion maybe an important step in platelet virus depletion and inactivation methods, filtration alone not has been shown to be satisfactory. Forbacteriaassociatedwithplateletproducts, it is unlikelythat leukodepletion will have much of a positive effect.(l06) Washing is not an
INFECTIVITY REDUCING
COMPONENTS OF BLOOD
61
acceptable method due to platelet activation, high losses and the strong associationwith HIV-1.(64,65) WhenUVBalonewas used toinactivate extracellular virus, platelet damage resulted.(107,108) Photochemical aDDroaches Several photochemical-based approaches have been studied. Those used which merocyanine 540 or aluminum phthalocyanine sulfonate in combination with visible light resulted in significant inactivation of viruses, but with platelet damage.(97,108-110) Considerable effort has been directed at studying the use ofpsoralens in conjunctionwithUVAlight.Withpsoralens,suchas 8-methoxypsoralen (8-MOP) or AMT, which form psoralen-nucleic acid adducts, inactivation of 5-6log,, o f several model viruses and 4-6 log,, inactivation of extracellular HIV-l(l11,ll~) hasbeen demonstrated, sometimes under conditions which do not impact in vitro plateletproperties.(105,109,113,114) Intracellular HIV-l is also inactivated by both 8-MOP and AMT.(112,115) In addition, 8-MOP has been shown to inactivate duck hepatitis B, a model for HBV.(116-118) Free radical quenchers were able to reduce platelet damage resulting fromofuseAMT under conditions which inactivated26 log,, cell-free virus and 15 log,, cellassociated virus in 100% plasma.(119,120) These studies used high UVA doses to compensateforreducedvirusinactivationduetoAMTbindingtoserum albumin.(l21) At such doses in the presence or absence of AMT, irreversible p1 atelet damage can occur. Preservation of hemostaticeffectivenessofAMT-UVAtreatedhuman platelets,usingvirucidalconditions,hasbeendemonstrated in arabbit model. (103) However, the light exposure to achieve this must be carefully controlled. In addition, AMT (in the dark) produces frameshift mutations in bacteria and the frequency of these mutations is greatly enhanced by an S9 metabolic activation systemused to simulate drug metabolism in the liver. (121) Thus, theresidual photochemical and its breakdown products may require removal. Efforts to synthesize less mutagenic psoralens which retain virucidal activity without harming the in vitro propertieso f platelets have recently been reported. Limited data regarding use of a brominated psoralen derivative (PSRBr) indicate r6 log,, inactivationofabacteriophagewithnoreported genotoxicity.(42,122) Data have also been presented on a series of novel primary amino-psoralens.(l23) In platelet concentrates, 5-6 log,,/mL of cell-free HIV-l wasinactivated by several of them.Littleornoframeshiftorbase substitutionmutagenicitywasobservedwiththesecompoundsortheir photoproducts. After 5 days of storage, normal aggregation was exhibited although platelet concentrate pH was lower than controls. One of them, S-59, inactivated >106/mL duck hepatitis B and >105/mL HCV based on a PCRassay.(124) It also inactivated >106/mL cell-associated HIV-l. Genotoxicity was examinedin both bacterial and mammalian systems and found acceptable. S-59 was weakly
62
STROMBERG, FRIEDMAN,
AND WAGNER
mutagenic in one Ames test strain (TA1537) without metabolic activation; this result disappeared after light activation. As pointed out previously, bacterial sepsis is also associated with platelet
products.
Studies
have
been
conducted
which
demonstrated
that
8-MOP
than
lo5
can
inactivate many bacterial species spiked into platelet concentrates while preserving cfu/mL Four
platelet
of5 gram of
function
positive,
and
27 gram of
the
more
protein
level
was
andthe
8-MOP concentrationincreased.
organism,
was
resistant
during 5 days
reduced
of
bacteria
to
15%
storage.(125-128)
negative were
inactivated
(through
use
of
a
were >lo5
new
inactivated. cfu/mL
when
synthetic
the
storage
pla
medi
P aeruginosa, the most resistant
inactivated loby 4.' cfu/mL. In conjunction with these studies, the
pharmacokinetics of intravenous 8-MOP was studied.(l29) Five infused
Greater
organisms,
over60 min
to
used for PUVA therapy.
normal
subjects;
peak
plasma
or 10 mg were
levels
were
less
than
those
No drug was measurable after 2 hrs no andside effects
or adverse reactions were observed. Summary
of~1 atelet
Drocesses
Although many approaches have been investigated, no process is yet available
for
the
inactivation
of
viruses
and/or
bacteria
in
platelets.
As
with
red cells, aphotochemicalprocess,possiblycoupledwithleukodepletion filtration, is likely to be successful. There is
a reasonable chance that a
novel photochemical will be developed which will permit concurrent virus and bacterial inactivation. Table V reviews some of the process considerations for known photochemicals and indicates data still needed.
ISSUES TO CONSIDER Over
the
years
we
have
learned
that
the
use
of
model
viruses
for
eva
may
imp
proposed depletion and/or inactivation approaches may not be suitable. Model virus interactions withred cells, leukocytes, platelets and plasma may differ significantly
from
those
of
the
viruses
of
concern.
These
differences
how efficiently a specific approach will work. As a result, many investigators have focused on HIV-l as their primary target virus. However, there may also be differences among laboratory strains HIV-I, of HIV-1 from newly sero-converted individuals latter
case,
andHIV-1 from which
antigen
represents
positive,
donors
in
antibody the
negative
"window
blood
period",
is
donors. most
The
relevant
the blood banking community. In addition to HIV-l, studies of removal and inactivation processes should include a broad range of transfusion transmitted pathogens. Another issue is how to demonstrate the safety and efficacy of a removal or inactivation process. Since disease transmission as a result of known blood borne
pathogens
is
infrequent,
controlled
clinical
trials
to
effectiveness would be virtually impossible to carry out in U.S. the
demonstrate Instead,
process
t
e
OF BLOOD COMPONENTS
INFECTIVITY REDUCING
63
Table V. Virus Inactivation of Platelets Photochemical Consideration Visible Light MC540, A1 Pc Extracell ul ar virus Intracellular virus Virus limitation only Bacteria Chimpanzee studies P1 ate1 et damage Plasma interferes Genotoxicity
UVA Light &MOP, AMT, PSR-Br,
$59
Yes No
Probably ?
Lipid No
No
Possibly 8-MOP
Depends on photochemical Depends on photochemical Depends on photochemical
we will most likely need to rely in on vitro virus and bacteria studies aswell as in vivo chimpanzee studies. A series ofin vitro and in vivo platelet and red cell studies o f treated components should be acceptable to demonstrate safety. In vitro genotoxicity studiesand pharmacokinetic studieswill also be required, but it may be hard to demonstrate with a high odegree f certainty, that the risk of a process is significantly lower than its benefit. Provided that sufficient data are available, the medical and scientific community may be able to decide on the relative safety and efficacy and of virus bacterialremoval orinactivationprocesses. In addition, in thecurrent atmosphere of concern for healthcare costs, the issue of cost-effectiveness of any process should be discussed. An analysis has been conducted to estimate the cost of blood borne infectious diseases per unit of red transfused.(l30) cells The medical costsand lost earnings potential due to morbidity and mortality associated with HIV-l, HBV, HCV, CMV, syphilis, Epstein-Barr virus, Yersinia, malaria, Babesia and Chagas were considered. Other inputs included the incidence of infection, the occurrence of subsequent clinical diseaseand the age and sex of thepatients. All costs were presentedin 1990 dollars. Using a5% discount rate to calculate net present value, the results indicated that the medical, morbidity and mortality cost to society was $18.78: $0.63 for HIV/AIDS; $0.05 for HBV; $17.34 for HCV; and $0.76 for all other infections combined. These figures are currently being recalculatedand the cost may be even lower, depending upon the mortality associated with HCVand HCV test improvements. Additional studies of this type need tobe undertaken.
64
STROMBERG, FRIEDMAN,
AND WAGNER
Table V I . Process A c c e p t a b i l i t y F a c t o r s
cceptable Less Acceptable Consideration Highly V i r u st y p e Inactivation/reduction Bacteria type Inactivation/reduction Component y i e l d Component s u r v i v a l Shelf-life Process type Singlevspooled Complexity Throughput Disposables Toxicity cost Process l o c a t i o n Suitability Hospital
It i su n l i k e l yt h a t
u l t i m a t ea c c e p t a b i l i t yo f
Generic
Se1ected
6 10970 Generic
< 6 loglo
log,, Unaffected Unaffected Unchanged One ( f o r a l l Single unit Easy High Current None
< 6 10910
2
Se1ected
2 6
Reduced
components)
Low Bloodcenter A ll p a t i e n t s No change
Reduced Reduced Several Pooled Complex Moderate New/additional Toxicity
any one process w i l be perfect.Therefore,the a v i r u s and/or b a c t e r i ad e p l e t i o no ri n a c t i v a t i o n
process w i l l i k e l y dependon many i n t e r r e l a t e d f a c t o r s and t r a d e - o f f s wil need Some examples otfh e s ae r e shown i n Table VI. Undoubtedly, differentpeople w i l have d i v e r g i n g v i e w s o f t h e i r r e l a t i v e i m p o r t a n c e .
t o be made.
WHAT HAVE WE LEARNED?
Extensivevirusremoval
and i n a c t i v a t i o ns t u d i e s
have been conducted i n
p1 asma, r e d c e l lS and p1a t e l e t s by many groups. Much has been learned about what w i l and w i l n o t be s u i t a b l ef o r use w i t hb l o o d components. For plasma, although some issuesremain,three methodshavebeen implementedoutsidethe pasteurization,solventdetergent and methyleneblue.Forredcells and possibly platelets, leukodepletion should be safe and e f f e c t i v feot rh e e l i m i n a t i o n o f CMV disease and may prove t o be b e n e f i c i a l f o r t h e r e d u c t i o n o f some b a c t e r i a ,a tl e a s ti nr e dc e l l s .S e v e r a ln o v e lp h o t o c h e m i c a l sa r eb e i n g developed f o r v i r u s i n a c t i v a t i o n i n p l a t e l e t s . They may a l s o have t h e p o t e n t i a l t oi n a c t i v a t eb a c t e r i a . However, since these photochemicals are new drugs,
U.S.;
REDUCINGOF INFECTIVITY
BLOOD COMPONENTS
65
licensing studies will take several years. Although several photochemicals may have have cells.
limited value in inactivating some viruses red cells, in no generic methods been identified which inactivate all species of viruses without red damaging However,
despite
the
difficulty
in
identifying
processes
which
inactivate
a broadrange of extracellular and intracellular pathogens without harming the cells being preserved or the patients who receive the blood components, significant knowledge has been accumulated. It is realistic to anticipate that virus and bacteria removal and inactivation processes will continue to be developed for future implementation.
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G. Moroff, S. Wagner, L.E. Benade and R.Y. Dodd, Blood Cells 18, 43-56 (1992).
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INFECTIVITY REDUCING
COMPONENTS OF BLOOD
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H. Londe and M. Janda, Blood (1994).(In press)
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This Page Intentionally Left Blank
PHOTODYNAMIC VIRUS INACTIVATION OF BLOOD COMPONENTS
H. Mohr, B. Lambrecht and A. Selz Blood Transfusion Service of Lower Saxony Springe Institute D31830 Springe, FRG
INTRODUCTION Photodynamic virus inactivation of cellular blood products (red cell, platelet concentrates and of fresh plasma is, in principle, easy to perform: after addition of a photosensitizing agent (which should be nontoxic and nonmutagenic), theblood component is exposed to visible light. This resultsin the inactivation of all viruses whichmight contaminate the productwithout compromising the properties of the plasma proteins. Unfortunately, things practice, so it is notsurprisingthat
are not as easy in
after years of activitiesin this field(1-12)only
one
photodynamic virus inactivation procedure for blood products has been established to date: decontamination of fresh frozen plasma by methylene blue (MB)/light treatment. PHOTOINACTIVATION OF PLASMA The virus inactivating properties of methylene blue (MB) and other phenothiazine dyes in combination with visible light have been knownfor many years. The list of virusesfor which this has beendemonstrated is long (13-27). The useofthesedyes
for the photodynamic
inactivation of viruses in plasma was also proposed: Heinmets described a flow-through device that was suitable to treat plasma pools (13). Perhaps becauseof its technical complexity this procedure was never routinely used. The German Red Cross Blood Transfusion Service of Lower Saxony developed another procedure especially designed to treat
single plasma units. It was
introduced in 1992 and is now being used by a number of other transfusion services ofthe Red Cross in Germany and in Switzerland.
By the end of 1994 approximately 250,000 units of 73
MOHR, LAMBRECHT, AND SELZ
74
MBllighttreatedfreshplasma
hadbeen distributed.Experience to date showsthat it is as
effective and as well tolerated as untreated plasma (31, 32). A schematic depiction of the procedure is shownin Fig. 1. Fresh plasma units in plastic
bags which were stored at -30°C or lower are thawed in a shaking water bath at 27°C within approx. 15 min. MB as a 50 pM aequous stock solution is added to these plasmas by use of a sterile connection device and a computer controlled balance system (Fig. 2).
The final dye
concentration of MB in plasma is 1 pM, i.e. 320 pgll. In the following working steps, about 20 plasma units are processed at the same time. The plasma containing bagsare preincubated in a thrombocyte agitator in the dark at 10°C for 1 h. To ensure mixing of plasma anddye, the bags are rotated over head during preincubation. Aftexwards, the plasma units are illuminated on an air-cooled light box equipped with fluorescent tubes (Fig. 3). The minimum light intensity has to be 45,000 Lux (31). Illumination timeis 1 h. It was found thatthe plastic containers fromall common suppliers can be used for MB/light treatmentof plasma (15). Finally, the plasma bags
are sealed into other plastic bags and again shock frozen to below -30°C within less than 45
min. In contrast to the manufacture of conventional fresh frozen plasma, that of the MBllight treated product involvesan additional thawinglfreezing step.Like others (33,34), we found that this does not influence the functional activitiesof plasma proteins, e.g. coagulation factors. INFLUENCE ON THE ACTIVITY OF PLASMA COMPONENTS It has beenreportedthattheseactivities
are moderatelyaffectedbyphotodynamic
treatment itself (28-32).The extent of losses in activity is directly dependenton the illumination time, as demonstrated for coagulationfactors I, VIII, IX and XI (Fig. 4). After 1
h in the
depicted experiments it ranged between 20-30%. However, not all plasma proteins suffer aloss in activity as a result of MBllight treatment: coagulation factor XIII, Cl inhibitor, AT III and other inhibitors and the complement factorsCl and C, are hardly altered (35).There is also only marginal alteration of inflammatory proteinsby MBllight treatment (not shown). Although a number of plasma proteins is functionally affected by MBllight treatment, no immunological modifications were detected: extensive animal and laboratory experiments have shownthatMBllighttreatmentunderroutineconditions
does notlead to the formation of
neoantigenic structures (36, 37) that might lead to the formation of antibodiesin the recipients
of the plasma. MB remains in the plasma after photodynamic treatment. This is justifiable because the toxicological propertiesof the dye are well known (38), and it has been in clinicaluse for a long time, e.g. in the treatment of methemoglobinemia and depressive illnesses(39,40). The dosage levels thatcan be administered to humans without remarkable side effects are in the range of 1-2
PHOTODYNAMIC VIRUS INACTIVATION
75
Fresh frozen plasma stored at 3 0 "C or lower
I
f Thawing of plasma in water bath (27 "C, approx. 17 min)
I
Check for usability (visual test)
molytic, lipemic,
Discarding of plasma or alternative use
\ (50 PM, 5.1 m11250 ml plasma)
I
Preincubation (mixing at 8-12 'C for 60 to 90 min)
l
(60 to 65 min, >45 000 LUX) I
I
Labelling, Vacuum sealing into additional plastic bag
1 Shock freezing within 30 40 min,
-
FIGURE 1 Schematic representationo f the production procedure for MB/light treated fresh plasma.
FIGURE 2
Equipment for the addition of MB-stem solution to single units of plasma.
FIGURE 3
Photodynamic treatment of single units of fresh plasma containing 1 pM MB on a light box equipped with fluorescent tubes.
I
0
PHOTODYNAMIC VIRUS INACTIVATION
i
0 0 0 0 0 0 h 1 O o O W d h l l-l-
0
L
S
LL
x
0
W
v)
d
E
.-c
Y
77
MOHR, LAMBRECHT, AND SELZ
78
mg per kg body weight per day, even for prolonged time periods (40), which means that an adult tolerates about 100-150 mg of MB per day. In contrast, the amount of MB required for photodynamic virus inactivation is not higher than approx.
320 pg per liter of plasma. This
indicates a considerable rangeof safety. The same is true with regard to the mutagenic properties of MB or MBllight treated plasma. In our studies the latter was found to be negative in all bacterial and eukaryotic test systems used.MB itself was positivein the Ames test insome strainsof Salmonella typhimuriwn at concentrations lOOOfold higher than those used for the photodynamic treatment ofthe plasma. These findings are in accord with published reports indicating that MB has some mutagenicity in prokaryotes (41-43). In contrast, published data indicate that MB is of non or very low our investigations: mutagenicity for eukaryotic cells (44-47). This is supported by the results of MB was negative in V79 cells (Chinese hamster lung fibroblasts) up to 100pglml. In human T-
lymphocytes without metabolic activation there was no indication of an increase in chromosomal damage up to 2 pglml (at higher concentrationsMB was cytotoxic). With metabolic activation, MB concentrations of more than 1 pg/ml led to chromosomal aberations whichwere significantly higher thanin the control cultures notexposed to thedye.Lower negative. Inan in vivo modelwhichwas
MB concentrations were
probablythe most relevanttestsystem,
MBwas
administered intravenously to rats at the maximally tolerated dosage (25 mglkg body weight). There were no indications of MB induced chromosomal abberations in bone marrow cells (6, 24) even 48 h post treatment.
VIRUS INACTIVATION MBllight treatment was reported to inactivate lipid enveloped as well as non enveloped viruses (13-27). We found, however, that at least in human plasma this is not quite true. Table
I shows a list of those enveloped viruses which were tested
to date. It includes retro, herpes,
toga, flavi viruses (hepatitis C virus belongs to this latter group (48)) and others. All were sensitive to MBllight treatment, although some had to be illuminated for longer times than others. This is demonstrated by Fig. 5 , in which the inactivation kinetics for Semliki Forest (SFV) and vesicular stomatis (VSV) virusesare shown. Whereas SFV was inactivated within 5-10 min, it took about 1 h to achieve complete inactivationof VSV. Retroviruses proved to be especially sensitive to MBllight treatment.The infectivity of HIV1 containing plasma could be removed within 10 min of illumination. The total reduction factor was more than 6.3 log,, steps (49). To look for the behaviors of Hepatitis B-like viruses, a working group from Baxter (Deerfield, Illinois) conducted an animal study, using the duck model. Duck hepatits-B virus could be inactivated, although it had to be exposed to high doses of light (J. Chapman, personal communication).
79
PHOTODYNAMIC VIRUS INACTIVATION
TABLE I Sensitivity of enveloped viruses to MBllight treatment Virus
Family
Character'ktics
HIV-1 HIV-2 SIV Herpes simplex Bovine herpes Suid herpes-type 1 Sindbis West Nile Hog cholera Semliki Forest Vesicular stomatitis
sRNA Retro
Influenza
Orthomyxo 60
Herpes
dsDNA
Toga
SSRNA
Inactivation Rate (log 10)
Illumination Tie (min)
> 6.32 ** > 3.81 * > 6.26 * > 5.50 * > 8.11 *
10 15 15 60 30 60 5 60 <60 10 60
4.43 *
Rhabdo
> 9.73 > 6.50 > 5.92 * > 7.00 * > 4.89 *
sRNA
+
5.1
SSRNA
* tested under production conditions ** 76 ml plasma in cell culture flask > inactivation below detection limit
+ During preincubation, inactivated more than 3.2 log steps below detection limit of small volume assay Virus Hter
(log10 TCIOSO)
7.00
T
"-
6.00
vsv
5.00
SW
4.00
3.00 2.00
l .oo 0.00
-l
Control
after FIT
0
10
20
30
Illuminationtime (mln)
FIGURE 5
40
50
I 60
vation acteristics Family
80
MOHR, LAMBRECHT, AND SELZ
TABLE II Sensitivity of non enveloped viruses to MB/light treatment Virus
nlumination Time
M C MEV Polio Hepatitis A Porcine parvo Adeno &DNA Calici SV 40 &DNA
Picorna
Rate (log 10)
sRNA
(min)
0
60 60 120 60 60
0 :
0 Parvo Adeno Calici Papova
=DNA
o * o *
sRNA
> 3.9 *
4
120 5 30
> 4
* tested under production conditions > inactivation below detection limit Virus titer
(log10TCIDIO)
7.00
T
Light source
6.00
Fluorescenttube
5.00
LED (660 nm)
4.00
Sodium lamp(590 nm)
3.00 2.00 l.oo 0.00 4 Control after
0
FIT
10
20
30
40
50
I 60
Illumination time (rnin)
FIGURE 6
Photodynamic inactivation of VSV in plasma containing 1 p M MB. Comparison of different light sources.
PHOTODYNAMIC VIRUS INACTIVATION
81
In contrast to lipid enveloped viruses, most non enveloped viruses were resistant to MB/light treatment (Table 11) - with some exceptions, e.g. SV 40, calici and to a certain degree adeno viruses. This is more or less identical to those results obtained with other photodynamic procedures (2-4, 7, 8, 10). The inability to inactivate non enveloped
viruses is also limitation a
of the
solventldetergent procedure which is frequently used to inactivate viruses in coagulation factor concentrates and also in plasma (50-52). INFLUENCE OF DYE CONCENTRATION AND LIGHT INTENSITY A critical parameter in photodynamic virus inactivation is, of course, dye concentration (28, 31). As alreadymentioned, an MB concentration of 1 pM is being routinely used for
plasma treatment. We found that in the
case of SFV, at 0.3 pM ofMB all infectivity was
inactivated below the detection limit within less than 15 min. This is, however, not sufficient for more resistant viruses. For example, for inactivation of VSV, 0.8-1.0 pM of MB were required; the necessary illumination time was 45-60 min (31), indicating the importance of light energy input, i.e. light intensity. Using the illumination device equipped with fluorescent tubes (Fig. 3), it wasfoundthat
at 1 pM MB for complete inactivation VSV within 1 h the light
strength had to be at least 40,000-50,000 Lux(31).At
lower values residual infectivity was
detectable after treatment. Therefore, for routine processing of fresh plasma, the minimal light strength has to be 45,000 Lux. If other light sources are used, this value will be different. MB has its main light absorption between about 600 and 700 nm, with a maximum at approx. 660
nm (22). A light source emitting lightat or near the absorption maximum of MB (or any other photosensitizer used) should be preferable to fluorescent tubes which emit light over a broad range of the spectrum. Indeed, VSV inactivation was much more effective when the virus containing plasma was illuminated with a device equipped with light emitting diodes (LEDs) that emitted lightwith a wave length of 660-670nm. As Fig. 6 demonstrates, not more than15 min wererequired to completelyeliminateviralinfectivityfromtheplasma.
In contrast, some
residual infectivity was detected even after h1of illumination by fluorescent tubes inthe control cultures. Another illumination device equipped with high energy sodium lamps emitting light at 590 nmwaseven
more effective than the LED system in inactivating
VSV (Fig. 6). It was
surprising tosee that the influenceof these two light sourceson coagulation factors and thrombin time (which in our experience is one of the parameters most sensitive to MB/light treatment (32)) was not more pronounced than that of the fluorescent tubes (Fig. 7).
These findings indicate that the efficacy of photodynamic virus inactivation can be further improved by selecting the optimal dyes and dye concentrations, light intensities, light sources and other parameters. This will be especially important for cellular blood components, which
MOHR, LAMBRECHT, AND SELZ
82 Fibrinogen (% of initial concentration)
4 200 1 01 0
4
15
45
60
45
60
:
:
,
30
45
60
30
Illurnination time (min)
Factor Vlll (% of initial concentration)
0
15
30
Illumination time (min)
Thrombin time (% of initial concentration)
:
20 0 0
15
Illurnination time (min)
FIGURE 7 Illumination of plasma containing 1 p M MB with different light sources. Influence on coagulation factors I and 111 and on thrombin time, respectively.
PHOTODYNAMIC VIRUS INACTIVATION
83
by themselves might adsorb dyes, more light than plasma and, in addition, are more sensitive
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LEUKOCYTE FILTRATION MECHANISMS. FACTORS INFLUENCINGTHE REMOVAL OF INFECTIOUS AGENTS FROM RED CELL CONCENTRATES.
I. Steneker, R.N.I. Pieterszand H.W. Reesink. Red Cross Blood Bank Amsterdam PO box 9137, 1006AC Amsterdam, The Netherlands
ABSTRACT The purpose ofthe present overview wasto determine the factorsinfluencing the removal of infectious agents fromred cell concentratesby filtration. In general, the efficacyof the filtration method depends on the physical as well as the fhnctional properties of blood cells. These properties are highly influenced by the changes exerted onthe blood cells during blood collection, processing and storageand the filtration method itself. In particular, the removal of infectious agents of red cell concentrates by filtration will be determined bythe type of virus and therewiththe binding towards leukocytes, the type of bacteria and holding period before filtration, the deformability of infected cells and the disintegration of cells in the filter INTRODUCTION
A serious complication ofthe transfusion of blood components is the transmission of infectious agents suchas viruses and bacteria(1-3). Virus infections can be transmitted free by virus presentin plasma, virus attachedto cell membranes and virus infected cells.The incidence of transhsion-associated viral infections can be reduced by selection of the donor and by virus specific screenings assays on the donated blood. Further reduction of virus transmission can be obtainedby inactivation of extracellular virusin blood components and removal of virus-bound or infected cells. Sources for bacterial contamination of blood products include the blood donor and the procedure for collection and processing. The risk of bacterial contamination of blood components was minimizedby the introduction of disposable plastic blood-processing systems, aseptic techniques and storage of blood components4°C at (4). However, noneof these precautions eliminatesthe risk of drawing blood from an asymptomatic donor with a transient bacteremia, causedby bacteria capableto grow at refrigerator temperatures (5). 87
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STENEKER, PIETERSZ, AND REESINK
Other risks of blood transfbsions suchnon as hemolytic febrile transfusion reactions and HLA-immunization can be preventedby removal of leukocytes from red cell concentrates @CC) by filtration (6,7). Several authors have suggested that removal of leukocytes containing virus or bacteria may also contributeto reducing transmissable disease. Leukocyte removal reduced or prevented transmission of leukocyte-associated viruses such as CMV and HTLV-I (8-10). However, filtration failedto remove the cell free HIV released from lysed cells (11). Recently, Pietersz eta1.(12) showed that growthof Yersinia enterocolitica in red cell concentrates could be prevented by a holding period of whole blood for6 to 24 hour at 22"C, during which leukocytes can phagocytize and kill the bacteria, followedby buf@ coat removal and filtration. There arealso some reports suggesting that leukocyte depletion filters also remove bacteria from RCC by direct binding of the bacteriato the fibers (13,14). The aim of this study isto give an overview ofthe factorsinfluencing the filtration mechanisms, with special emphasisto the factorswhich could influencethe removal of infectious agents from blood componentsby filtration. Knowledge aboutthe filtration characteristics (mechanisms) would beof value for fbrtherdevelopment and optimalization of leukocyte depletion filters andthe optimalization and standardization of filtration procedures in research and routine practice.
FILTERS AND MECHANISMS Leukocyte depletion filters. The introduction of the first commercially available leukocyte depletion filter was based on the results of Diepenhorst et al.( 1S), who used cottonwool for the removal of leukocytes from red cell concentrates. Since that time the knowledge about biomaterials, technical proceduresto produce nonwoven webs of synthetic fibers and modification of fiber material has dramatically increased, which to ledthe introduction of a wide scala of commercially available leukocyte depletion filters. These filters can be divided in two types; column- and flat-bed filters. Columnfilters. Column filters aremainly made of a polycarbonate tube filled with identical skeinsof cellulose-acetate or cotton-wool fibers. This construction provides a fiber network with an equal distribution of pore sizes. Although these column filters were designed for the removal of leukocytes from RCC, they were also used for the removal of leukocytes from platelet concentrates (PC). Hut bedfilfers.Flat-bed filtersare composed of a flat plastic container filled with several thin layers made of nonwoven webs of polyester fibers. The construction of the filter is such that the blood flow is directed throughthe layers from inletto outlet. The filter layers can be divided in a pre-filter layer with coarse fibers (upstream), made for the removal of storage generated micro-aggregatesand a main filter with medium andfine fibers (downstream), madefor the removal of leukocytes. The flat bed filters are designed for use with either RCC or PC. The fiber material can be modified to improve efficacy (16). Filtration mechanisms. The removal of leukocytes from blood components filtraby tion is based on depth filtration. The porous structure in leukocyte depletionfilters with a
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89
wide distribution of pore sizes throughout the filter matrix allows capture of leukocytes at any place insidethe filter by adhesion or mechanical sieving depending onthe deformability and adhesive capacity of blood cells (17,19). It was found thatthe major mechanisms in commercially available filters were: mechanical sieving, direct adhesion of leukocytes onto the fiber surface and adhesion of leukocytes by cell-cell interactions onto platelet covered fibers (20). Adhesion. Electronphotomicrographs of fibers from celluloseacetate column filters and polyester flat bed filters after filtration of RCC showed granulocytes, monocytes and platelets adhering directlyonto fibers via pseudopod formation (20). Most the of leukocytes and platelets had undergone a shape change, indicating that activation was needed for adhesion. The underlying mechanismof leukocyte and platelet adhesion ontothe fibers is hardly known. Takemoto et a1.(21) described two mechanisms involvedin blood cell adhesiononto artificial surfaces. Oneis complement-mediated and the other is adhesive-protein-mediated. In vivo, leukocytes and platelets have a variety of specific adhesion molecules on their plasma membrane to promote surface contact and adherence (22). The most common are CD62 (GMP140) and CD41 (GPIIbDIa) for platelets andCDIl/CDlS for granulocytes.Two observations suggest that direct adhesion of granulocytesonto fibers inside leukocyte depletion filters is mostlikely complement-mediated. Firstly, a preliminary study showed that granulocytes, which were incubated with a monoclonal antibody (CD18) against the betachain of the complement-receptor 3 (CR3), hardly adheredto polyester fibers [personal observation]. Secondly, artificial devices made of cellulose acetate are known to cause complement activation and subsequent granulocyte adhesion and aggregation (21). The CR3 receptor hasbeen found to be the main molecule mediating both adherence andaggregation of granulocytes (23). Steneker et a1.(20) indeed showed adhered and aggregated granulocytesin the top of a celluloseacetate column filter. Mechanical sieving. Mechanical entrapmentin a filter can occur by various mechanisms: a cell can be blocked insidea pore; twoor more cells (aggregates) can be entrapped by bridging when the cells simultaneously reach a poreor a cell can be interceptedin dead ends of the filter fibers. In leukocyte depletion filters leukocytes and red are cells captured by blocking insidethe pores. Light- and electronphotomicrographs revealed that lymphocytes, red cells (mostly sphero-echinocytes) and some monocytes were captured in small pores of the filters without contactwith the surrounding fibers (20,24). According to their morphology the lymphocytes and monocytes were not activated. Furthermore, some small aggregates of leukocytes and platelets werefound inside pores of the polyester pre-filter (20). Based on their deformability and adhesive capacity it was not expected that granulocytes would be captured by this mechanism. However, a small number of thesecells, which were swollen, were captured inside small pores(20). Cell-cell interactions (indirect adhesion). Granulocyte-platelet interactions were observed in red cell filters by Steneker et aL(20).It was suggested that platelets presentin RCC adhereto the fibers, become activated, and undergo shape change with pseudopod
90
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PIETERSZ, STENEKER,
REESINK
formation. These plateletsmay cause increased bindingof leukocytes. The interaction of leukocytes with platelets has been noted in several models of tissue injury. Neutrophils accumulate around platelet-rich thrombiat sites of hemorrhage (25). Platelets can alsoattracted be by neutrophils during migration across the endothelium (26). Although activated platelets release proteins thatare chemotactic for leukocytes there is also evidence for direct adhesive interactions between these two types of cells. Activated and spreaded platelets have inan creased expressionofthe selectin GMP-140, which is known to mediate adhesion of granulocytes onto platelets in the presence of Ca2+ (18). However, preliminary experiments showed thatthe removal of leukocytes from RCCby platelet-granulocyte interaction could not be blocked by incubation of the platelets with monoclonal antibody CD62 (GMP-140). Furthermore, adhered platelets are known to release granule bound materials suchas fibrinogen, fibronectin and von Wlllebrand factor, which may also act as bridging moleculesto granulocytes (27). However this effect is not systematically studied with leukocyte depletion filters. Since platelet-granulocyte interactions were found with filters designed for leukocyte removal from RCC,the influence of red cells must be considered. There is evidence that red cells transport plateletsand leukocytes towards a surface (vessel wall) and that this is dependent on the local shear rate (28). This is the so called margination effect. The effect of filter materirl. The influence of thefiber material onleukocyte adhesion, activation and disintegration depends on the physico-chemical properties the of fibers (surface charge, surface wettability, surface chemistry and surface free energy). Leukocytes capturedby longlasting adhesion showed a flat morphology and surrounded the fibers (29). Leukocytes captured by transient adhesion showed a round morphology with small pseudopods (29). These cells may be vulnerable for detachment. Following filtration of RCC in a celluloseacetate filter and a modified polyester flat bed filter the leukocytes adhered by longlasting adhesion, whereas most leukocytes in an unmodified polyester filterwere captured by transient adhesion(20). Although leukocytes captured by longlasting adhesion are less vulnerablefor detachment, intense cell-fiber contact may lead to leukocyte disintegration (cell fragments) or blocking of the filter. The hydrophilicity of the fiber is important for optimal contact between blood cells and the fiber. An optimal contact is only possible if the fiber is surrounded by the medium in which the cells are suspended. For example, an air bubble will induce an uneven distribution of the blood flow through the filter, which will reducethe leukocyte depletion capacity. Coating or chemical treatment ofthe fibers will increase the hydrophilicity and allow self-priming the of filters withthe blood component.An other possibility is primingof the filters with protein free medium used for storage ofRCC or PC. By this method air and decomposition products or possible contaminantswill be removed from the filter bed. Most of the effects ofthe physico-chemical properties of synthetic materials are described in biomaterial research. However its very hard to draw conclusions ofthe effect of one single parameter onthe leukocyte adhesion, activation or disintegration.
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The effect of storage at 4OC. The adhesive capacity and deformability of blood cells will change during storage conditions (19,30-33). Already after 24 hour storage under blood
bank conditions granulocytes show profound alterations of their bactericidal activity, chemotaxis, aggregation and superoxide production (34,35). The deformability of granulocytes will also decrease during storage 4°C at and after approximately24 hour disintegration starts (30). Thus, one may conclude that the direct adhesion capacity of granulocytes will decrease (3 1). In contrast, themechanical entrapment of granulocytes may increase after storage overnight at4OC due to a decreasein deformability. However, prolonged storage at 4°C will induce fragmentation. The effect of plasma proteins.During the filtration there will be a gradual displacement of plasma proteins at the fiber surface, known as the Vroman effect (36). This will occur in the following sequence: albumin, immunoglobinG, fibrinogen, fibronectin, high molecular weight kininogen and factor XI1 (36). Thus, leukocyte adhesion onto fiber surfaces will be largely influenced by preadsorped proteins of the blood components (29). Recently, Szuflad and Dzik (37), using a polyester flat bed platelet filter showed that the retention of granulocytes suspendedin platelet rich plasma differed from granulocytes suspendedin a crystalloid solution. The effect of platelets. The number of leukocytesand platelets in RCC will depend on the preparation method of PC (24). Steneker et a1.(24) showed a positive correlation between leukocyte depletion capacity (the number of leukocytes in the RCC applied to the filter which resulted ina leukocyte amount of 5.0 x lo6) of the filters and the platelet count in the RCC prior to filtration (24). This phenomenon was dueto a diminished capacity ofthe filters for granulocyte depletion. These results were confirmed by Pietersz etaL(38). The effect of temperature. Based on the literature,it was expected that a temperature of 20°C would favor the deformability of red cells (19) and an optimal adhesive capacity of leukocytes and platelets(24). There areonly a few publications concerning the optimal temperature of filtration and these studies differin type ofRCC, storage history and filter type and brand (39,40). Thus, itis impossible to draw a conclusion concerning the optimal temperature for filtration. The effect of flow time. The adhesion of leukocytes onto fibers is a process which requires a sufficient contact time. A flow rate of more than 100 ml per min may prevent adherence and,in addition, may induce disruption of blood cells.A longer filtration timemay increase the riskof leakage of cell remnants or intact leukocytes from the filter because the strength of adherence declines over the incubation period (41). Ledent and Berlin(42) showed, regardlessof the filter used, that the number of residual leukocytes was significantly higher in units filteredat slow flow(2 hours) compared to fast flow(10 minutes). This difference in leukocyte contamination was mainly due to an increase of granulocytes in the units filtered at slow flow.
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DISCUSSION AND CONCLUSIONS Leukocyte removal of RCC by filtration is the result of several filtration mechanisms, i.e mechanical sieving, adhesion and indirect adhesion (cell-cell interactions). The result of any filtration is highly dependent onthe adhesive capacityand deformability of leukocytes, platelets and red cells and by the fiber material(16). On their turn, the deformability and adhesive capacity of blood cells maybe influenced by cell-cell interactions(1 7,lS), age and temperature (19,30-33),the compositionof the blood components (plasma proteins(29),divalent ions (29)and type of blood cells(24).Moreover, the flow(42)and temperature (39,40)during filtration will also affectthe physical as well as the functional properties of blood cells. If filtration would be appliedto remove infectious agents from RCC the clinical efficacy will be determined bythe type of virus and therewiththe binding towards leukocytes, the type of bacteriaand holding period before filtration, the deformability of infected cells the and disintegration of cellsin the filter. Thus, optimal leukocyte depletion and therewith the removal of infectious agents requires a filter with a leukocyte depetion capacity adapted to the cellular composition ofthe RCC and the type of infectious agent that to hasbe removed.The latter implicates that the application of leukocyte-poorRCC by filtration needs a well-considered choiceof a leukocyte depletion filter based on a validation in routine of the filter with standardization of the blood collection, storage, processing of blood components and filtration method. REFERENCES 1. J.P. Soulier, Vox Sang, 47, 1-6 (1984). 2. R.Y. Dodd, L.F. Barker, Infection. Immunitv and blood transfusion, Alan R. Liss Inc (1 984). 3. P.L. Mollison, C.P. Engelfiiet, M. Contreras, in Blood transfusion in clinical medicine, Vol. S.,Blackwell Scientific Publications,(1987)pp. 764-806. 4. M. Goldman, M.A. Blajchman, Trans Med Rev, 5,73-83 (1991). 5. M.J. Arduino, L.A. Bland, M.A. Tipple, S.M. Aguero, M.S. Favero, W.R. Jarvis, J Clin Microbiol, 2 7 , 1483-1485 (1989). 6. G. Sirchia, P. Rebulla, L. Mascaretti, N. Greppi, C. Andreis, S. Rivolta, A. Parravacini, Vox Sang, 3,2-8(1986). 7. M.F. Murphy, P.Metcalfe, H. Thomas, J.Eve, J. Ord, T.A. Lister, A.H. Waters.Br J Haematol, 529-534 (1986). 8. G.L. Gilbert, K.Hayes, I.L. Hudson, J. James, and the Neonatal Cytomegalovirus study group, Lancet, i, 1228 (1989). 9. Y.C.E. de Graan-Hentzen, J.W. Gratama, G.C. Mudde,L.F. Verdonck, J.G.A. Houbiers, A. Brand, F.W. Sebens, A.M. van Loon, T.H. The, R. Willerrue, G.C. de Gast, Transfusion, B,757-760 (1989). 10. K.Okochi, H. Sato. AIDS research, 2, 5157-5161 (1986). 11. B. Rawal, T.S. Benedict Yen, G.N. M. Bush, Vox Sang, 60,214-218 (1990).
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USE OF LEUKODEPLETION FILTERS FOR THE REMOVAL OF BACTERIA W. Dzik Blood Bank and Tissue Typing Laboratory Departments of Pathology and Medicine Deaconess Hospital and Harvard Medical School Boston, MA 022 15
ABSTRACT Recipient exposure to allogeneic donor leukocytescan mediate a number of immunologic complications of transfusion or can transmit leukotropic viruses carried by the donor. Leukocyte depletion of cellular blood components has been shown to reduce the incidence of such complications. In recent years, prestorage leukocyte depletion by filtration has also been suggested as a means of decreasing the incidence of bacterial overgrowth in cellular blood components. This review analyzes published studies on the use of leukodepletion filters for removal of Stuphylococcur epidennidis and Yersiniu enterocoliticufrom blood. Although ineffective for removal of S. epidennidis from Platelet Concentrates, inoculation studies demonstrate removal of low levelsY.ofenterocoliticu from Red Cell Concentrates. Based on these studies, four possible mechanism(s) for removal of bacteria by leukodepletionfilters are analyzed:phagocytosisbyleukocytesduring aprefiltration holding period; complement-mediated bacterial killing enhanced by filtration; adherence of bacteria to leukocyte surfaces retained within the filter; and direct removal of bacteria by the filter media. Just as multiple mechanisms appear to account for the efficiency with which these filters deplete blood of leukocytes, it is likely that more than one mechanism accountsfortheexperimentalobservationthatleukocytedepletionfilterscanreduce overgrowth of Y. enterocoliticu in stored Red Cell Concentrates. BACTERIAL OVERGROWTH
IN BLOOD COMPONENTS
Among the more devastating acute complications of transfusion is the infusion aofblood component in which bacterial overgrowth has occurred. The clinical consequences of such a transfusion depend upon the particular bacteria, the degree
of contamination, and the
clinical state of the recipient and can vary from mild chill reactions to overt shock and
95
DZIK
96
of bacterialovergrowthisuncertain.
death.Theincidenceofclinicalcomplications
However, studies have provided data on the incidence
of detectable bacteria in blood
components. An earlyreport byBuchholz etalfoundthat
0.9 % to 2.4 % of 4,500
Platelet Concentrates were bacterially contaminated.’ More recently, a report from the Canadian Red Cross found approximately
0.43% of 10,730 Red Cell Concentrates and
0.35% of 11,740 PlateletConcentratestohavedetectablebacterialcontamination?In
addition, two studies from large US transfusion services found that approximately0.18%
of 3853 pooled Platelet Concentrates had detectable bacterial c~ntamination?.~ Concernoverclinicalconsequencesofbacterialcontaminationresultedin
a 1986
recommendation that the storage period for Platelet Concentrates in the U.S. be reduced from 7 daysto 5 days.In
1991 considerationwasgiveninthe
US toshorteningthe
storageperiodforRedCellsaswell.AlthoughtheexpirationdatingonRedCell Concentrateswasnotchanged,thereiscontinuinginterestintechnologythatwould decrease the incidence of substantial bacterial contamination
within blood components.
Such technology includes the development of devices which could rapidly blood screenfor bacterialovergrowth,nontoxicbloodbagadditivesthatcouldkillbacteriaorinhibit bacterial growth, and changes decreasethechance
in blood collection or component preparation that would
of bacterialovergrowth.Prestorageleukocytedepletion
is one
proposed method to reduce the incidence of bacterial overgrowth:
fiperimental studies of leukocyte jiltration and bacterial overgrowth: The ideal approach to demonstrating whether filtrationwilldecrease
or not prestorage leukocyte depletion by
the incidenceoftransfusioncomplicationsduetobacterial
overgrowth would be to examine outcomes
in patients who were randomly assigned to
receive either unmodified or leukodepleted blood. Observations would include clinical assessmentbyanobserverblindedtothepresenceorabsenceoffiltrationandwould includebothsurveillance
and targetedbacterialcultures
of thepatientsandthe
components. Because the prevalence of bacterial overgrowth is low, such a study would require a very large number of patients to accurately discriminate between unmodified and filtered blood components. Intheabsenceofsuchanenormousclinicalstudy,experimentalinvestigationof bacterialovergrowthhasfocusedondeliberateinoculation
of bloodcomponentswith
LEUKODEPLETION
97
bacteria.These"spiked"specimensarethensplitandeitherunmodified(control)or leukocyte-depleted by filtration or centrifugation. The units are then checked for bacterial overgrowth during storage. Numerous variables, listed in Table suchexperimentalinvestigations.Becauseofthesevariables,onemust
1, affect the results of use caution in
interpreting the results of these experiments or in formulating policy based on them. PLATELETCONCENTRATES AND
STAPHXOCOCCUS EPIDERMIDIS
Although many different organisms may contaminate Platelet Concentrates, S. epidennidis isthemostcommonlyisolated
~rganism.~.~ S. epidennidis presumably originates from
donor skin as a result of inadequate topical sterilization, skin scarring,' or skin plugs that enter the bag.' A few experimental studies have inoculated blood with S. epidennidis and evaluated
thegrowthofbacteriaduringplateletstorage.Wenzandcolleaguesinoculatedwhole blood with two different doses ofS. epidennidis (ATCC strain #12228, doses10 CFU/mL or 50 CFU/mL) and prepared PRP within 30 minute^.^ Cultures were not obtained after the 30 minute period in PRP and so any direct antibacterial effect occumng during this
The PRP was then either made directly into short holding period could not be determined. Platelet Concentrates (control group) or was first passed through a leukodepletion filter designedforPRP(Autostop,PallCorp)priortopreparationasfilteredPlatelet Concentrates.ThePlateletConcentrateswerestoredatroomtemperature.Rapidlog phase bacterial growth was observed in both filtered and unfiltered units. No beneficial effect of leukocyte filtration was demonstrated. Brecher et alalsostudiedthegrowthof
S. epidennidis inPlateletConcentrates
prepared from inoculated whole blood." Using a pool-and-split paired study design, they prepared 7 two unit pools of AB0 identical whole blood. Each pool was inoculated with S. epidennidis at 5 CFU/mL (clinical isolate prepared at 37 C). No holding time was
used. The pools were then split into paired units and centrifuged to prepare PRP. The
PRP from one member of each pair was filtered through a filter designed for one unit of PRP. Platelet Concentrates were prepared from the PRPand stored at room temperature for for 9 days. The development of bacterial overgrowth during storage was not different filteredversusunfilteredPlateletConcentrates.Thisstudyconfirmedthefindingsof
wen^.^
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TABLE 1: Variables in the experimental study of bacteria in blood components Strain of bacteria source conditions of culture prior touse Blood component characteristics volume and cellular content anticoagulant-preservative medium plastic bag formulation Filter selected appropriate to theblood component appropriate volumeof blood filtered Details of experimental design paired study design stage of component preparation when blood is spiked with bacteria inoculation dose(s) in CFU/mL holding time between inoculation and filtration assay of bacterial concentration after holding time just prior to filtration time between filtration and subsequent assays of bacterial concentration Sensitivity of culture assay used to detect bacteria Kind of isolator system used to culture bacteria Statistics
Gong et al demonstrated that the combination of a long holding period and filtration S. epidennidis."
mightinhibitgrowthofevenarelativelyhighdoseof investigatorsinoculated
100 mLaliquotsofwholebloodwith
100 CFU/mLof
epidennidis (clinicalisolate)andthenheldthebloodatroomtemperature lengthsoftime
(5 minutesto 20 hours)priortofiltering
These S.
for varying
the wholebloodthrough
a
Sepacell PL-SA filter (Asahi Medical CO, LTD) designedfor Platelet Concentrates. PRP wasthenpreparedandstoredatroomtemperaturefor
10 days.Althoughlogphase
growth occurred in nearly all aliquots, no growth O C C U K ~in ~ the two aliquots stored for 20 hours prior to filtration.
REDCELL CONCENTRATES AND Reports from the last several years suggest that encounteredseriousbacterialcontaminantin
YERSINIA ENTEROCOLITICA
Y. enterocoliticu is the most commonly
Red CellConcentrates.Theincreasing
frequency with which Y. enterocoliticu is identified as a contaminant may result from the growing number of individuals with asymptomatic infection and from the propensity for
LEUKODEPLETION FILTERS
99
Y. enterocoliticu to grow at low temperatures in an iron rich environment. Because ofthe
sensitivity of Y.enterocoliticu to killing by complement, Gibbet al have suggested that the change to plasma-reduced additive solution storage of
Red Cell Concentrates may have
promoted the occurence of Yersiniu overgrowth.'* Arduino et al demonstrated in 1989 that Y. enterocoliticu present in Red Cell Concentrates at a concentration of only 0.1 - 1 2
CFU/mLwouldenterlogphasegrowthafter
-
3 weeksin 4 C tora age.'^ Several
experimental studies of bacterial removal by filtration have focused on Y. enterocoliticu. Buchholz etal did a careful in vitro evaluation of the effect of leukocyte filtration on thegrowthof
Y. enterocoliticu instoredblood.I4Theinvestigatorsused
an excellent
experimental designin which units of A B 0 identical whole blood were first pooled (n=24 two unit pools) and then split thus allowing for
a strict paired comparison of filtered vs
unfiltered blood as well as allowing for filtration of wereinoculatedwith
an entire unit of blood. The pairs
Y. enferocoliticu,held a room temperature for 7 hours to mimic
processing and then made into Red Cell Concentrates. These concentrates were Red Cell Concentrates refrigerated overnightand then filtered througha filter designed for (Sepacell R-500,AsahiMedicalCo.,LTD).
The investigatorsseparatelystudied two
serotypes (0:3 and 0:8) and studied four different inocula doses.
In all cases there was
a dramatic decline in the concentration of bacteria from the time of original inoculation to the timeof filtration/storage as a result of the natural antibacterial properties of blood. A keydetailofthisstudyisthattheconcentration
of bacteria justpriorto
filtration/storage (post-holding period) was measured usinga sensitive culture assay. Units were then stored for 42 days. The results were slightly different for the
two serotypes.
Serotype 0:8 which was obtained from a case of transfusion-transmitted Y. enterocoliticu and maintained at 37 C did not grow as avidly as serotype 0:3 which was maintained at room temperature. For serotype 0:8 at the lowest original inoculum dose(0.3 CFU/mL), no organisms were recovered after the holding period or during either the filtered or the unfiltered units.
42 days of storage in
At mid-range inocula (5 - 43 CFU/mL), the
bacterial concentration of unfiltered units fell to0 - 3.1 CFU/mL after the holding period and 2 of 6 units grew bacteria, whereas filtered units with post-hold concentrations of 0-
0.6 CFU/mL remained sterile. At the high dose inoculum (99 CFU/mL) unfiltered units had a post-hold concentration of 0 - 0.3 CFU/mL and 3 ,of 3 units grew bacteria, but filtered units witha post-hold concentration of 0.1 CFU/mL remained sterile.In contrast,
100
DZIK
serotype 0:3 grew in nearly all unfiltered units (post-hold concentrations: 0 - 2 CFU/mL). 0 - 2.7 CFU/mL, but grew Filtered units showedno growth at post-hold concentrations of
bacteriaatpost-holdconcentrationsof
2.0
-
3.7 CFU/mL.Thisstudydemonstrated
(withintheconstraintsoftheexperimentalmodel)thattheholdingtimeresultsin
a
significant reduction in bacterial concentration and that prestorage filtration of Red Cell Concentratespreventsbacterialovergrowthwith
Y. enterocoliticu whenunits contain
(< 2 CFU/mL)priortofiltration.Filtration failed to sterilize the units when the prefiltration concentration was > 3 CFU/mL.
relativelylowconcentrationsofbacteria
Wenz et al also inoculated whole bloodwith four different dosesof Y. enterocoliticu serotype 0:3.15 After a 3 hourhold at room temperature, Red Cell Concentrates were prepared, stored overnight (12 hours) at 4 C and then filtered. They split individual units (Pall Corp). Cultures of whole bloodso that half-units were filtered through a BPF-4 filter
were not taken just prior to filtration, but according to the findings of Buchholz et al the prefiltrationconcentrationsinWenz’sstudyprobablyrangedfrom
0
- 4CFU/mL. In
agreement with the study by Buchholz, Wenz found that at midrange bacterial concentrations unfiltered units grew bacteria during storage but filtered units did not. At the higher concentrations of bacteria, both filtered and unfiltered units grew bacteria. Because only half units were filtered in this study, the effect of filtration may have been slightly magnified. ThereportofKimetal
also examinedtheeffectoffiltration
on growthof
Y.
enterocoliticu serotype 0 : 3 (37 C clinical isolate) in stored Red Cell Concentrates.16 The initial whole blood inoculum wasa single dose of 65 CFU/mL. After a 5-6 hour holding period at room temperature, packed Red Cells were prepared.
No assessment was made
of the concentration of bacteria after the holding period just prior to the
filtration. The
packedcellswereeitherunfiltered(control)orfilteredthroughleukodepletionfilters designed for Red Cell Concentrates (RC300, Pall Corp; Leukotrap, Miles Corp). All four unfiltered units grew bacteria whereas only2 of 10 filtered units grew bacteria. The study reported byHogman
etalalsodemonstratedthatprestorageleukocyte
filtration would reduce the incidence of Yersiniu overgrowth in experimentally inoculated blood components.” This report, however, differed from those of Buchholz, Wenz and Kim in several important respects.
A singleoriginalinoculumof
100 CFU/mLof Y.
enterocoliticu 0 : 3 (clinical isolate, preparation temperature not stated) was addedmL to 50
LEUKODEPLETION
101
aliquots of whole bloodor buffy coat preparations and heldfor S hours prior to filtration. The whole blood or buffy coats were filtered througha leukodepletion filter designed for Asahi Medical CO,LTD). The use of small volume aliquots Platelet Concentrates (PL-SA,
and a leukodepletion filter not designed for whole blood complicates the interpretation of their results. The following year, Pieterszet al published yet another study ofY. enterocolificuand filtration. They used serotype
0:3 prepared at room temperature culture and inoculated
into whole blood at five different doses. Inoculated pooled units were split into matched
the buffy coat pairs, held for 20 hours at22 C, and made into Red Cell Concentrates using removal technique. One member of each pair of buffycoat depleted Red Cells was then filteredthrough
a leukodepletionfilterdesignedforRedCells(CellSelect,
NPBI,
Netherlands). Cultures were not taken after the holding period, but were done periodically during refrigerated storage of the Red Cell Concentrates. Over the entire dose range, bacterial growth during storage was less frequent among buffycoat depleted units that were filtered compared with unfiltered buffycoat depleted units.
Y. enferocolificugrew during
storage in filtered units only among those units with an initial inoculum of 3,000 CFU/mL or higher. This study differs from the others in that higherinitialbacterialinoculawereused 3,000 CFU/mL and
a longer holding period and much
(20 CFU/mL, 100 CFU/mL, 300 CFU/mL,
30,000 CFU/mL). Given these very high initial bacterial
concentrations, it is unfortunate that quantitative cultures were not obtained after the room temperature holding period and after the buffycoat removal just prior to the filtration. Such cultures may have documented the presence of extracellular bacteria that were either removed by filtration, killed as a result of filtration, or killed after filtration. The role of plasmids in the survival ofY. enferocolificucomplicates interpretationof experimentalstudiesinvolvingleukocytefilters.Thevirulenceof
Y. enterocolitica is
associated with aplasmid,pYVe.Plasmidencodedproteinswhenexpressedpromote adherence of the bacteria to tissue, increase the resistance of the bacteria to phagocytosis, and decrease the susceptibility of the organism to complement.'*.'* Expression of plasmid
virulenceproteinsishighlydependent
on thetemperatureatwhichthebacteria
are
growing. In addition, the plasmid is not stable and may be lost from stock cultures used forinvitrostudies.As
aresult,concernhasbeenexpressedthatpublished
in vitro
experiments may not sufficiently mimic conditions that occur with donors who harborY.
102
DZIK
entero~olitica.~~ Indeed, plasmid expression was noted in
a case report inwhich a unit
of blood leukodepleted on day 7 of storage by filtration (cotton wool) was unexpectedly foundduringroutinepost-filtrationsterilitytestingtobepositivefor
Y. enterocolitica
serotype 0:3.20 MECHANISMS OF BACTERIALREMOVALBYLEUKOCYTEFILTRATION
A betterunderstandingofthemechanismsbywhichleukocytefiltrationmayprevent
bacterial overgrowth is essential for the proper use of this technology in blood component preparation. At issue is thetimingoffiltrationduringtheprocessingofblood,the expectation of the utility of filtration asa mechanism to reduce bacterial overgrowth, and the development of improved methods of bacterial clearance during blood filtration.
Hypothesis #I:Bacteria are ingestedby leukocytes during a holding period after which the intracellular bacteria are removed by leukocytefiltration. Thishypothesis,putforwardbyHogman
and others, is that bacteria are ingestedby
leukocytes during a prefiltration holding period and then removed
by filtration. In the
absence of filtration, the leukocytes gradually disintegrate during and storage are presumed then to release viable bacteria which can proliferate. For bacteria such as Y.entercolitica, it is assumed that the leukocytes have already ingested the majority of bacteria before the blood is removed from the donor. In the
case of bacteria such as S. epidennidis which
are introduced into the blood bag at the time of collection, Hogman suggests a holding that period is essential to allow leukocytes sufficient time to ingest bacteria before filtration. The temperatureand duration of this holding period is not strictly defined and range from 4 hours at 22 C in Swedento 20 hours at 22 C in the Netherlands.21 In favor of this
hypothesis is the fact that polymorphonuclear leukocytes are well known to degenerate during refrigerated storage; the expectation that filters would not be capable of efficient direct removal of bacteria; and the finding that inoculated blood became sterile after
a
holding period thereby suggesting that extracellular organisms must have been consumed by leukocytes. In the study by Hogman et
al,” no Y. enterocolitica were recovered by
culture after the5 hour holding period prior to filtration. The investigators attributed this to phagocytosis of the bacteria by leukocytes which if not removed by filtration would go on to release the bacteria during subsequent refrigerated storage. However, according to
LEUKODEPLETION
103
the results of Buchholtz, the inoculum and holding period used by Hogman might be expected to result in a low but detectable residual post-hold bacterial concentration of < 1 - 1.5 mLofblood
1 CFU/mL.BecausetheHogmanstudysampledonly cultureassay,they
fortheir
maynothavereliablydetectedresidualextracellularbacterial
concentrations < 1 CFU/mL.Nevertheless,suchlowconcentrations result in bacterial overgrowth after
aresufficientto
Thus, complete inhibition a 3 week lag
of bacterial growth might depend upon either direct removal of the residual extracellular bacteria by filtration or upon subsequent killing of these bacteria after filtration.
Hypothesis #2: Complement proteins destroy bacteria, mediate
binding of bacteria to
filters, or killing of bacteria as a result offiltration. Rawal and Vyas pointed to a role for complement during bacterial removalby leukocyte filtration.=Theyadded stated) at 7.7 x
Y. encerocolicica serotype 0:3 (preparationtemperaturenot
lo6 CFUs/mL to either fresh-frozen plasma or heat-inactivated(56 C for
30 min) fresh-frozen plasma. After an unspecified period of time, only were recovered from the normal plasma (99.2% reduction) compared
1207 CFU/mL with no reduction
in heat-inactivated plasma. When the two preparations were then filtered, the suspension of bacteria in normal plasma was further reduced to 8 CFUs/mL. This suggested a 99% removal of bacteria by filtration that did not depend upon the presence of leukocytes and occurred in a plasma environment. In contrast, filtration of heat-inactivated plasma failed to reduce the concentration of bacteria. This suggested that a heat-labile plasma protein was required for killing or removal of Y. enterocolitica as a result of filtration. The effect of complement on survival ofY. enterocolitica serotype 0:8 was shown by Wagneret
al.24
Using an initialinoculumof
preparation), they examined bacterial killing over
200
-
400 CFU/mLofbacteria
(30 C
20 minutes at room temperature in a
varietyofconditions.Theyfirstshowedthattheanticoagulant-preservative
medium
ADSOL had no antibacterial effect by itself. Packed Red Cells suspended in ADSOL or the supernatant of additive solution Red Cells dramatically reduced the concentration of bacteria within 20 minutes at rmm temperature. Moreover, leukodepleted Red Cells in ADSOL or the supernatant of the leukodepleted Red Cells also dramatically reduced the concentrationofbacteria
(384 CFU/mLto 6 CFU/mLin20minutes).Incontrast,
bacteriasurvivedafterinoculationintoWashedRedCellConcentrates.Inaddition,
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104
bacteria survived after inoculation into the heat treated (56 C for 1 hour) supernatant of leukodepleted Red Cells in ADSOL. These findings
are consistent with those of Rawal
and demonstrate rapid complement-mediated bacterial killing that occurs in the absence of leukocytes by the residual plasma present despite its dilutionby additive solution. The poor survival ofY. enterocolitica serotype 0:3 in a plasma rich environment was also shown by Gong et
al.=
They inoculated whole blood with
80 CFUlmL and after
two hours at room temperature separated the blood into packed Red Cells, Buffycoat, and Plasma. All three components were then stored at 4 C. Although Y. enterocolitica grew in the packed Red Cells and Buffycoat, there was no growth in
the plasma. In addition,
Y. enterocoliticu (100 CFU/mL) did not survive after direct inoculation into plasma. Therole ofcomplementopsonizationordestructionof Y. enterocoliticu during survival in blood bags is complex. Proteins encoded on plasmid genes play important roles. For example, the ail gene is present in pathogenic strains of Yersinia and confers somedegreeofresistancetocomplementdestruction.Serumsensitivestrainswhen transfected with the ail gene demonstrate serum resistance.26 Expression of the ail gene productdependsonthetemperatureofgrowth
of Y. enterocolitica. Onestudyfound
bacteria grown at37 C to be 10,OOO fold more resistant to serum killing than cells grown at 30 C.26 Thus, the temperature at which the organism is prepared for use in inoculation yadA codes for the studies may influence the sensitivity to complement. The plasmid gene
cell surface protein YadA which is synthesized only by bacteria growing at YadA protein was recently shown to bind the complement downregulator protein
37 C. The
H.n
As a result YadA+ bacteria bind H protein, develop lower concentrations of C3b on their surface, and are less opsonized for leukocyte phagocytosis. These studies document that conditionsofgrowthandplasmidcontentmayaffectexperimentalobservations
of
complement-mediated destruction of bacteria during filtration. The influence of complement resistance coded by temperature-sensitive plasmids was considered in a recent inoculation study by Gibb
et
al.”
They studied Y. enterocolitica
serotype 09 (clinical isolate). The presence of the virulence plasmid pYVe was verified at the time of the experiment. Blood without antibodies to Yersinia was used. They found 5,000 CFU/mL that bacterial killingin cell-free plasma was temperature dependent. When
wereinoculatedintofreshplasmaandincubatedat complementresistanceandbacterialnumbersincreasedover
37 C, theorganismdemonstrated
24 hours.
In contrast,
LEUKODEPLETION
105
Yersiniu were not detected at 24 hours when incubated at 20 C-- a temperature at which complement proteins are active but at which the virulence plasmid would be lost. When inoculated plasma was incubated at4 C, complement activity was impaired and the initial inoculum of 5,000 CFU/mL remained unchangedat 24 hours. By modifying the growth conditions used to propagate the bacteria, they prepared both complement sensitive and complement resistant versions of the same strain. These were inoculated at 50 CFU/mL into split units of fresh whole blood, kept for storedat
24 hours at either 4 C or 20 C, and then
4 C.Bacterialovergrowthoccurredmorerapidlyintheunitscontaining
complement resistant bacteria particularly those units which omitted
the one day room
temperature holding period. Their report highlights the effect of plasmid expression on the results of inoculation experiments.
Hypothesis #3: Bacteria adhere to leukocyte sufaces retained in the jilter. In addition to opsonized phagocytosis by leukocytes and direct killing by complement,
it
is possible that bacteria adhere to the surface of leukocytes which are retained in the filter. There is strong host evolutionary pressure for leukocytes to bind bacteria
and the high
concentration of leukocyte surfaces within the media of the filter may establish a unique
of bacteria. IgG or C3b coated bacteria which were not biosurface that promotes retention cleared in donor blood may be brought into close physical contact with leukocytes during filtration. Although the opsonic effects of IgG and C3b are well described, other plasma proteins may also play an important role in mediating bacterial adherence to leukocyte surfaces. Thecollectins may represent an importantgroupofproteinsmediatingbacterial adherence to leukocytes. Collectins comprise a family of proteins which are named for theircollagen-typestructureandlectinstructure.”Examplesofcollectins includethecomplementprotein
in humans
Clq andmannosebindingprotein(MBP).MBPis
a
bouquet-shaped glycoprotein synthesizedin the liver. Upstream transcriptional regulatory elements result in increased synthesis of MBP as
an acute phase reactant. The plasma
concentration of MBP varies widely among individuals(10 ng/mL to 10 pg/mL) andmay account for individual differences in killing of bacteria
byplasma.Humanleukocytes
possess a cell surface receptor (collectin receptor) that binds MBP andClq. Thus, MBP can servetolinkbacteriato
leukocyte^.^^ Inaddition,MBPboundtobacteria
can
DZIK
106
Y.
independently initiate the complement cascade. Enterobacteriaceae including
encerocoliticu possess D-mannose residues in the 0-polysaccharide region that makesup the outermost region of LPS. These sites may serve as binding sites for MBP. Fibronectin is another plasma protein which may play
a role in bacterial-leukocyte
interactions. A varietyofgrampositiveorganismsincluding surfacestructuresthatbind
S. epidermidis havecell
fibr~nectin.~’ Bothleukocytesandplateletsalsobind
fibronectin through P,-integrin receptors. In addition, leukocytes adherent to fibronectincoated surfaces undergo spreading and increase the number and affinity of the p-integrin CR3 (MAC-l). It is possible that leukocytes physically retained within the filter undergo some degree of activation of surface receptors suchas integrins which may increase their ability to capture bacteria. Indeed, evidence for granulocyte activation during filtration exist.31 Van Oss reported that
Direct binding of bacteria to leukocytes has been described.
hydrophobic forces can result in the direct binding of bacteria to leukocytes provided that the bacteria had a higher contact angle (greater hydrophobicityand surface tension) than the le~kocyte.’~ Bacterial cell surface proteins coded by virulence genes of Enterobacteriaceaepromoteattachmentandinvasionoftheorganism.Studiesby Wrighe3 with E. coli andby binddirectlyto
Isbere with Yersiniu suggestthattheseorganisms
PI integrinreceptorsfoundon
can
many cellsincludingleukocytes.In
addition to these virulence proteins, Enterobacteriaceae express Type
I fimbriae which
haveattheiroutertipalectindomainthatbindstoDrnannose
polysaccharide^.^^
Fimbriae represent the major structure for attachment of Enterobacteriaceae to host tissue. Research has suggested that type I fimbriae attach to leukocyte 6-integrins which are cell surface glycoproteins with N-linked mannose residues.36 Adhesion of Y. encerocoliticu to leukocyte surfaces has been examined in vitro. China
Y.
et al used fluorescence microscopy and flow cytometry to demonstrate that
encerocolicicu attached to the external surface of polymorphonuclear leukocytes.” Strains of Y. encerocolitcu that expressed the surface protein YadA bound poorly to leukocytes however.Theirresultagainemphasizestheroleofplasmid-codedproteinsinthe interactions betweenY. encerocolicicu and leukocytes. The YadA protein also may mediate binding of Y. encerocoliticu to fibronectin-coated surfaces. Tertti et al measured adherence of Y. enterocoliticu serotype 0:3 toglasscoverslipscoated
with eitheralbuminor
LEUKODEPLETION
107
fibronectin. Bacteria did not bind to albumin-coated surfaces, but did bind to fibronectin coatedglassprovidedthattheorganismexpressedtheYadAprotein.38
A reportby
Wuorela et al suggested that Y. enterocolitica adhere for prolonged periods to the outer surface of leukocytes. They fed peripheral blood monocytes in vitro with heat-killed
Y.
enterocolitica serotype 0:3.39 Using immunofluorescence staining of bacteria antigens, the investigators were able to detect bacterial lipopolysaccharide both within cytoplasmic vacuoles and on the outer surface of monocytes up to 1 day after exposure. Experimental studies of blood inoculation clearly demonstrate that bacteria associate with leukocytes although it is difficult to be certain whether bacteria were attached to the
surface of leukocytes, were ingested by them, or both. Gong et al inoculated whole blood with 80 CFU/mLof
Y. enterocolitica andwithin
2 hoursseparatedthebloodby
centrifugationintopackedRedCells,Buffycoat,andPlasma.Althoughthebuoyant densityofbacteriawouldcausethemtoseparatewiththeplasma,thegreatest concentration of bacteria was associated with the leukocytes.2s
Hypothesis #4: Bacteria are directly removed by the Plter media. Removal of leukocytes by filtration is considered to result from a combination of factors including barrier retention; surface phenomena such as charge and surface tension; and biologicfactorssuchascell-proteinorcell-cellinteractions.40Althoughtheeffective pore size of leukocyte depletion filters (approx4 micron diameter) is too large to remove bacteriabasedonbarrierretention,bothphysical
and biologicforceswhichpromote
leukocyte retentionby filters may contribute to retention of bacteria. Phvsical forces: An extensive literature exists concerning mechanisms of bacterial adheren~e.~'Bacteria have evolved different mechanisms to increase their adherence to biological surfaces and thereby promote colonization or invasion. Both gram positive and gramnegativeorganismshavesurfacesmadehydrophobicbystructuressuchas lipoteichoic acid (gram positives) or fimbriae (gram negatives). For example, there may be evolutionary pressure for organisms such as Yersinia to leave the water phase of the intestinallumenandassociatewithhosttissues.Theabilityoforganismssuch
as
Staphylococcus tocolonizesurfacesonmedicaldevicesorindwellingcatheters
is
dependent in part on the cells' hydrophobic surface characteristics. Techniques such as hydrophobic interaction chromatography and phase partition studies have documented the
DZIK
108
affinityofvariousbacteriaforhydrophobicsurfaces?2Hydrophobicinteractionsmay promote adherence of bacteria to the synthetic microfibers found in leukocyte depletion filters.Thesurfacecoatingofthesesyntheticmaterials adherence. For example, Kawabata et
may furtheraffectbacterial
al studied the removal of
variety a of
or absence of a pyridinium-type microorganisms through non-woven cloth in the presence polymer coating(N-benzyl-4-vinylpyridinium). A suspension of E. coli (6 x l@cells/mL) in water was filtered through the cloth. Bacterial removal was only
10% using uncoated
cloth but rose to over 99.9% using treated ~10th."~ Surface chargemay also affect physical adhesion of bacteria to the filter media. Most fimbrial adhesions on Enterobacteriaceae are negatively charged moleculeswith isoelectric points in the range of pH
3.7 - 5.6.3' Thus, the charge on suspensions of bacteria in
normal saline @H 5.5) or stored blood may be different from that found atpH 7.4. This may have influenced experimental observations made with bacteria suspended in(see saline below). The surface charge of synthetic microfibers used in blood filters is controlled by the manufacturer. For example, in some leukocyte depletion filters the fibers have been deliberately coated with a negatively charged Evidence exists for the importance of hydrophobic and/or charge interactions between
Y. enterocoliticu andpolystyrene.Paerregaardetalexaminedmultiplestrains
of Y,
enterocoliticu. Theyfoundthatthesurfacesofnonpathogenic,plasmid-negative,and plasmid-positivestrainswereincreasinglyhydrophobicrespectively.Thedegreeof hydrophobicity (asmeasured by two phase partitioning) directly correlated with increased adherence to polystyrene." Similar results were found by Mantle and Husar who also noted that adherence of Y. enterocoliticu to polystyrene could be reduced by80% in the presence of a chemical agent (tetramethyl urea) that disrupts hydrophobic interaction^.^' Biologic forces: Although Y. enterocoZitica suspended in 5% albumin do not adhere to leukodepletion filters,the sameorganismssuspendedinplasmadoadhere.This suggeststhatplasmaproteinsotherthanalbumin
maymediateadhesion.Candidate
proteinswouldincludefibronectin,complement,oranotherheat-labileprotein.
Local
activation of complement on the surface of the filter media might produce a filter coated with sufficient C3 to promote bacterial binding.
LEUKODEPLETION
109
Exmrimental evidence for removal of bacteria bv filtration indeuendent of leukocvtes: Experimental evidence for direct removal of bacteria by leukodepletion filters has been pursued by several investigators. AuBuchon prepared suspensions of
enterocoliticu serotype 0:3 ineithersaline,in
25 CFU/mL of
Y.
5% albumin, or inplasma.&Bacteria
suspended in saline appeared to be completely removed by filtration. In contrast, there was no reduction by filtration of bacteria suspended in albumin. Bacteria suspended in plasma were killed even before filtration. Their experiment suggested that bacteria in saline directly adhered to the filter media. To test this, they prepared suspensions in saline of four different strains of Y. enrercoliticu (approximately 100 CFU/mL), three different speciesofgramnegativebacteria
(1 CFU/mL),andonespeciesofDiphtheroids
(1
CFU/mL). There was nearly complete removal of these bacteria when suspended in saline andfilteredthrough
a leukocyte filter designed for
Red Cells(Sepacell R500, Baxter
Healthcare Corp). Wagner also filtered Y.enterocoliricu suspended in saline and observed a 1.5 logreductionintheconcentrationofbacteria."Theseexperimentssuggestthat physical forces such as hydrophobicity or charge can cause bacteria to directly adhere to filtermedia.However,whenalbuminwassubstitutedforsaline,bacteriawerenot retained suggesting that the media fibers became coated with albumin which neutralized any hydrophobic/charge attractive force. It should be noted that previous investigators haveused
5% albumintoblockreactivesitesonpolystyrenewellswhenstudying
hydrophobic adherence of Y. enterocoliticu.qo Such neutralization would also presumably occur under conditions of routine blood donor filtration. Rawalreportedtheinterestingobservationthat
Y. enterocoliticu serotype 0:3
suspended in plasma were retained by filtration through a polyester leukodepletion filter. When the bacteria were suspended in heat-treated plasma, however, they were not retained by thefilter.DThisexperimentsuggestedthatplasmaproteinssuchascomplement proteins might play a role in direct adherence of Y. enterocoliricu to the filter media. An alternative interpretation is that the process of filtration activated complement sufficiently to kill the bacteria. BlajchmanandAliusedanexperimentaldesignwhichdidnotdependonblood
leukocyte^.^'^^ They studied removal of S. xylosur by filtration (filter not specified by
110
DZIK
authors). Eight blood packs containing AS-3 solution without blood were inoculated with 100 CFU/mL of S. xylosus. Although bacteria grew in 6 of 8 unfiltered packs, none of
8 filteredpacksgrewbacteria.Theabsenceofproteininthisexperimentalmodelis similar to the observations of AuBuchon and Wagner (cited above) and may have led to adherence of bacteria to the filter by hydrophobic interactions. In additional experiments, eight units of packed Red Cells were first leukodepleted by filtration. The eight units were then inoculated with 100 CFU/mL of S. xylosus and split into equal aliquots” one which served as a control and one which was filtered
a second time through a leukodepletion
filter. Compared with control units, bacteria overgrowth was delayed in the units that were refiltered and 2 of 8 re-filtered units did not grow bacteria up throughtwo weeks of storage at 4 C. The latter experimental model restored
the presence of plasma proteins
including complement. CONCLUSION Whether or not the prevalence of bacterially contaminated blood components increases will depend on the complex interactions of donor demographics, microbial conditions, and details of the techniques of component preparation and storage. Although we cannot be certain of the effect that prestorage leukodepletion filters would haveon Y. enferuculificu contamination from asymptomatic donors, the evidence provided by inoculation experiments strongly suggests that the combination of a room temperature holding period in the presence of plasma plus filtration through
a leukodepletion filters decreases the
likelihood of subsequentYersiniu overgrowth during prolonged refrigerated storage of Red CellConcentrates.Experimentalevidencesuggeststhatthiseffectresultsfroma combination of phagocytosis, complement-mediated bacterial killing, and direct retention of bacteria on either the surface of leukocytes retained within the filter media itself (Table 2). In contrast to the results of studies employing
or on the filter
Y. enferucoliricu,
experimental inoculation studies usingS. epidennidis suggest that leukodepletion filters do not alter bacterial growth during storage. There is insufficient experimental data regarding other bacteria and there are two published reports of Y. enferucu2itica contamination found in Red Cells which had already been filtered through
a leukodepletion
111
LEUKODEPLETION FILTERS
TABLE 2: Possible mechanisms in the removal of certain bacteria by leukodepletion filters I. Phagocytosis
complement opsonization other protein opsonins 11. Complement
activated by antibacterial immunoglobulins or alternate pathway activated by mannose binding protein activated by filtration III. Adherence to leukocyte surfaces mediated by opsonins mediated by collectins enhanced by leukocyte activation within the filter
IV. Direct binding to filter hydrophobicitykharge protein-mediated adherence
Leukocytedepletionfiltershaveundergoneprogressivedesignmodificationsto increase the efficiency with which they retain leukocytes from specific blood components. Retentionofdonorleukocytesis
a single focused goal of this technology. Conditions
affecting removal of bacteria by filtration appear to vary amongst different bacteria. It would seem unlikely that any single filter formulation could provide high performance leukocyte depletion and at the same time retain themany different strainsof bacteria which have been implicated in cases of transfusion sepsis due to bacterial contamination.
It is
unlikely therefore that leukocyte depletion filters should be regarded as a primary strategy for dealing with bacterial overgrowth inblood components. New technologies such as the detection of bacterial ribosomalRNA'O or the use of photochemical bacterial inactivatorsSO hold promiseto prevent the collection, storage and distribution of bacterially contaminated blood components. REFERENCES 1. D. H.Buchholz, V. M. Young, N. R. Friedman, J. A. ReillyandM. N. Engl. J. Med., 2 8 5 , 429-433 (1971).
R. Mardiney,
112
DZIK
2. M. A. Blajchman and A. M. Ali, in: Blood safetv: current challenpes, S. J. Nance, ed. American Association of Blood Banks, Bethesda, (1992) pp 213-228. 3. R. Yomtovian, H. M. Lazarus, L. T. Goodnough, N. V. Hirschler, A. M. Momssey and M. R. Jacobs, Transfusion, 3,902-909 (1993). 4. B. B. Barrett, J. W. Andersen and K. C. Anderson, Transfusion, 3 3 , 228-233 (1993).
5. J. M. Heal, Transfusion, 2 1 , 581-583 (1991). 6. J. F. Morrow, H. G. Braine, T. S. Kickler, P. M. Ness, J. D. Dick and A. K. Fuller, J.A.M.A., 2 6 6 , 555-558 (1991). 7. K. C. Anderson, M. A. Lew, B. C. Gorgone, 3. Martel, C. B. Leamy and B. Sullivan, Am. J. Med., 8 1 , 405-411 (1986).
8. T. Gibson and W. Nods, Lancet,
2, 983-985 (1958).
9. B. Wenz, D. Ciavarella and L. Freundlich, Transfusion, 3,520-523 (1993). 10. M. E. Brecher, G. Boothe and A. Kerr, Transfusion,
11. J.Gong,C. Transfusion,
B,450-457 (1993).
F. Hogman, A. Hambraeus, C. S. Johanssonand
L. Eriksson,
12. A. P. Gibb, K. M. Martin, G. A. Davidson, B. WalkerandW. Transfusion, 3, 304-310 (1994).
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a,802-808 (1993).
13. M. J. Arduino, L. A. Bland, M. A. Tipple, S. M. Aguero, M. S. Favero and W. R. Jarvis, J. Clin. Micro., 2 2 , 1483-1485(1989). 14. D. H. Buchholz,J. P. AuBuchon, E. L. Snyder, et al, Transfusion, (1992).
3, 667-672
15. B. Wenz, E. R. Bums and L. F. Freundlich, Transfusion, 3 2 , 663-666 (1992).
LEUKODEPLETION FILTERS 16. D. M. Kim, M.
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E. Brecher, L. A. Bland, et al, Transfusion, 2,658-662 (1992).
17. C. F. Hogman, J. Gong, A. Hambraeus,C. Transfusion, 2,654-657 (1992).
S. Johanssonand
L. Eriksson,
18. T. L. Cover and R. C. Aber, N. Engl. J. Med., 321, 16-24 (1989). 19. A. P. Gibb, K. M. Martin, G. A. Davidson, B. Walker and W. G . Murphy, Lancet,
340, 1222-1223(1992).
20. J. Jacobs, D. Jamaer, J. Vandeven, M. Wouters, C.Vermylenand Clin. Micro., 2 2 , 1119-1121 (1989).
J. Vandepitte,
21. R.N. I. Pietersz, H. W. Reesink, W. Pauw, W. J. A. Dekker and L. Buisman, Lancet, 3 4 0 , 755-756 (1992). 22. L. Franzin and P. Gioannini, Transfusion,
z, 673-676 (1992).
23. B. D. Rawal and G. N. Vyas, Transfusion, 3,536 (1993). 24. S. J. Wagner, D. Robinette and R. Dodd, Transfusion, 3 3 , 713-716 (1993).
25. J.Gong,C. F. Hogman, A. Hambraeus, C. S. Johanssonand L. Eriksson, Vox. Sang., 65, 42-46 (1993). 26. D. E. Pierson and S. Falkow, Infect. Immun., Ll, 1846-1852 (1993). 27. B. China, M. P. Sory, B. T. N’guyen,M.deBruyereandG.R. Immun., 61, 3129-3136 (1993).
Cornelis, Infect.
28. U. Holmskov, R. Malhotra, R. B. Sim and J. C. Jensenius, Immunol. Today, 15,6774 (1994). 29. M. Kuhlman, K. Joiner and A. B. Ezekowitz, J. Exp. Med.,
m, 1733-1745 (1989).
30. M. Paulsson, A. Ljungh and T. Wadstrom, J. Clin. Micro., 3,2006-2012 (1992).
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31. E. J. M. Al, S. C. E. Visser, H. K. Prins, R. N. I. Pietersz, H. W. Reesink and J. 835-842 (1991). G. Huisman, Transfusion,
x,
32. C. J. van Oss and C.
F. Gillman, J. Reticuloendo. Soc., Q, 283-292, (1972).
33. S. D. Wright and M. T. Jong, J. Exp. Med., 34. R. R. Isberg and J. M. Leong, Cell,
35. G.
m,1876-1888 (1986).
B,861-871 (1990).
W.Jones and R. E. Isaacson, CRC Crit. Rev. Micro.,
36. N. Sharon, F.E.B.S. Letters,
217,
D,229-260 (1983).
145-157 (1987).
37. B. China, B. T. N’Guyen, M. deBruyere and G. R. Cornelis, Infect. Immun., 1275-1281 (1994). 38. R. Tertti, M. Skurnik, T. Vartio and P. Kuusela, Infect. Immun., (1992).
a,3021-3024
39. M. Wuorela, S. Jalkanen, P. Toivanen and K. Granfors, Infect. Immun., 5270 (1993). 40. S. Dzik, Transf. Med. Rev.,
62,
61, 5261-
2, 65-77 (1993).
41. L.
M.Baddour, G. D. Christensen, W. A. Simpson and E. H. Beachey, in Principles and Practice of Infectious Diseases, 3rd edition, G. L. Mandell, R. G. Douglas and J. E. Bennett JE, eds. Churchill Livingstone, New York, (1990) pp 9-25. 42. M. C. M. van Loosdrecht, J. LyMema, W. Norde, G. Schraa and A. J. B. Zehnder, Appl. Environ. Microbiol., B, 1893-1897 (1987). 43.
N. Kawabata, T. Inoue and H. Tomita, Epidemiol. Infect., U, 123-134 (1992).
44. A. Paerregaard, F. Espersen and N. Baker, APMIS, 45. M. Mantle and S. D. Husar, Infect. Immun.,
B,927-932 (1990).
a,2340-2346 (1993).
LEUKODEPLETION FILTERS
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46. J. P. AuBuchon and C. Pickard, Transfusion, B, 533-534 (1993). 47. J. J. Freedman, M. A.BlajchmanandN.McCombie, (1994). 48. M. A. Blajchman, A. M. Ali and H.
Transf. Med. Rev.,
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1-14
L. Richardson, Vox. Sang. (in press)
49. D. M. Kim, M. E. Brecher, L. A. Bland, T. J.Estes, R. A. Carmenand E. J. 221-225 (1992). Nelson, Transfusion,
z,
50. L. Lin, H. Londe, M. Janda, C. V. Hanson and L. Corash, Blood,
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This Page Intentionally Left Blank
TRANSFUSION-TRANSMITTED CYTOMEGALOVIRUS INFECTION Raleigh A. Bowden, M.D. Acting Head, Program in Infectious Diseases, and Associate Member, Fred Hutchinson Cancer Research Center,
WA. Associate Professor of Pediatrics, University of Washington, Seattle Program in Infectious Diseases Fred Hutchinson Cancer Research Center Seattle, WA
98104
INTRODUCTION Cytomegalovirus (CMV) remains a major threat to the immunocompromised host. While the major risk for CMV infection and disease in immunocompromised patients results from reactivation of latent virus in the seropositive individual, there remains a significant risk for acquisition of primary infectionin the seronegative patient who receives the blood
(1). Exposure is products or an organ allograft from a seropositive donor degree of the major determinant CMV of infection while the immunosuppression is the major determinant for the development for symptomatic CMV disease. Although effective antiviral therapyis now
CMV infection, this treatment is available for the treatment of established not without toxicity and associated morbidity and cost. Therefore,
CMV infection through the manipulation prevention of transfusion-acquired of blood products by serologic screening or leukocyte depletion remains the preferable approachin the CMV seronegative patient. This review will present some general aspects pertaining to the biology, a description of which cells in blood and which blood products are
CMV infection, and an overview of populations at most likely to transmit risk for transfusion-acquired CMV infection. Options for prevention by the
CMV seronegative or leukocyte depleted blood products, use of either CMVincluding their limitations as well astheir advantages for providing safe blood products to individuals at high risk CMV for infection and
117
BOWDEN
118
disease will be reviewed. Finally, the risk for transmission of a second
CMV strain infection to an already seropositive individual be will discussed.
GENERAL ASPECTS OF CYTOMEGALOVIRUS
CMV is a ubiquitousDNA virus which is acquired as a primary infection through contact of body secretions, blood products or organ in allografts. The acquisition of CMV in the normal host usually results an asymptomatic infection. Once infection has occurred, CMV remains latent for life and can cause recurrent infection it whenreactivates. In the
CMV or reactivationof immunocompromised patient, acquisition of primary latent virus result in an increased likelihood of clinically symptomatic infection. The risk for development ofCMV disease depends on the degree of immunosuppression of the host. The prevalence ofCMV infection in the population depends in part on geographic location and socioeconomic factors (2). In highly developed but 20% less densely populated areas, the seropositivityrate may be as low as
in the general population. In contrast, the seropositivityrate approaches
100% in some developing countries or in large urban areas which are densely popul
ated
and/or
underdevel
oped.
THE RISK OF PARTICULAR BLOOD PRODUCTS While epidemiologic factors help identify the patients at highest risk for transfusion-acquiredCMV infection, factors that might distinguish a potentially infectious "healthy" seropositive blood donor remain poorly understood. Culturing urine or IgM screening of healthy blood donors has not proved helpful(3,4).
What is clear is that therisk of acquiring
transfusion-associated CMV infection increases as the exposure increases, particularly exposure to blood from multiple, different seropositive donors
(5). The volume of blood, the age of the blood, the number of leukocytes and characteristics of the patient and the blood donors presumably play a role but their specific risks have not been clearly defined.
CMV is a highlycell associated virus and the leukocyte has been shown to be the vehicle of transmission of CMV in blood ( 6 , 7 ) . The risk of CMV for particular types of blood products has not been well characterized, although therisk should be predictably higher for products containing relatively more leukocytes. Granulocyte transfusions from seropositive donors carry a very high risk of CMV transmission (8), as one would expect
TRANSFUSION-TRANSMITTED CMV INFECTION
119
when giving a concentrated unit of leukocytes. By conventional pheresis techniques, such units contain10'o--lO" leukocytes/unit. Whether the risk of CMV transmission
Si
lower
for
platelets red or blood
cell S has
not
been
clearly defined, although it is presumed that risk the of CMV from the
1 atter two is approximately the same. An average red blood cell unit contains approximately10' leukocytes and a unit of platelets contains approximately 10'.
Since platelets are most often given in transfusions of
4-6 units, the differencein the number of leukocytes betweenreda blood
a platelet transfusion is less than a half log,,.a cell transfusion and If there is an appreciable difference in risk the of CMV infection between the two, it has not been appreciated. Fresh frozen plasma does not transmit CMV infection (g), presumably because this product contains very low numbers of leukocytes which are disrupted during the freezing procedure. Thi S latter mechanism is presumable what also makes frozen deglycerolized red blood cells non-infectious(IO). While it isclear that the leukocyte is the vehicle for transmission responsible and the of CMV from blood products, the specific leukocyte specific mechanism of transmission from blood products remains unclear.
CMV can be cultured( 1 1 ) or detected byDNA probing (12) from the granulocyte or mononuclear fraction of patients actively infected patients, however, this is generally not true of latently, non-productively infected seropositive healthy individuals. Experimental data have shown that using
CD8 lymphocytes high enough titers of lab-adapted CMV, monocytes as well as are, (13,14). Different cells may carry CMV in the latent infected individual thanin actively infectedor experimental situations. Polymerase chain reaction studies of normal healthy seropositive
CMV genome canbe detected in the leukocytes of individuals have shown that healthy seropositive individuals (15,16), identified in one study by
CMV genome in the CD4 lymphocytes in 2 of 8 immediate early proteins of the subjects with a frequency of one positive cell per 500 cells (15). Culture of these cells failed to produce replicating virus.
POPULATIONS AT RISK FOR CMV INFECTION Historically, the incidence of transfusion-associated CMV infections was defined for surgical patients (17), for patients undergoing cardiac bypass surgical procedures (18), and for newborns undergoing exchange transfusion (19). These studies described the onset of infection, which was usually asymptomatic, occurring 4-12 weeks after exposure from blood products (37,18). The risk of infection appeared to increase with the
BOWDEN
120
number of blood products(19) as well as the number of seropositive blood risk varies donors (5). Calculations in surgical patients showed that the (20). between 3 and 12 infections per100 units of blood transfused
Table I outlines therisk for and manifestations of CMV infections in four highrisk groups of immunocompromised patients risk at for morbidity and mortality ofCMV.
The severity ofCMV disease and risk for associated
mortality is directly correlated with the degree of immunosuppression characteristic of each individual transplant setting. In somehigh risk settings, the majorityof serious infections in these groups results from reactivation of CMV infection in previously seropositive recipients. For example, the marrow transplant setting has historically been the setting where patients are at the most significant risk from death from CMV, usually fromCMV pneumonia which occurs in as many 35%as of seropositive allogeneic transplant patients if antiviral prophylaxis is not (1). given
In the sol id organ transplant setting, the most severe disease as a rule results frominfection transmitted to the seronegative recipient of a seropositive organ allograft(21). CMV infection in these patients comes either from the unscreened blood product or seropositiveallograft. organ The risk for morbidity, and the manifestation CMV of infection focus on the
CMV organ of transplant. For example, in the liver transplant setting, hepatitis is quite common (22), a finding rarely seen after marrow transplant. Likewise in lung transplant recipients,CMV pneumonia may be more common. Most patients infected with the human immunodeficiency virus (HIV) are CMV seropositive and the reactivation rate of CMV approaches 100% in these individuals. CMV infection can be associated with a variety of clinical syndromes and although death is uncommon, sight-threatening infection may lead to blindness in up 25%to of patients notreceiving antiviral therapy. Because the endogenous reactivation rate of CMV is so high in these settings, it is difficult to determine role the that virus transmitted from unscreened blood products may contribute both to the incidence and severity of infection. However, therare CMV seronegative
CMVpatient is obviously at risk for the same syndromes and should receive safe blood to prevent these complications.
CMV is in the seronegative infant For newborns, the major risk for who acquires CMV during the mother’s primary infection when it occurs during pregnancy. All seronegative pregnant women requiring blood products during pregnancy should receive some form of CMV-safe bloodproduct. The
CMV seronegative blood. standard of care has been to provide
121
TRANSFUSION-TRANSMITTED CMV INFECTION
TABLE I.
Cytomegalovirus Infection High Risk Settings Incidence ofCMV
Settinq
(CMV serol Transplantation(1) 70% 50% Marrow
itv
MortalDisease Infection ogy
pati
ent/donor)
in t/t
Pneumonia
3
in -/t
Enteritis
30%
30-50"/.
15%? Hepatitis Fever/Leukopenia 15%? Solid organ(21,22) 77-83% in t/-/+
1WO
Hepatitis 43% Pneumonia 17%-38% Fever/leukopenia
15%
Organ Rejections EnteritiS
10%
CMV SERONEGATIVE BLOOD PRODUCTS CMV seronegative blood has become the standard of care for the CMV infection in seronegative high prevention of transfusion-associated risk patients (Table11). While both seronegative and seropositive individuals are presumably
CMV from blood products, the incidence CMV of at risk for acquisition of infection acquired from blood products is easier to decipher in the CMV exposure is 1 imited to blood products. seronegative recipient where
(5), there are now First demonstrated in a controlled trial in newborns reports in both marrow transplant (23,24) and solid organ transplant donor (or mother) is patients (25) that show as long as the organ seronegative, that therisk for CMV infection is lessthan 7%. This small but continuedrisk is presumablydue to the insensitivity of the serologic screening methods used for either the patient, organ donor of blood donor allowing false negative testing to allow transmission in this setting. The rate of false negative test results canaffect the incidence of
CMV infection in several ways. First, if the seropositive blood donor is CMV in identified as falsely negative, his donated blood may result infection in the seronegative recipient. Secondly, the recipientmay be falsely identified as seronegative either when he is in the early stages of
122
BOWDEN
TABLE 11. Percent of Patients Infected with CMV Among Patients Receiving CMV-Seronegative Blood Products Donor (mother) Patient Powlation
1.
Newborns
2.
BMT (23, 24)
3.
*
SeroDositive Seroneqative
(5)
Cardiac Transplant
0
(25)
(14%)*
18% (15%)
3% (25%)
25% (31%)
6% (37%)
62% (42%)
083% (20%)
( 100%)
% infected patients when receiving unscreened standard blood products
a primary infection and not yet mounted a detectable antibody response or has such poor immunity from his underlying condition or its therapy that he is unable to maintainor mount a positive response. Because such patients may reactivate this "previously acquired" virus, they will appear as a failure CMV-safe blood products. Any screening method will falsely define a certain number of true positive patientsas being seronegative. In a recent study at our Center
(CF) was compared to latex where two test the complement fixation agglutination (LA) in 409 sera, we observed equivocalresults in 4.1% and false positive or false negative results in 1.1%(26).
Table I 1 also
illustrates that when the mother or organ donor are seropositive, that infection continues to occur from transmission of infection from the seropositive mother or allograft despite the use of seronegative blood products. Because few data are available to discern the relative contribution of unscreened blood from the contribution of infection from the seropositive organin the transplant setting, seronegative blood is generally reserved for seronegative patients.
It is also1 i kely that the use of large numbers CMV seronegative of blood products from a center for a particular group risk of patients high will shift in theremaining available blood pool to a higher percentage of seropositive products. The risk ofCMV in these recipients has not been
TRANSFUSION-TRANSMITTEDC W INFECTION
123
well documented but is presumably increased. For example, a center such as the Puget Sound Blood Center in Seattle that serves a community 1 of million people, 50% of the blood donor population is CMV seronegative. If
50 seronegative marrow transplant patients per this center provides for year, the estimated percent of seropositive bloodthe in remaining blood pool goes up to approximately 55% (personal communication, Merlin Sayers,
M.D.).
If the numberof seronegative marrow transplant patients goes upto
150 per year in that same center, calculations show that the seropositive rate in the remaining blood pool may approach 75%.
LEUKOCYTE DEPLETION FOR PREVENTION OF TRANSFUSION-ACQUIRED CMV INFECTION Because of the limited supply of seronegative blood, alternative methods for prevention of transfusion-associated CMV infection have been explored. While leukocyte depletion is based on the concept that the leukocyte is the vehicle of transmission CMV, of the number of cells required to transmit infection and therefore the number of cells required to be removed to prevent infection is unknown. Table I11 shows a summary of published studies to date in a variety of clinical settings using a variety of methods for leukocyte depletion (27-32). While the majorityof these studies were not controlled, the results strongly suggest that leukocyte depletion was an effective alternative for seronegative blood in high risk settings. Most of these studies used methods resulting in a 2-3 log,, depletion of leukocytes. In our center, we began a series of studies to determine the ability
of leukocyte depleted blood by filtration to prevent transfusion-associated
CMV infection in marrow transplant patients. The first study was a study comparing leukocyte-depleted platelets plus CMV seronegative red cells in one group with the control group who received standard, unscreened or filtered products (32). The next study was an open-labelled studyin autologous patients to determine if rate the of infection was comparable to seronegative blood before proceeding to randomized studies (30). In this study, there was one infection in32 patients. Encouraged by these studies, we wanted to determine if this approach could protect allogeneic
CMV infection and transplant patients as well, who are higher at risk for disease. We therefore began a controlled, randomized trial comparing leukocyte-depletion by filtration of both red cells and platelets with seronegative blood in both autologous and allogeneic marrow transplant patients to determine if the two methods were equivalent for prevention of
BOWDEN
124
TABLE 111.
CMV Infection In Patients Receiving Depleted Blood Products
Study Blood Products
1.
2.
(28)
(27)
Patients
Leukocyte-Door
RBC: Fi1 tered
Leukemia/
Plts: Centrifuged
Lymphoma
RBC: Filtered
Newborn
0/42
Plts: Not given
ControlS
9/59
0/59
005 p=O.
3.
(29)
RBC: F i 1 tered Plts: CMV -
BMT
0/29
4.
(30)
RBC: Fi1 tered
BM1
1/32
BM1
0/28
RBC: CMV -
BM1
Plts: Centrifuged
ControlS
0/25 p=O.OOI 9/20
Plts: Filtered 5.
(31)
RBC : Fi1 tered Plts: Centrifuged
6.
(32)
transfusion-associated CMV infection (33). Final report of these results are forthcoming. If it proves to be confirmed that filtration is equivalent for seronegative blood, the impact could be very broad reaching for patients who need CMV-safe blood in communities where there is not enough CMV seronegative blood to meet the demand.
RISK FOR SECOND CMV STRAIN INFECTION IN THE SEROPOSITIVE PATIENT With the availability of an effective alternative for prevention of role of second transfusion-acquired CMV infection, the question of what the strain infection becomes increasingly relevant. As alternative means of providing CMV-safe blood are becoming available, the issue of whether a
CMV (i.e. is a patient can become infected with more than one strain of seropositive patient atrisk for second strain infection from blood), has become of increasing interest. Detection of second strain infection in the
DNA restriction seropositive individuals is difficult because requires it enzyme analysis of replicating virus (34, 35). Earlier studies suggested
INFECTION TRANSFUSION-TRANSMITTED CMV
125
that in some patients, such as those undergoing marrow transplant, risk the so high that the additional risk of blood of endogenous reactivation is products, including seropositive granulocyte transfusions could not be appreciated clinically (8). The important questions that affect the decision to provide CMV-safe
1) the blood to the seropositive patient must consider two factors; evidence that second strain infection can occur2)and the associated morbidity and mortality associated with second strain infection. Recent data in bothHIV-infected individuals(35), and organ
( 3 6 , 37), patients have shown quite transplant (34), and marrow transplant clearly that patients can be infected with more than one strain. The clinical impact of these second strain infections, however, is less well defined.
Since the seropositive individual already has immunity CMV to
from exposure during their primary infection, one might expect their infection with a second strain to have less clinical impact since they have existing immunity which should have cross-reactivity to the second strain. However, several pieces of evidence suggest that risk the for severe CMV disease may be greater with second strain infection. Grundy et al. reported that seropositive renal transplant recipients receiving seropositive kidneys, had more severe CMV disease thandid recipients of seronegative allographs, suggesting second strain transmission from the allograft resultedin more severe infection than reactivation of the primary strain in the seropositive recipient (38). A small study in seronegative 1 iver transplant recipients organ showed that unscreened blood increased the severity of CMV infection rate in patients receiving seropositive compared to seronegative liver allografts (39). However, it has also been reported that seropositive marrow transplant patients
CMV disease (40, 41) than did receiving seronegative marrow had more severe recipients of a seropositive marrow(42). They, and others have proposed transfer of immunity provided protection against severe disease in the
1 ater
case(42).
FUTURE AREAS OF INVESTIGATION There are many areas where more information i S needed regarding the
CMV is transmission of CMV by blood products. While data supports that transmission by leukocytes, the exact number of leukocytes required to transmit infection is unknown. The specific leukocyte and the mechanism of to recipient are also unknown. The transfer of CMV from organ donor significance of second strain infection requires further study before the
126
BOWDEN
use of CMV-safe blood products becan routinely recommended for high risk seropositive populations. We also need more sensitive screening methods for identification of the seropositive individual. Perhaps polymerase chain reaction techniques will of bevalue in this regardin the future. And finally, means of determining what factors in the seropositive blood donor are critical for transmission of infection might allow one to transfuse seropositive blood that is CMV-safe, expanding the pool of blood for
highrisk patients. In conclusion, patients at high risk forCMV infection are increasing
with the growing number of transplant and cancer patients. Recent studies in the useof leukocyte filtration in the past five years are expanding our ability to provide CMV-safe blood to this growing population of patients. Future workis needed to determine the various factors and mechanisms that result in transmission ofCMV from blood products.
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1.
J.D. Meyers, P. Ljungman, L.D. Fisher. J. Inf. Dis.
2.
W.L. Bayer, G.E. Tegtmeier. Yale J. 8iol. Med., 49, 5(1976).
3.
J.S. Beneke, G.E. Tegtmeier, H.J. Alter, et al.
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R.C. Kane, W.E. Rousseau, G.R. Noble, et al. ASM ll, 719-723(1975).
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A.S. Yeager, F.C. Grumet, E.B. Hafleigh. J. Ped. 98, 281-287(1981).
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(1990).
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G.P.A. Rice, R.D. Schrier, M.8.A. Oldstone. Proc.
81, 6134-6138 (1984).
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Natl Acad. Sci
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7.
L. Einhorn, A. ost. J. Infec. Dis. 149, 207-214(1984).
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3. Hersman, J.D. Meyers, E.D. Thomas, et al. Ann. Intern. Med. 1491982.
9.
R.A. Bowden, M. Sayers. Transfusion
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N.E. Tolkoff-Rubin, R.H. Rubin, E.E. Keller, et al. Ann. Intern. Med.
11.
H.M. Garnett. J. Lab. Cl in. Med.99, 92-97(1982).
12.
S.A. Spector, J.A. Rua, D.H. Spector, et al. J. Infect. Dis. 150,
13.
R.W. Braun, H.C. Reiser. J. Virol. 6 0 , 29-36(1986).
14.
C. Soderberg, S. Larsson, S. Bergstedt-Lindqvist, et al. J. Virol.
30,
96,
762-763(1990).
(Part 1) 8 9 , 625(1978).
121-126(1984).
6 7 , 3166-3175(1993).
INFECTION TRANSFUSION-TRANSMITTED CMY
15. R.D. Schrier, J.A. (1985).
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Nelson, M.B.A.
Oldstone.Science
16. P. Stanier, A. D. Kitchen, D. L. Taylor, A.S. Probes., 6 , 51-58 (1992).
230,
1048-1051
Tyms. Moll. and C e l l .
17. W. L. Drew, R.C. Miner. JAMA 247, 2389 (1982). 18. D.J. Lang, E.M. Scolnick, J.T. Willerson. (1968).
N. Engl. J. Med., 278, 1147
19. S.P. Adler, T. Chandrika, L. Lawrence, e ta l .P e d i a t r .I n f e c t .D i s . -2, 1150 (1968). 20. H.V. Lamberson. 21. R.H. Rubin.
Vox Sang,
46, 398 (1984).
Rev. I n f e c t .D i s .
12
(suppl. 7), S754-S766 (1990). 51,
22. R.J. S t r a t t a , M.S. Shaefer, K.A. Cushing, e t a l . T r a n s p l a n t a t i o n 90-97 (1991). 23. R.A. Bowden, M. Sayers, N. Flournoy, e t a l . 1006-1010 (1986).
New Engl
24. W.J. M i l l e r , J. McCullough, H.H. B a l f o u r ,e ta l . 7, 227-234 (1991). 25. J.K. P r e i k s a i t i s , S. Rosno, C. Grumet. (1983). 26. R.A. Bowden, M. Sayers, C.A. (1987).
deGraan-Hentzen,
757-760
31. T. De Witte, A. Schattenberg, B.A.Van 50, 964-968 (1990).
Mudde, e ta l .T r a n s f u s i o n Dekker AW, e t a l .
S l i c h t e r , M.H.
J. I n f e c t .D i s .
Bone
3 , 5205
D i j k ,e ta l .T r a n s p l a n t a t i o n
Sayers, e ta l .B l o o d
33. R.A. Bowden, M. Cays, G. Schoch, e ta l .B l o o d (1993). 34. S. Chou.
478-481
Lancet 1, 1228-1231
30. R.A. Bowden, M.H. Sayers, M. Cays, e ta l .T r a n s f u s i o n (1989).
32. R.A. Bowden, S.J. (1991).
21,
(1989).
29. L.F. Verdonck, Y.C.E. de Graan-Hentzen, A.W. Marrow Transpl . 2, 73-78 (1987).
18, 246-250
82 (suppl .l), 204a
155, 1054-1055 (1987).
35. S.H. Chandler, H.H. Handsfield, J.K. McDougall JK. 155, 655-660 (1987).
.
147, 974-981
Gleaves, e ta l .T r a n s f u s i o n
J.W. Gratama, G.C.
Med. 3 1 4 ,
Bone Marrow Transpl
J. Infec.Dis.
27. G.L. G i l b e r t , K. Hayes, I.L. Hudson, e t a l . (1989). 28. Y.C.E. 29,
. J.
J. I n f e c t .D i s .
12s
36. B. Fries, et al.
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J. Infect. Dis.
169,
769-774 (1994).
37. J.A. Zaia, G. Gallez-Hawkins, M.A. Churchill. J. Cl in. Micro. 28, 2602-2607 (1990). 38. J.E. Grundy, S.F. Lui, M. Super, et al. Lancet 39. R. Mafiez, S. Jusne, M. Martin, et a l .
fi, 132-135 (1988).
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B, 594-597 (1993).
40. J.F. Grob, J.E. Grundy, H.G. Prentice, et. al. Lancet 776 (1987). 41. C-R. Li, et al. Blood
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42. G. J. Boland, et al. Clin. exp. Immunol.
88,
506-511 (1992).
PART 11: TESTING FOR INFECTIOUS AGENTS
This Page Intentionally Left Blank
EXISTING PROBLEMS IN THE TESTING FOR INFECTIOUS DISEASES
Kathleen Sazama, MD, JD Department of Pathology and Laboratory Medicine Medical College of Pennsylvania Philadelphia, PA, 19129, USA
ABSTRACT Current methods for testing donated blood for presence of infectious viral agents in the USA differ from those used in other countries because of the USA Food and Drug Administration's (FDA) control which inhibits rapid introduction of testing methods or improvements. Delays in FDA approval may occur because of concerns about methodology or the state of knowledge about the disease it is intendedto detect as wellas due to variability between manufacturers. Despite strict FDA control, testing problems continue to occur in the USA. No approved method detects infectious agents during the "window period," and variations in detection, i.e., false positives and false negatives (even with confirmatory testing), continue to occur. The effect of physical and chemical changes (e.g., various anticoagulants) on samples has not been thoroughly evaluated. Test performance problems include lapses in sample identification, failure to use routine laboratory controls, improper calculation and reporting of results, improper acceptance of test runs and failure to properly detect and retest samples when carryover from very reactive samples occurs. For these reasons, transfusion-related disease transmission continues to occur. The current USA emphasis on good manufacturing practices should provide continuous improvements.
Although infectious agents other than viruses are transmissible by blood, most laboratory testing of donated blood in the United States (US) is intended to detect (and eliminate) virally transmitted diseases, specifically for infections caused by human retroviruses (human immunodeficiency viruses, types 1 and 2 [HIV-1/21 and human T-lymphotropic viruses, types 1 and I1 [HTLV-1/11]) and the hepatitis viruses, hepatitis B and C. US blood donations are tested for hepatitis B virus surface antigen (HBsAg), antibody to hepatitis B virus core antigen (anti-HBc), antibody to hepatitis C virus (antiHCV), antibody to HIV-112 (anti-HIV-1/2), antibody to HTLV-1/11 (anti-HTLV-1/11), syphilis, and occasionally for antibody to cytomegalovirus (CMV). Non-specific testing
131
SAZAMA
132
for evidence of liver damage (alanine aminotransferase [ALT])is also performed to exclude possible non-A, non-B, non-C hepatitis. Current methods for testing donated blood for presence of infectious viral agents in the US differ from those used in Europe, Japan and other countries. The US Food and Drug Administration (FDA), which controls release of both original and revised methodologies and instrumentation, requires manufacturers to meet stringent regulatory compliance before allowing any test for infectious diseasesto be applied to donated blood. These tight controls inhibit rapid introduction of testing methods or improvements, causing the US blood banking profession to lag behind its international counterparts in implementing new or revised testing.(l) However, US blood banking performs donor testing using standardized approaches so that results of clinical investigations of newer methods can be predictably applied to routine use once regulatory permission is obtained. Frequently, delays
in regulatory approval occur because the
relationship between the proposed testing methods and whether they actually detect the disease condition for which they are intended is uncertain or needs further definition.(2-5) As knowledge of new diseases, e.g., AIDS and Hepatitis C , advances, methods for
detecting the infectious agents associated with them also improve. With strict FDA regulatory control over testing for infectious viral agents, it is tempting to conclude that few problems occur. Unfortunately, such is not the case. In addition to individual biological variability, some samplesare repeat reactive with one manufacturer's screening method and completely negative with another, with variable results on further or supplemental testing. None of
the currently FDA approved methods
employ true "controls", i.e., samples that are repeatedly tested over a time period to establish the predictability of sample reactivity from day to day or month to month, especially samples reacting within the "grey zone." external controls for viral disease testing.
Blood centers do not routinely use
All testing currently in use in US blood banks
fails to detect infectious agents during the "window period" of infection when the organism is present but in low titer or before it has elicited a detectable host immune response. Variable levels of seroreactivity also occur. There have been few studies on the effect of various physical conditions and agents on detection of infectious agents or antibodies to them. There are also problems with performance of testing, including lapses in sample identification (6), failure to calculate correctly and report accurately results, improper acceptance of "runs" and failure to detect and retest properly samples when carryover from very reactive samples occurs. (Table I) For these and other reasons, unfortunately, transfusion-related disease transmission continues
to occur, albeit at a lower rate than before testing was performed.(7) (Table
11)
133
PROBLEMS IN INFECTIOUS DISEASE TESTING
TABLE I Reasons for Failure to Test Correctly for Infectious Disease Methodologic differences between manufacturers Inadequate control of testing Sensitivity of current assay version Biologic variability - the "window period" Sample handling variables Sample identification Improper test performance or calculations Other reasons
TABLE I1 Risk of Transfusion-Transmitted Viral Infection US - 1992
f&gg
Risk/Unit
Relative
Hepatitis C
1/3,300
Hepatitis B
1/200,000
HIV- 1 HTLV-1/11
11225,000 c 1/50,000
METHODS FOR DETECTING INFECTIOUS AGENTS Methods for detecting infectious agents include assays that test for antibody, viral antigens, enzymes, and even combinations of antibodies. Test formats may include microtiter plates, beads, microbeads, dipsticks, dots, slots, paper or plastic strips, swabs, cartridges, etc. Indicator systems include production or inhibition of color, radioactivity, fluorescence and chemiluminescence. There are at least five versions of enzyme-linked immunoassays (ELBA) available, including indirect, competitive, antibody sandwich, antigen sandwich and antibody- capture techniques.@)
SAZAMA
134
A two-tiered strategy is used for testing US donor blood for presence of infectious agents: use of a screening method, usually an enzyme or radio-immunoassay, with generally high sensitivity, and a supplemental, more specific method such as an immunoblot, immunofluorescent or immunoprecipitationmethod.Except
for testingforanti-HCV, most
methods originally relied on use of viral lysates, with subsequent versions or followup testing evolving to include the use of recombinant antigens expressed in bacterial or fungal systems or chemically synthesized peptides or other methodology.(9,10) For HCV, the virus has not
been isolated. Instead, molecular cloning (11) has been used to establish testing (12). Each manufacturer relies on its own internal expertise to devise and formulate new or improved test methodologies calculated to
meet the FDA approval requirements, which
include correctly identifying a pedigreed panel of samples (housed in an FDA repository and used for lot-by-lot release of approved products). Variations in detection of these pedigreed samples are permitted during the FDA review process, so that some discrepancies between manufacturers’ kits can be anticipated even with stringent manufacturing controls. Hepatitis B virus testing, the original (and for a decade the only) virus testing method used in US donors, consists of testing for both HBsAg and anti-HBc, with positive confirmatory testing available only for HBsAg reactive samples. Methods currently available for screening permit several incubation options as part of the same kit instructions. However, even sophisticated automated and fully computerized techniques have not eliminated the possibility of operator error and incorrect interpretations when the instructions for one method are mistakenly applied to another. Such
errors havereceivedrenewed
attention during recent FDA investigations. Retroviral testing, triggered by reports of transfusion-transmitted AIDS (13), was first implemented in March 1985 when the Abbott method
for anti-HTLV-111 screening (now
called anti-HIV-l) was FDA approved. Other manufacturers quickly obtained similar approval, even though no manufacturer performed perfectly on the FDA panel.(l4) (Table 111). According to a recent comprehensive review, there are at least 130 tests for retrovirus detection currently manufactured by at least 40 companies worldwide,(8) including an increasing number of rapid methods. (15) Information
is available from the World Health
Organization (WHO) about test performance of many of these assays.(l6) Testing for retroviruses in US donor blood (in 1994) is still manufacturer-dependent, i.e., some samples will react only in one, but not in another, manufacturer’s test system. Although the number of such samples is quite small, each U S blood bank generally uses only a single manufacturer’s method and can anticipate missing an occasional reactive sample based on the limitations of that manufacturer’s kit, no matter how carefully testing is performed.
PROBLEMS IN INFECTIOUS DISEASE TESTING
135
TABLE 111 Claims by Two Manufacturers of anti-HTLV-111 Tests in 1986 %Specificitv % Sensitivitv
Manufacturer A
98.3%
99.8%
B
100.0%
99.2%
LIMITS OF SEROREACTIVITY Testing limitations Use of FDA approved methods of detecting viral diseases in
US blood banking
facilities has lacked one component of standard laboratory practices, the use of an "external" control which provides some assurance that results would be reproducible for each sample over a period of time or after prolonged storage or exposure to repetitive freeze-thaw cycles. The US regulatory scheme for blood banking explicitly mandates use of FDA-approved testing following the manufacturer's directions. The language incorporated into the manufacturer's directions has inappropriately applied the terminology "controls" for materials used to calibrate or standardize the test run (providing the "go"/"no-go" signal to continue with testing that day or for that run). Consequently, laboratories performing these tests have rarely incorporated an "external control" into these test runs (even though such control materials are uniformly used for all other laboratory methods). The predictability of result reproducibility, particularly for samples that react at or near the cutoff of a run, is uncertain. The US Centers for Disease Control, concerned over the quality of HIV-1 antibody testing, established a Model Performance Evaluation Program to assess laboratory reproducibility for HIV-I testing. The most recent study demonstrates continued performance problems evenin laboratories having tested
> 10,000 specimens. (17) Some
data are available from referral laboratories where a 3.7% discrepancy rate for 27 anti-HCV test results was recently found.(l8) A disturbing aspect of this study was that the discrepant results were explained as "imprecision at cutoff level." One referral laboratory was unable to repeat a very high (95.0 pg/mL) hepatitis B viral DNA level, and overall there was a 25% failure rate for HBV DNA testing.
The FDA-approved testing scheme, to repeat the testing
in duplicate for reactive samples only, may still permit infective units that are falsely negative in single (or even worse, in duplicate repeat) testing to be transfused. In addition,
136
SAZAMA
despite continuing efforts to improve specificity of screening methods, problems with false positive results remain. False Positive Results From the first approved test in March 1985 until
the present time, HIV testing has
identified donors whose test results are falsely positive, with original estimates of approximately 0.17% to 0.25% EIA repeat reactive samples obtained from more than
1
million American Red Cross donations.(l4,19) Even then, however, only 0.038% (n
=
1455) could be confirmed with the Western Blot, a more specific supplemental test.(20) When polymerase chain reaction (PCR) was initially applied for HIV detection, there was demonstrable poor sensitivity, specificity and reproducibility.(21) Five laboratories had participated in a proficiency trial that substantiated the need for standardization of procedures and for adequate use of controls to permit valid result interpretation. (22)
The results of
these studies, illustrating problems with possible endogenous human DNA sequences and
cross-contamination,(23) may explain the confusing results regarding predictability of seroconversion from time of infection. With improvements in reagents and better understanding of the disease process, the false positive (FP) rate has been decreasing. Interestingly, when the FDA mandated HIV-2 antibody testing for US blood donors in the summer of 1992, there was little opposition voiced by US blood bankers, even though data suggested that current HIV-1 methods were probably adequate to detect HIV-2 antibodie~(24)In the nearly two years that have lapsed since, over 20 million units of blood have been tested with not
one HIV-2 infected unit
detected.(25) However, the total number of false positive anti-HIV (both anti-HIV-l and -2) testing reports in blood donors has doubled, to nearly 5,000 annually. Cross-reactive epitopes against p24 (26) and gp 41 (27) antigens continued to contribute to the FP rate in blood donors.
HTLV-1/11 antibody testing was initiated in US blood banks in November 1988. Recently Busch et al. (28) evaluated 994 repeat reactive samples obtained from five different blood center donors between 11/88 and 12/91. There were only 3 false positives out of the 410 PCR- or repeat serologically-confirmed samples, for a FP rate of 0.73%, all occurring within the first 12 months of testing and attributable to overreading of one of the supplementalmethods(radioimmunoprecipitationassay or RIPA).Another 1.4% (6 of 425) indeterminate samples were PCR positive, suggesting that continued emphasis on improving non-PCR supplemental testing is desirable. The relatively new anti-HCV testing methods continue to evolve, with up to 50% of first generation EIA repeat reactive results reported to be false positive in low-risk
ECTIOUS E IN
137
TESTING
PROBLEMS
populations like volunteer blood donors.(29). Even these second-generation assays, while clearly an improvement in reducing post-transfusion HCV infection, (30) are plagued with continued unacceptable levelsof FP reactions,(3l) particularly those directed against the capsid protein, c22-3.(1) Additional supplemental testing using immunoblotting
and/or
polymerase chain reaction (PCR) methods is helpful in clarifying some results(32), particularly when donorlrecipient pairs are evaluated.(Table IV)(33,34) However,
the
presence of anti-c22 alone is apparently indicativeof false positivity in blood donors(35,36) while different interpretations appear true in high-risk persons.(37,38) A disturbing observation that HCV FP apparently occurred following administration of intravenous immunoglobulin (39) has l e d to changes in US manufacturing specifications.(40) Recently, some investigators have suggested using the signal to cutoff ratio of < 2 for EIA-l as indicative of FP reactions (41); others recommend removing cl00 antigen from HCV antibody assays to improve specificity without adversely affecting sensitivity. (Table V)(42-44) Recent US studies using the still-investigational EIA-3 method demonstrated its superiority for resolving 81% of 73 EIA-2 false positive (RIBA-2 negative) donor samples.(45) However,
as is also true for retroviral testing, PCR continues to be
problematic, with only 5 of 31 laboratories performing satisfactorily for all samples in a coded panel of 4 HCV-positive plasma samples, 6 HCV-negative samples and two dilution series of HCV-positive samples.(46) False NePative Results Part of the regulatory strategy when HIV testing began was to include all possible reactive samples, to ensure that no truly HIV-infected person who might donate blood would be missed and subsequently transmit AIDS. Consequently, the limit
for approval of the
testing methods was deliberately set to include a very high rate of false positivity, virtually eliminating the possibility of false negative (FN) reactions. Subsequent transmission of AIDS by transfusion (TAAIDS), when studied by the CDC, demonstrated that, in fact, such transmission only occurred when the donor was in the "window period" between acquisition
of the virus and development of detectable immune response.(47). Obviously, timing of sample collection is vital to avoid or minimize this reason for FN results.(48) Although
US
cases of TAAIDS have continued to occur, even after universal screening began in March 1985 (49-SI), no more than 5-24 casedyear have occurred following receipt of test-negative blood.(52,53). The debate over prolonged immunosilent infection for periods up to 45 months (54,55) appears in recent studies to favor appearance of antibody soon after infection, generally within 1(56) to six (57) months after PCR positivity can be detected. In a high-
138
SAZAMA TABLE IV Representative Patterns of HCV Reactivity in 64 Blood Donors
EIA-2
EIA-1 N
N f%)
E
Positive
47
0
0
47
17
0
17
0
Ind
Negative
39(83%) 7(15%) 1(2%) 5(29%) 4(24%) 8(47%)
TABLE V Sensitivity and Specificity of Recombinant Antigens Used in Current Assays to Detect Anti-HCV in 1,101 Donors Antiyen 5-1-1
Sensitivity
C 100-3
63
55 %
SDecificity 95 % 76
c33c
95
91
~22-3
97
69
prevalence area, no persistent silent HIV-1 infection has been found.(58) PCR testing
is
exquisitely sensitive to a variety of conditions, with FN to HIV reported due to Contamination by glove powder (59), inhibition by serum proteins, hemoglobin and certain anticoagulants (60,61), suboptimal storage conditionsand possibly insufficient volume of test sample. (23) While there have been relatively fewer reports of testing data for HTLV assays, Kleinman and associates reported on the inability to exclude HTLV-I1 infection in 3/46 recipients who were FN by EIA.(62) Even more ominous is the failure to identify 43% of HTLV-I1 infections using the currently available HTLV-I secreening methods.(63) For anti-HCV testing, both EIA and recombinant immunoblot assays (RIBA) as well as synthetic peptide neutralization methods,(64) have been improved several times during
the
past 4 years, with additions of recombinant antigens from non-structural region NS3 and from the core to the original single non-structural protein, NS4.(65) Although improvements have been found, problems with FN continue.(66) In Europe, the newest version of anti-
139
PROBLEMS IN INFECTIOUS DISEASE TESTING
HCV EIA, with an additional antigen from the NS5 region (coding for an RNA polymerase) and replacement of c22-3 and c100-3 with synthetic peptides [c-22(p) and c100(p)], was adopted in some regions in May 1993. With this third generation EIA, containing antigens from NS3,4,5 and the core region, increased sensitivity has permitted identification of at least one donor whose sample is falsely negative by prior versions of testing from at least two manufacturers.(4) Contrarily, when applied to screening 1,560 blood donors and 47 hemodialysis patients in Belgium, no such correlation was seen, rather confirmatory patterns were attributed to the specific elimination of false-positive c22-3 and c100-3 reactions(1) However, Japanese researchers recently detected post-transfusion hepatitis in 5/5 recipients including one in which the donor blood was reactive only for anti-c22-3.(67). HCV has been detected recently by PCR blood donors with complete non-reactivity to
in plasma from 17% (68) and 32% (69) of EIA and RIBA-2 methods.
The recognition of
at least three HCV variants raises new concerns over FN due to lack of antibody production to existing antigens in test kits.(70) However, PCR results can also be FN due to primer mismatching, apparently circumvented by using primers in the highly conserved coding region (71,72). Also, in addition to variations in both IgM and IgG
5' non-
response to
HCV,(73,74) the classical pattern (IgM preceding IgG production) does not occur.(75) Seroreversion With the advent of HIV in the US blood supply, concerns over the limits of detection of the human immunodeficiency virus (HIV) resulted
jn
unwarranted paranoia. Recent
studies in US army active-duty personnel demonstrate that once antibodyto HIV is detected, it is rarely lost.(6). In fact, of the six persons (out of nearly 2.6 million tested, of whom 4,911 were found to be repeat reactive on two separate occasions) who might have been seroreverters, four were found to be due to specimens collected in error from different persons, the fifth was a testing error, and the sixth never had a sample drawn.
ERRORS IN SAMPLE IDENTIFICATION Careful analysis of possible errors in blood banking, including errors in sample identification, have been made for more than 20 years by Taswell et al. at the Mayo Clinic,(76) with an observed overall rate of 27 to 29 errors per 10,oOO procedures. However, other studies in which viral disease detection was closely monitored in relatively small populations reported a much higher incidence of improper sample identification.
In
Coutlee's study (54), one patient of 79 (1.3%) tested for HIV by PCR was falsely identified as positive while 5 of 62 (8.1 %) possible HIV seroreverters among US Army personnel
SAZAMA
140
studied by Roy et al. (6) were sample identification problems. Sheppard et al.(22) found at least 2 of 138 (1.5%) seronegative samples to be falsely positive(by PCR and/or viral culture) when samples taken at different time intervals from the same subject were subjected to HLA analysis. These errors were attributed to problems in identification during prolonged storage. These rates of between 1.3 and 8.1 % errors in sample identification are troubling if they are indicative of such rates in blood donor populations. Using antibody fingerprinting techniques to establish the true identity of the person in serially obtained samples, Ascher and Roberts demonstrated that half (8115) of such possible "seroreverters" represented errors in sample identity.(77) Bar-coded sample identification
is not universally available, nor have
robotics been fully explored in this field of laboratory testing. Clearly greater efforts in applying continuous quality improvement measures should achieve significant benefitsin this vulnerable area of laboratory testing. EFFECTS OF PHYSICAL AGENTS ON VIRAL DISEASE TESTING
Few studies have been undertaken to evaluate the
effect of various physical
conditions, such as changes in temperature and the type of anticoagulant used to collect the test sample, on the results of viral disease testing. Our laboratory demonstrated complete
loss of reactivity by anti-HCV enzyme immunoassay in 6/316 (1.9%) serum samples subjected to a single freeze-thaw cycle.(78) Busch
et a1.(79), using PCR, cautioned that
well-controlled sample processingand storage conditions are critical. Cuypers and colleagues found that storage conditions of samples as well as primer selection affected their PCR yields.(80) Although Wang et al.'s results differed (81) insufficient data exist in the literature to unequivocally establish the reliability of the current practice of storing frozen serum samples for subsequent retesting. Concerns over the stability of HIV-l antibodies in various tropical conditions (82) might extend to include questions about the practice of some US facilities of sending plasma samples for testing in non-insulated containers across the country by regular air cargo shipment has never been the subject of a well-controlled prospective published study to determine the effects of several freeze-thaw cycles on results obtained from these samples. Studies using PCR raise questions about the interchangeability of various anticoagulants in the collection of samples for testing.(60,61) PERFORMANCE AND CALCULATION ERRORS
By written request to the FDA's Freedom of Information Act Office, anyone
can
obtain copies of recent inspection reports (FDA Form 483) citing problems with performance
PROBLEMS IN INFECTIOUS DlSEASE TESTING
141
TABLE VI Overall Error Rate by Year
Year
Errors/10,000 Procedures
1982
28
Year 1988
1983 1984
Errors/lO,rn Procedures 20 24
29
22
1990
1985
20
1991
28
1986
28
1992
27
1987
of and correctly calculating the results from viral marker testing by blood collecting organizations. Recent citations included failure to properly respond to low control values, failure to pipette correct volumes of reagents, and failure to change pipette tips.(83) Particularly for the hepatitis B testing in which several methods of calculation are possible, errors in such calculations have caused centers to close their testing laboratories and send samples across the country for testing. The likelihood and frequency of such errors has been the subject of ongoing review at the Mayo Clinic (84) for a number of years. While Taswell’s observations of the various functions within blood collecting and transfusing activities do not specifically address calculation errors, he has detailed the various opportunities and frequencies of errors, finding a remarkably stable 1 in 500 error rate, first reported in 1973 and remaining reasonably consistent at 28/10,000 in 1993. (Table
VI)
There is little doubt that introduction and increasing dependenceon automation and robotics will improve this rate while no human being can.
CONCLUSIONS While it is indisputable that testing blood for transfusion to exclude infectious disease is vital to enhance safety, as long as standard methods rely on detection of antibody, the unpredictability of human response to infections will remain a confounding variable.
In IOW
incidence populations suchas blood donors, the use of increasingly sensitive techniques may become prohibitively costly due to increased frequency of fake positivity. However, efforts to eliminate cross-reactivi~and to improve specificity and sensitivity of existing methods will likely continue.
142
SAZAMA Further automation of basic testing techniques with bar code sample identification
systems and robotics for aliquoting can further reduce possibilities for human error, particularly when properly controlled and coupled with a fully validated information system. Innovations such as PCR detection technology and the molecular cloning of the hepatitis C virus continue to stimulate improvements in disease detection and elimination. Studies should be undertaken to elucidate the effect of various physical conditions such as repeated freezing and thawing of samples, the various anticoagulants and other possible variables that affect the sample itself. Closer attention to personnel competency and performance is included in current US regulatory requirements for clinical laboratories, including those performing tests on donated blood for transfusion. In addition to the variables presented by individual immune response to infection and of various manufacturers in their tests to detect such antibodies, the predictability of human performance error in sample handling, testing and interpretations makes the goal of zero defects, while highly desirable, virtually unattainable. We can only continue to try. REFERENCES 1. S. Uyttendaele, H. Claeys, W. Mertens, H. Verhaert and C. Vermylen. Vox Sang 6 6 , 122-129 (1994). 2. M.J.Alter.
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TESTING BLOOD DONORS FOR HIV: CURRENT
CONTROVERSIES
M. P. Busch Department of Laboratory Medicine University of California, San Francisco and Irwin Memorial Blood Centers 2 7 0 Masonic Avenue San Francisco, California 94118
ABSTRACT Despite an estimated riskof HIV infection from anti-HIV screened blood transfusions of less than one in 2 2 5 , 0 0 0 per unit, there continues to be strong pressure to implement additional donor screening and viral inactivation procedures. Decisions to implement such procedures must be based on analyses that incorporate accurate estimates of residual risk, and data-based projections for the reduction in risk that would result is from eachmeasure. Since the residual risk of HIV primarily due to donations given in the infectious pre-seroconversionwindow, effort must be directed at: reducing donationsby persons inthis window; employing tests that narrowthe window; and development and implementation of procedures that inactivate viral compartments that predominate the during window. Unfortunately, as the risk of HIV has declined to nearundetectable levels,the challenge of generating appropriate data to evaluate new measures, and thereby support rational policy decisions, has increased inversely. To meet this challenge,we must refine our understanding ofthe virological characteristics of early HIV seroconversion, and of the types of donors who present in the seroconversion window. Thoughtful applicationof a thorough understanding of the seroconversion window, inthe context of accurate HIV incidence data in the donor settings, should enable us to assure the public of a safe blood supply while resisting inappropriate implementation of unnecessary and usually non-specific procedures. INTRODUCTION The discovery of the human immunodeficiency virus (HIV) and rapid development of sensitive enzyme immunoassays (EIAs) for screening the blood supply represent a great success of modern medical science. Compared to the slow pace of progress in most other areas of clinical
A modified version of this paper was presented the at 1994 International Society of Blood Transfusion, in Amsterdam, July 1994, with publication as a supplementto Vox Sanguinis.
147
AIDS
BUSCH research, one would think that the remarkable improvements in the safety of blood transfusions achieved over the past decade would be touted as a job well done an area with little need for further improvement or concern, and certainly not one warranting significant allocation of scarce public health resources. Yet, the political and media focus on HIV infection from blood transfusion remains intense. There are strong pressures to doeverything conceivable to further reduce the risk of acquiring HIV from transfusions. In response, behavioral scientists have proposed new donor selection and screening procedures, reagent manufacturers have developed new tests for narrowing the seronegative window period following infection, and development continues on viral inactivation procedures, particularly for cellular blood components. Unfortunately, development of sound approaches for evaluating the need for, and utility of, proposed new measures has not kept pace with the cries for action or the advances in technology. Ironically, part of the problem is that the current risk of HIV infection from contemporary, screened blood transfusions in developed countries is so low that documentation of the residual risk is exceedingly difficult. In fact, all of the currently published estimates (Table I)are based on data from the
-
late 1980s (1-9). To my knowledge, at the present time there are no prospective studies of the riskof HIV from screened transfusions eitherongoing or planned in any country, with the possible exception of Thailand. Without being able to monitor the residual risk directly, how are we to evaluate the efficacy of proposed new procedures to reduce risk? And just as important, how can we determine if measures implemented in some countriesearly in the epidemic, such as permanent deferral of certain risk groups (e.g., men), confidential unit exclusion (CUE) procedures, or surrogate
gay
"lifestyle" markers (e.g. syphilis or antibody to hepatitis B core protein [anti-HBc] tests) warrant retention. In response to thisdilemma, there has been a tendency to use "surrogate endpoints" to project the utility of proposed new procedures or the residual value of old ones (8). For example, in the area of viral inactivation, approaches are being developed and evaluated using either model viruses studied in in vitro systems or in animal models, or studies in which HIV (or other agents)has been spiked into blood components (10). Few studies have investigated naturally infected blood from seropositive donors, and none have focused on the most relevant target, i.e., blood collected from donors during the seronegative window phase of infection. In employing model systems to evaluate approaches for viral inactivation, investigators have sought to recreate conditions believed to exist in naturally infected blood. Although one might presume that the distributions in blood of major human pathogens such as HIV would have been well characterized by now, relatively little data is available on the kinetics or compartmentalization of viremia during primary infection (11) the stage most relevant to viral inactivation studies. Another example is the widespread misuse of HIV prevalence rates as surrogates for incidence rates. It is frequently argued that higher rates
-
149
TESTING BLOOD DONORS FOR HIV TABLE I Estimates of Risk of HIV-1 Infection from Anti-HIV-l-Screened Blood Transfusions. Risk of HIV-1 Infection TYDe Of Study Time Period Reqion Statistical Models: 1985 USA USA 3/85 - 2/87 USA 2/81 3/85 Lookback Models:
Der Unit Transfused
-
11 in 99,000 1 in 38,000 1 in 153,000
-
12/86 L o s Angeles CA 1 in 68,000 1/91 USA 1 in 225,000 Prospective Recipient Seroconversion: 12/89 Baltimore MD 4/85 Houston TX 1 in 60,000 6 3/05 1/88
-
Reference
2 3
4 5
-
ProsDective Donor Cell CultureIPCR: 11/87
-
12/89
San Francisco CA
1 in 160,000
7 (updated)
of confirmed seropositivity among demographically-defined subgroups of donors, or among donors who have tested "positive" on a surrogatetest (e.g., anti-HBc, syphilis serology, or CUE) is adequate evidence for exclusion of this subgroup or retention of that test (8). This approach
is specious, however, since test-positive units are already detected and discarded. We must remember that the risk of HIV from blood transfusions is almost solely attributable to donations given during the windowphase between initial infection and EIA-detectable seroconversion. To reduce risk, we must: reduce donations by persons in the infectious seronegative window; employ tests that narrow this window; and implement procedures of proven efficacy for inactivation of viral compartments and titers that exist during this window phase. The challenge, therefore, lies in expanding our understanding of the temporal, virological, and serological characteristics of the window. Current Understanding of the HIV Seroconversion Window: Our understanding of the antibody negative window phase of HIV infection, and of the relativesensitivities of viral and antibody assays for detection of evolving primary HIV-1 infections, has been limited by a lack of optimal specimens for characterizing this dynamic phaseof infection. Current information comes primarily from three sources: seroconverting plasma donors; patients with symptomatic primary HIV infections (the so-called acute HIV syndrome); and seroconvertors in highrisk cohort studies. Analyses of closely spaced serial bleeds from paid plasmapheresis donors later determined to have seroconverted, have proven very useful for developing new serological assays and for documenting
BUSCH their enhanced sensitivity (12-14). However, because cryopreserved leukocytes are not available from these donors, these seroconversion panels do not allow assessment of DNA detection techniques. Studies of cell and plasma samples from persons presenting with the acuteHIV-1 syndrome have enabled detailed characterization of virological and immunological events coinciding with early seroconversion (11,15-17). However, because symptoms first appear only days to a week prior to seroconversion, these data are restricted to the later stages of viral dissemination. Moreover, only 20-50% of infected persons manifest symptomatic seroconversions, and results from these subjects may be biased in that symptomatic cases appear to have higher concentrations of virus during the dissemination phase compared to asymptomatic seroconvertors (18). Analyses of preseroconversion samples from subjects who seroconverted in prospective cohort studies avoids several of these limitations, since cryopreserved peripheral blood mononuclear cell (PBMC) as well as serum and plasma specimens are available, and asymptomatic seroconvertors are represented. Several cohort studies have reported early detection of infection in 7% to 16% of seroconvertors using direct viral assays (i.e., p24 antigen in plasma and HIV DNA in PBMC) (12,19-22). However, the numbers of seroconvertors observed in individual cohort studies are limited and the sampling intervals long (3 to6 months), precluding detailed characterization of the reductionin the window achieved by these assays. We recently completed two studies which together define the duration and virologic progression of the seronegative window phase of HIV infection to a level of precision not previously possible. The first study was designed to determine the overall duration of the infectious window. In a collaborative study sponsored by the U.S. Centers for Disease Control and Prevention (CDC), previous recipients of 701 blood donors who seroconverted to HIV positivity prior to 1991 were traced (23). of pre-seroconversion donations were identified and One or more recipients tested for 179 seroconverting donors. The most recent seronegative donation resulted in infection in 36 (20%) of the cases. The recipient infection rate correlated with the time interval between the seropositive and seronegative donations (e.g., interval c90 days, 76%; 91-180 days, 28%; >l80 days, 11%). Mathematical modeling of these data indicated a median 45-day infectious window period (95% CI: 34-55 days) for the overall period, 1985-1990. Separate analysis of donations given priorand subsequent to March, 1987 (when improved sensitivity EIAs were implemented in US donor centers) indicated a reduction in the median infectious seronegative window of from 55 to 42 days. In a second study (24) designed to characterize theearly virologic and serologic events preceding asymptomatic SC, we analysed pre-SC specimens from 81 SCs observed in 3 high-risk cohort studies (19-21). PCR investigation of cryopreserved pre-SC PBMC identified 13/81 SCs in whom the sample collected on the study visit prior to seroconversion wasHIV DNA-PCR-positive. These 13 DNA-PCR-positive pre-SC samples were tested with 10 2nd- and 3rd-generation anti-HIV EIAs, 6 supplemental anti-HIV
TESTING BLOOD DONORS FOR HIV
151
assays, p24 Ag EIA, and immunocapture RT-PCR. Of the 13 lst-generationEIA-negative, DNA-PCR-positive specimens, 4 were 2nd-generation-EIAEight of the 13 samples were positive and 9 3rd-generation-EIA-positive. p24 Ag-positive, including all 4 3rd-generation-EIA-negative samples. HIV RNA was detected in 11 of the 13 DNA PCR-positive sera (including all 4 3rd-generation EIA-negative sera) aswell as in 1 DNA-PCR-negative pre-SC this sample wasnegative by p24 antigen and 3rd generation antisample HIV EIAs. Based on a model in which these data were analyzed relative to the inter-bleed intervals for all 81 SC, we estimated the following reductions in the window period (relative to anti-HIV E I A s employed in
-
1989-90): 3rd generation anti-HIV-l/HIV-Z EIAs, -19.7 days (95% confidence interval (CI]: 7.7-31.6 days); DNA PCR and p24 antigen, -25.6 days (95% CI: 12.6-38.7); RNA PCR, -28.1 days (95% CI: 14.5-41.7). Quantitative DNA and RNA PCR were also performed on serial pre- and post-SC samples (18.25). These studies documented a peak of high-titer plasma viremia, as well as virus bound to platelets, preceding antibody seroconversion; virus declined 2-4 logs post-SC. In contrast, concentrations of infected PBMC were low and stable following initial detection. These results indicate that virus disseminates days to weeks following exposure (26) from a focus of primary infection in regional lymphoid tissue (ll), and that this dissemination is primarily as cell-free (and platelet-associated) virions rather than as infected PBMC. Figure 1 summarizes our current model of the kineticsof viral dissemination and seroconversion. Applications of Seroconversion Data to P o l i c y Decisions To illustrate how an improved understanding of seroconversion is critical for addressing policy decisions in donor screening, let's consider the issue of adding direct viral detection assays (e.g., p24 antigen or PCR) to routine donor screening. By combining estimates of the duration of the infectious seronegative window with estimates of the incidence of seroconversions among donors, we can calculate the probability of donors presenting in the infectious window (i.e., the residual risk), as well as the incremental reduction of that risk which additional measures would achieve (8,27). For example, we estimated that p24 antigen testing would reduce theresidual infectious window by approximately 5 days (relativeto 3rd-generation anti-HIV-l/HIV-2 EIAs), an estimate thatis consistent with recent results from several other groups based on analysis of seroconverting plasma donors (13,14). Based on this window reduction and the estimated incidence of HIV seroconversions in the donor setting (50 seroconversions per lo6 donor years) (27), we projected a detection rate of 0.7 antigen-positive, antiHIV-negative units per million donations (5-10 units out of 12 million Such a projection is consistent with donations per year in theU . S . ) . studies showing lack of utility of HIV-antigen screening in low incidence donor populations (28,29), while in high incidence settings up to1% of infected persons are detected only by the antigen test (30-32). We can also estimatethat introducing this test would result in reduction of the residual infectious window by 1/4 (from approximately 20 days to 15 days),
152
9
P cu n
L' I
BUSCH
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TESTING BLOOD DONORS FOR HIV thereby reducing the residual risk of HIV infection from transfusions proportionately. Although PCR tests (andparticularly RT-PCR for cellfree RNA) could narrow thewindow to a slightly greater extent, these assays are not yet practical for large scale screening. CONCLUSIONS Decisions to implement or discontinue tests or proceduresin blood banking should be based on cost-benefit analyses that incorporate accurate estimates of residual risk for each agent, and accurate projectionsfor the reduction or increase in risk that would result from each measure. As the riskof HIV from transfusions hasdeclined to near undetectable levels, there has beenan inverse increase in the challengeof generating relevant data to support rational decisions. To meet this challenge, we must continue to enhance ourunderstanding of theseroconversion window and seroconverting donors, for therein lies the risk. REFERENCES 1.
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Schorr, and R.Y.
Transfusion,
Dodd.
N Engl J
20, 499-501 (1988).
Donahue, and A. Munoz, et al.
Annnals Int Med,
1 1 7 , 554-559 (1992). 7.
M.P.
Busch, B.E. Eble, and H. Khayam-Bashi, et al.
N Engl J Med,
3 2 5 , 1-5 (1991).
8.
M.P. Busch, in Blood Safetv: Current Challenaes, S.J. Nance, ed, Amer Assn of Blood Banks, Bethesda MD, (1992) pp. 1-44.
9.
R.Y.
Dodd.
N Engl J Med, 3 2 7 , 419-421 (1992).
5, 18-
10.
S. J. Wagner, L.I. 1991).
11.
A.S.
12.
M.T. Niu, D.S. Stein, and S.M. Schnmittman. 1490-1501 (1993).
13.
H.L. Zaaijer, P. v Exel-Oehlers, T. Kraaijeveld, E. Altena, and P.N. Lelie. Lancet, 3 4 0 , 7 7 0 - 7 7 2 (1992).
14.
J.L.
30,
Fauci.
Friedman, and R.Y.
Dodd.
Transfus Med Rev,
Science, 2 6 2 , 1011-1018, (1993). J Inf Disease,
Gallarda, D.R. Henrard, and D. Liu, et al. 2379-2384 (1992).
15.
S.J. Clark, M.S. Saag, and W.D. 954-960 (1991).
16.
E.S. Daar, T. Moudgil, R.D. 961-964 (1991).
Decker, et al.
Meyer, and D.D. Ho.
168,
J Clin Microbiol, N Eng J Med, N Eng J Med,
324, 324,
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M.T.L. Roos, J.M.A. Lange, and R.E.Y. Dis, 165, 427-432 (1992).
18.
D.R. Henrard, E. Daar, and H. Farzadegan, et al. 1994 (submitted).
19.
S. Read, S. Cassol, and R. Coates, et al. (1992).
20.
de Goede, et al.
J Infect
J Infect Dis,
J AIDS,
H. Farzadegan, D. Vlahov, and L. Solomon, et al. 327-331 (1993).
5, 1075-1079, J Infect Dis,
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H.W. Sheppard, M.P. Busch, P.H. J AIDS, 6, 1339-1346 (1993).
22.
A.R. Lifson, M. Stanley, and J. Pane J, et al. 436-439 (1990).
23.
L.R. Petersen, G.A. 283-289 (1994).
24.
M.P. Busch, L.L.L. 1994).
25.
T-H. Lee, H.W. (1994).
26.
C.R. Horsburgh Jr, C.Y. 640 (1989).
27.
L. Petersen, M. Busch, G. Satten, R. Dodd, and D. Henrard.
28.
Louie, R. Madej, and G.C.
Satten, and R.Y.
J Infect Dis, 1 61,
Dodd, et al.
Lee, and G. Satten, et al.
Sheppard, and M. Reis, et al.
Rodgers.
Transfusion,
3,
( m 5 in preparation,
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Ou, and J. Jason, et al.
1, 381-388
Lancet,
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Abstract #PO-C17-3001, in IX Intl Conf on AIDS/IV STD World Conuress, V01 11, Berlin, (1993) p. 717. H.J.
323,
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M.P. Busch, C. Stevens, and B.A. 1308-1312 (1990).
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P. Chiewsilp, P. Isarangkura, and A. Poonkasem, et al. 1341 (1991).
31.
C. Nuchprayoon, S. Tanpraset, and S. Thanomchat. to XXIII Conaress, ISBT, Amsterdam, (1994).
32.
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Lancet, 3 3 8 ,
Abstract submitted
HEPATITIS TESTING Celso Bianco New York Blood Center New York, New York 10021
ABSTRACT There has been remarkable progress in hepatitis testingin recent years. This article reviews the transmission of Hepatitis A by blood products indicating that it is a rare event. However, it has been recently observed with certain preparations of Factor VI11 concentrate. Hepatitis B transmission has been effectively reduced by the application of HBsAg screening tests, and screening for antibodies to HBcAb. Hepatitis C transmission has been substantially reduced by the used of the second generation tests for antibodies to HCV. A new generation of screening tests for HCV is expected to be licensed for use in the United States in the near future, and should further reduce the risk of transmission of HCV. At the present time, the incidence of the incidence of post-transfusionhepatitis in hospitalizedpatientsisindistinguishablefrom hepatitis among patients who did not receive blood transfusion. INTRODUCTION Prevention of hepatitistransmission is one of themajorsuccess
stories inTransfusion
Medicine. More than 25% of patients who received multiple transfusions in the 60’s developed post-transfusion hepatitis . Today, the incidence of post-transfusion hepatitisis so low that it is barely distinguishable from the incidence of hepatitis among hospitalized patients who did not receive blood transfusion (1). This remarkable decrease resulted from both administrative and scientific developments that took place in the past30 years.
The first major factor was the change in the population of blood donors. In the W s , most of the blood was collected from paid donors who depended on the donation for some of their basic needs. Paid donors lived in poverty and were in poor health. A number of visionaries at the timerecognized the seriousnessof the problem,andcreatedcommunity,regionaland national blood centers which relied entirely on volunteer blood donors, changing the character
of the blood donations. The volunteer blood donor population has a much lower prevalence of infectious disease markers than the general population. The identification of the Hepatitis BVirus (HBV) allowed the development and introduction of blood donor screening assaysfor Hepatitis B surface antigen (HBsAg) in the late 1960’s and 155
BIANCO
156
early 70's. The format and sensitivityof these tests were greatly improved withthe introduction of third generation screening tests able to detect less than 1 ng of HBsAg in the late 70's and early 80's. Unfortunately, the introduction of HBsAg screening did not eliminate hepatitis B transmission bytransfusion.While
the incidence of overt clinicalhepatitis
with jaundice declined,a
substantial numberof blood recipients continuedto develop transaminase elevation and evidence
of liver damage by infectious agents,an entity classifiedas Non-A, Non-B Hepatitis becauseof the lack of markers
for the twoknown hepatitis virus at the time (2). A major study, the
Transfusion Transmitted Viruses was carried out
in the late 70's (3). This prospective study
showed that between 5 and 10% of recipients of multiple transfusions developed elevationsof alanine aminotransferase (ALT) in the weeks following the transfusion event.
The study also
showed correlation between ALT elevation in blood donors, and or the presence of antibodies to the core antigen of HBV (HBcAb), with transmission of
the Non-A, Non-B agent. These
findings were confirmed by otherstudies (4), andled
to the creation of the concept of
"surrogate" tests. Essentially, these studies predicted that screening of blood donors for elevation of ALT or for HBcAb could reduce the transmission of the Non-A, Non-B agent by about a third. A few blood centers introduced ALT screening in 1982. By 1987, all blood donors in the United States were being screened for both surrogate markers of Non-A, Non-B hepatitis. Continuous research efforts were applied to the identification of the elusive Non-A, Non-B agent in the 70's and early 80's. These efforts culminated with the identification and cloning of sequences of the Hepatitis C Virus followed by the development of the first antibody assays. There were moments of great excitement when testing of specimen panels from the NIH study in prototypescreeningassays
showedhigh degree of correlationbetweenantibodies
to the
recombinant viral proteinbased on the cloned sequences and the transmission of Non-A, Non-B hepatitis (5). Blood donor screeningfor antibodies toHCV using first generation screening tests wasimplementedin
1990. A secondgenerationassay,includingadditionalviralproteins
generated by DNA recombinant technology were introduced in 1992(6).Third generation assays with improved sensitivity havebeen in use in Europe since early 1993. FDA licensure of these tests for use in the US is imminent. Other factors, not directly related
to assay technology, also contributed to the decline of
hepatitis transmission by transfusion, particularly the better understanding of the conceptrisk of behavior by prospective blood donors, and the criteria for the acceptability of blood donations which were enforced after the discovery that AIDS could be transmitted by blood transfusion. Figure 1 shows estimates of the decline in the incidence of post-transfusion hepatitis which followed the introduction of donor selection procedures and blood donor screening assays. The following sections summarize essential aspects of the major hepatitis viruses associated with transfusion, the screening assaysfor their detection,and current proceduresfor the handling of donors with reactive test results.
HEPATITIS
TESTING157 HEPATITIS A VIRUS (HAV)
HAV is not usually listed as a virus transmissibleby blood transfusion. It is almost always transmitted by the oral-fecal route (7). However, rare cases of transmission have been reported among recipients of blood from donors who were in the incubation period and among IV drug users. Infectious virus particlesmay be found in serum for up to three weeks after exposure to Hepatitis A. HAV is a single stranded RNA virus from the Picornaviridae family (8). It does not have alipidenvelope,andconsequently
is notinactivatedby
current solvent-detergent viral
inactivation procedures for plasma and plasma derived proteins. Rare incidents of Hepatitis A transmission byFactor VI11 concentrate have been reported and this issue was recently reviewed.
(9). Transmission is rare because over 20% of the population has been exposed to the virus and carries protective antibodieswhich neutralize viruswhich may have been potentially introduced into the production plasma pools. Most of the hepatitis infections occur during the childhood yearsare due to HAV, and this is the reason why FDA
Guidelines allow blood donations from individuals with a history of
Hepatitis prior to the age of 11. The infection produced by HAV is self-limiting, neutralizing antibodies appear within 4 weeks of infection, and immunity is permanent. There is no carrier state following HAV infection. Intramuscular immunoglobulins are highly protective and are recommended for travelers to high prevalence areas. FDA licensureof a highly effective HAV vaccine is expected in the near future. HEPATITIS B VIRUS Hepatitis B is a double stranded DNA virus of the Hepadnaviridiae family and has
the
smallest genome of a virus known to infect man (10). It has a lipid envelope, and is highly susceptible to inactivationby the solvent-detergentprocedure currently appliedtoplasma derivatives. HBV virions found in serum (Dane particles) containa surface protein codedby the S and pre-S genes, named surface antigen or HBsAg, and a nucleocapsid polypeptide coded by
the C gene, which is called the "core" antigen. Assays designed for blood donor screening are based on the capture of HBsAg by specific antibodies. HBsAg is detectable during the acute stage of infection, and in the chronic or carrier stage. It disappears when the acute infection is resolved, concomitantlywith the appearanceof antibodies to thesurface antigen (HBsAb). These antibodies are highly protective, and denote immunity. HBV was called "serum" hepatitis becauseof its characteristic parenteral transmission.The virus is also transmitted perinatally, and by sexual contact. The illness typically develops between 12 and 18 weeks after exposure. About 5-10% of infected individuals have persistent infection and about 1% become asymptomaticcarriers, remaining negativefor HBsAb.
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The prevalence of carriers isveryhighwhen
the infection occurs perinatally, and this is a
serious public health issue in underdeveloped areas of the world. Antibodiesthe tocore of HBV (HBcAb) appear about8 weeks after infection and remain detectablefor many years. About 70% of individuals who are positive for HBcAb are also positive for HBsAb. The prevalence of HBsAg positive individuals among blood donors in New York
is 0.04%. The prevalence of
HBcAb is 1.5%. Use of HBcAb for the screening of blood donors was initiated in 1986-87, ostensibly to reduce further the transmission ofHBV by transfusion andas a surrogate test for Non-A, Non-N hepatitis. Some individuals believe thatHBsAb is also a surrogate test for HIV, because of the relatively high prevalence ofHBV infection among certain groupsat risk of the exposure to the virus includingIV drug users, male homosexuals, patients with hereditary deficiencies of clotting factors etc. Consideringthe current availability of highly sensitive assays for HIV and all other blood donor screening stepsin effect, there is no clear value in the use of HBcAb as a surrogate test for HIV infection. However,HBcAb may be useful for the prevention of HBV transmission in certain rare cases of HBV mutants withaltered HBsAg sequences (11). Countries with a high prevalence of HBV infection have chosen to accept donors who are negative for HBsAg, and have high titers of HBcAb and HBsAb
. These donors do not transmit HBV to their recipients.
Blood donors are screened for HBsAgbyELISA
typeassays.Donorswithrepeatedly
reactive results are subjected to confirmation by a neutralization assay.
The donated unit is
discarded. Individualsare considered positivewhen reactivity is inhibited by specific antibodies to HBsAg. According to FDA Guidelines, individuals with confirmed positive HBsAg test results are permanently deferred from donating blood. Individuals repeatedly reactive on the screening test can be re-entered and may continue to donate blood. However, they must be permanently deferred if reactive for HBcAb in the same or in a subsequent donation. Otherassays
for HBVbasedon
genetictechnology
are available,including
hybridizationandamplification by the polymerasechainreaction.Theyappearto
DNA be more
sensitive that HBsAg. However, because of their complexity, they application for blood donor screening awaits further developments. HEPATITIS D VIRUS (HDV, Delta) HDV is a defective viruswhich requires HBV for its transmission, and causes a distinct form of clinical liver disease, more serious than that caused by HBV.
It is a single stranded RNA
satellite virus. While thereare reports of transmission by transfusion,it is extremely rare in the
are effective US. Since it depends on HBV, methods of inactivation of plasma derivatives which against HBV are likely to be effective against HDV.
HEPATITIS TESTING
159
NON-A, NON-B HEPATITIS, HEPATITIS C (HCV) As previously mentioned, the developmentof screening assays for HCV had major impact on the safety of the blood supply. These tests also made great contributions tothe understanding of the epidemiology of the virus and the clinical impact of
the infection. A recent review of
serologic testing for HCV has been published (14). Acute HCV infection is usually mild and non-specific. Most cases are suspected because of routine determinationof transaminase levels. Unfortunately, 50-75% of the cases of HCV infection progress tochronicity, presenting chronic persistent hepatitis or chronic active hepatitis. About 20% of the HCV infected patients with chronic hepatitis may develop liver cirrhosis. There is association between HCV infection and hepatocellular carcinoma. Recent re-evaluation of patients infected for long periods of time have shown increased morbidity, without apparent increase in mortality (15). There appears to be substantial difference in the clinical course and incidence of complications of HCV infection in different parts of the world. More recently, these differences have been attributed to different viral subtypes (1 6). The first generation assays for antibodies to HCV were based on the recombinant protein c100-3, representing a small portion
of the NS3regionofthevirus.
The windowbetween
infection and appearance of antibodies was on average 6 months and could extend up to one year. Insomecasesitappearedthatantibodiesdisappeared individuals. The estimatedsensitivity ofthesetestswas sequencingof
the entire virus,andtothedevelopmentof
in some chronicallyinfected
80%. Intensive efforts led to the other recombinantproteins
corresponding to HCV sequences. In early 1992 multiantigen second generation HCV screening tests were licensed by FDA. The difference in sensitivity between first and second generation tests was such that blood banking organizations made an overnight transition to the new tests. The sensitivity of the second generation tests exceeds90%. HCV 2.0 detects antibodies toHCV 4-6 weeks after infection and, differently from
first generation tests, these antibodies remain
detectable for several years after infection. At the presenttime,
only one supplementalassayhasbeenlicensedbytheFDA
for
confirmation of repeatedly reactive resultson screening tests. The RIBA test is a nitrocellulose strip to which each of the recombinant proteins used in the screening test was immobilized in separatebands(c22-3,c33c,c100-3).Inaddition,thereisabandcontainingsuperoxide dismutase to detect non-specific antibodies against the fusion protein of the recombinant antigens, and low and high level IgG controls. Reactivity against two bands indicates a positive test result. In our experience, approximately55%of the blood donor samples whichare repeatedly reactive on EIA screening are positive on RIBA. Donors who have repeatedlyreactive results on ELISA screening must be indefinitely deferred. FDA Guidelines allow the reentry of donors who were reactive once on the second generation multiantigen test, and are negative on EIA screening and on RIBA on a specimen collected at least six moths after the repeatedly reactive results. This
160
BIANCO
interval allows sufficient time for seroconversion of individuals whoare truly infected by HCV. The HCV assay had majorimpact on thetransmissionofHCVbytransfusion,andrecent prospective studies indicate that it became a very rare event (1) Third generation assaysfor HCV are already in use in several European countries and their licensure in the US is imminent. These assays contain clear improvements in the sensitivity for antibodies to c33c which appear to be important in the detection of early seroconversions. The assays also contain recombinants corresponding to the NS5 region of the virus. Unfortunately, NS5 does notappear to contribute substantially to the sensitivity or specificity of these tests. The RIBA 3.0 assay has also been improved in terms of sensitivity and specificity, but licensed in the US. Other supplemental assays
is not yet
are available in Europe, including the Matrix
HCV and the Murex. These assays have been recently compared (16). Assays based on genetic technology have become very important inthe diagnosis of HCV, particularlyamplification by the polymerasechainreactionusing sequences of the 5'non-coding region of the virus.
primers corresponding to
These assays are still in the development
stage, and are not yet suited for blood donor screening (17). HCV is a highly susceptible to mutations, particularly in
its envelope region. In addition,
many different subtypes of HCV have been identified. There is also evidence that quasi species of the virus coexist ininfected individuals (18). These changes in viral structure mayhave implications in chronicity, seriousness of diseaseand susceptibility to treatmentwith interferon alpha have also been identified, and may become important in terms of donor follow up. The sensitivity of currently available screening assays does not appear tobe substantially affectedby these changes in viral sequences. CONCLUSION In conclusion,riskofpost-transfusionhepatitishasbeensubstantiallyreducedfrom
the
extremely high levels observed in the 60's and ~ O ' S , to the remarkable low levels observed today. The risk of transmission of HBV by
transfusion has been estimated at 1:200,000 (19).
The risk of transmission of HCV by transfusion was estimated at 0.06% per unit transfused (14). This remarkable reduction was the result of substantial progressthe in understanding of hepatitis viruses and hepatitis epidemiology, the introduction of a number of effective
donor selection
procedures, and the development of new and improved donor screening tests. REFERENCES 1. H.J. Alter, in Viral Hemtitis and Liver Disease, K. Nishioka, H. Susuki, S. Mshiro and T. Oda, eds, Springer-Verlag, Tokyo, (1994) pp. 551-553.
2. A.M. Prince, B. Brotman,
C.F. Grady et al. Lancet 2241-246 (1974).
HEPATITIS
TESTING161
3. Lemon, SM. Type A viral hepatitis: New developments in an old disease. N Engl J Med m 1 0 5 9 (1985). 4. R. D. Aach, W Szmuness, JW Mosley et al. N Engl J Med 304.989-994 (1981) 5. HJ Alter, RH Purcell, SM Feinstone, PV Holland et al. in Viral HeDatitis, GM Vyas, SN Cohen, R Schmid eds, Franklin Institute Press, Philadelphia, (1978) pp. 383-396.
6. HJ Alter, RH Purcell, JW Shih et al. N Engl J Med 321:1494-1500 (1989). 7. RD Aach, C.S. Stevens, F B Hollinger et al. N Engl J Med 325, 1325-1329 (1991). 8. SM Lemon and S P Day.in Infectious Diseases, SL Gorbach,JGBartlett Blacklow eds, WB Saunders Philadelphia, (1992) 705-709.
and N
9. Melnick JL and Howard, CR. Classification and Taxonomy of Hepatitis Viruses: Summary of a Workshop. in Viral Hemtitis and Liver Disease,K. Nshioka, H. Susuki, S. Mishiro and T. Oda, eds, Springer-Verlag, Tokyo, (1994) pp 47-49. 10. Hepatitis A Virus Transmission by Blood Products. Prowse, E, Follet E and Prince, A, eds. Vox Sang Q Suppl, 1-85 (1994). 11. Miller, RH, Kaneko S, Chung CT et al. Compact Organization of the Hepatitis B genome. Hepatology 9,322-327 (1989). 12. Esteban, JL, Camps J, Genesca J and Alter H. Hepatitis C and B New developments, in Blood Safety: Current Challenges, ed. Nance, S. American Association of Blood Banks. Bethesda, MD. (1992) pp 45-96. 13. Japanese Red Cross non-A, non-B Research Group. Effects of screening for hepatitis
C virus antibody and hepatitis B core antibody on the incidence of post-transfusion
hepatitis. Lancet: 338,1040-1 (1991). 14. J L Gerin, JL Casey and K F Bergmann, in Viral Heoatits and Liver Disease K. Nishioka, H. Susuki, S. Mishiro and T. Oda, eds, Springer-Verlag, Tokyo, (1994) pp 38-41. 15. MJ Alter, Arch Pathol Lab Med 118,342-345 (1994). 16. LB Seef, Z Buskell-Bales, E C Wright et al. N Engl J Med 327, 1906-1911 (1992). 17. H L Zaaijer, H Vrielink, PJ van Exel-Ohelem, et al. Transfusion 304, 603-607 (1994). 18. M Rios, M Duran, M Hempstead et al., in Viral Hemtitis and Liver Disease.K. Nishioka, H. Susuki, S. Mishiro and T. Oda, eds, Springer-Verlag, Tokyo, (1994) pp. 569573. 19. G Dusheiko, H Schmilovitz-Weiss, D Brown et al., in Viral Hemtitis andLiver Disease, Nishioka, H. Susuki, S. Mishiro and T. Oda, eds, Springer-Verlag, Tokyo, (1994) pp. 301305. 20. R.Y. Dodd. N Engl J Med 327,419-420 (1992).
This Page Intentionally Left Blank
BACI'ERIAL CONTAMINATIONOF BLOOD PRODUCTS AND THE VALUE OF PRE-TRANSFUSION TESTING
M. A. Blajchman The Canadian Red Cross Society; and the Departments of Medicine and Pathology, McMaster University, Faculty of Health Sciences; Hamilton,Ontario,Canada U N 325. ABSTRACT
There has been a dramatic increase recently in the number of reports of septic episodes associated with both red cell and platelet concentrate transfusions. These reports suggest that transfusion-associated septic reactions mayoccur as often as 1 per 4000 platelet transfusions,however, thetrue incidence of the bacterialcontamination of stored cellularbloodcomponentshas not yetbeen established. Recently developed automated techniques for the detection of bacteria are much more rapid than direct plating techniques. Such rapid techniques can be used to monitor the sterility of cellular blood products with greater sensitivity than Gram staining; using a small aliquot of the blood product takensoon after collection.Usingsuchequipment, the incidence of bacterial contamination of 15,838 random donor platelet concentrates collected over a sixmonth period was determined and evidence of bacterial contamination was found in32.Sevenwereclassified as confirmed, 10 as unconfirmedand 12 as nonconfirmedpositives. The confirmedpositivity rate was thus 4.4 per 10,000. This rate represents the minimumincidence of bacterialcontamination of platelet concentrates and thetrue rate is likelyhigher, as some of the unconfirmed positives are likely to have been found to bepositives,had the original platelet concentrate beenavailableforculture. Thetrue positive rate is therefore estimated to be between 4.4 and10.7 per 10,OOO. Giventhis rate of bacterial contamination, it is our contention that all platelet concentrate units be monitored for bacteriologic sterility prior to theirissue. The cost of suchscreeningwould be of the same order of magnitude as that for each of the testscurrently performed to screen donors for transfusion-transmitted diseases. The addition of abacteriologicsurveillanceprogramcouldcontributesignificantly to ablood supplywithreducedriskandwould enable relevantscientificadvances,such as the prolongation of the storage time to be fully implemented to optimize patient care as well as blood product utilization. 163
164
BLAJCHMAN
TRANSFUSION ASSOCIATED SEPSIS Transfusionassociated potentialcomplication
septic reactions(TAS)havebeenrecognizedasa
ofblood
producttransfusionsince
century.Thus,bacterialsepsiswasprobably
the earlypart of this
the firsttransfusiontransmitted
disease to have been recognized. In the 1940s, TAS were often observed with red cell transfusions when reusable containers and "open" systems were employed the collection and processing
of donor whole blood and
for
the prevalence of TAS
was reported to be as high as 25%. Thus, the catastrophic effect of the infusion of a blood product containing bacteria and their products has been recognized for many years
(1-6).
With the advent of "closed" systems,
transfusion medicine believed
that they no longer have
those involved in to worry about TAS and
the occasional report of aseptictransfusionreactionwas unavoidable occurrence.
deemed a rare and
was thus
The "closed" blood collection system
responsible for lullingthoseinvolvedin
the provisionofbloodproducts
into a
false sense of security about the sterility of the blood supply. Over the past decade, the AIDS epidemic has forced more attention to be paid to the quality of the blood products provided for patient care (7). The main focus of this attention has, however, until recently, been on viral transmission with little attention given to the questionwhetherbacteria
of red celland platelet concentrates (8-15). TAS due to
problem for recipients
contaminated red cellconcentrateshavebeencaused
Yersiniuenterocolitica
represents asignificant
(13-18).
mainlyby
This enteric organismiscapable
proliferation and even selective growth
at refrigeration
the organism of efficient temperature.
Interestingly, most TAS reactions associated with Yersinia enterocoliticahave been reported to have occurred after red cell concentrate storage for at least 21 days. For thisreason,
it hadbeensuggested
that the storage period of redcell
concentrates be reduced from six weeks to three weeks to prevent the occurrence of such episodes. Such an approach
was not adopted because of concern that a
reduction in red cell storage time would exacerbate existing shortages. It is important to note that TAS is not only apotentialproblem
for
allogeneic (homologous) blood product recipients, but also for potential recipients
OF PRODUCTS BLOOD
BACTERIAL CONTAMINATION
165
of syngeneic (autologous) blood products. Recently, a
case of septic shock due
to Yersinia enterocolitica was reported in arecipient
of asyngeneicredcell
transfusion who developed septic shock during surgery, while receiving autologous blood (18). Thus, while syngeneic blood donor programs may prevent most of the immunological consequences of allogeneic blood transfusions, they do not ensure bacteriologicsafety.
It is important to keep thisinmind,particularlywhen
realizes that manyelectivesurgerypatientshavesepticfociassociatedwith conditionwhichnecessitated
one the
the requiredsurgery.
During the past few years, there has been an increased awareness of TAS associatedwith platelet concentrate transfusions (8-11). Suchepisodesprobably had gone unrecognized previously, because
of the frequent Occurrence of the so-
called febrile non-hemolytic transfusion reactions
(m) in patients receiving
random donor platelet concentrates. Up to 30% of patients receiving platelet concentrates experience an
FNHTR during or immediately following
the
transfusion of a platelet concentrate pool (19). Fortunately, most FNHTR are not due to bacteria and over the past few years, there has been an evolution
in our
understanding of the pathogenesis of FNHTR. Thus, until recently FNHTR have been attributed to the recipient alloantibodies reacting with alloantigens on donor leukocytes. Most FNHTR are now being attributed to the presence of cytokines, elaborated by the allogeneicleukocytes,
present in most platelet concentrate
preparations. It has been postulated that the cytokines elaborated during storage cause many of the signs and symptoms associated with FWHTR (20). The specific cytokines causing FNHTR have not been elucidated, nor has it been proven that their absence will ameliorate the frequency of FNHTR in patientsreceiving platelet concentrates. Because
the signsandsymptomsassociatedwith
FNHTR
may be similar to those seen in patients with TAS, the latter may not be suspected and maygo
unrecognized,evenwhen
due to bacteria.Thus,only
the most
clinically severe cases of TAS may be recognized. The incidence of FNHTR tend to increasewith
storage time (19).
Similarly, the magnitude of the severity of TAS increases with storage time (8,lO). The latter phenomenon has been known for many years. Indeed, the current fiveday storage period for platelet concentrates, in the United States, was established
166
BLAJCHMAN
because it had been realized that longer platelet concentrate storage times were associated with an increased prevalence relevantly, the FDA registryreceiving
of TAS (21,22).
Interestingly and
reports of transfusionassociated
deaths
foundasignificantlyincreasedincidenceintransfusionassociatedseptic over the pastdecade.
Thus for the three yearperiod,1976
deaths
to 1978, 4% ofall
transfusion-associated fatalities appear to have been associated with contaminated bloodproductswhereasadecade
10% of transfusion
later (1986 to1988),
associated deaths wereattributed to bacterialsepsis(23,24). Recent reports of TAS have refocussedattention to the problem of bacteria in the blood supply (5,6,8,10,12,20).
It is clear from
these reports that
contaminated platelet concentrates are a much more frequent Occurrencethan heretofore appreciated.
One recent report suggests that TAS may Occur
frequently as one per 4,200 transfusions (8). perspectiveblood complacencyabout
may be saferthan
as
Thus,whilefromavirologic
it haseverbeen,
there is no room for
other typesoftransfusion-associatedinfection;particularly
that due to transfusionassociatedsepsis.Mostimportantly,TAS
is preventable
using currentlyavailabletechnology.
Two recent studies provide relevant information concerning
the incidence
of the bacterialcontamination of platelet concentrates (8,lO).Inone, occurring in asingleinstitutionweremonitoredperspectivelyovera
FNHTR 42month
period (1987 to 1990).All platelet concentrate transfusions associated with such reactionswereevaluated
by Gramstainandculture.SevenTASepisodeswere
observed with one of the seven causing a fatality. Two other recipients developed septic shock but recovered. The rate of TAS for this study was thus estimated as one per 13,460 donor platelet concentrate units, or one per 4,200transfusion episodes. In this report, the authors acknowledge that bacterial contamination of platelet concentrates is significant a clinical problem and suggest
that the
incidence of such episodes could be reduced by shortening the storage period for platelet concentrates.Thisrecommendation
wasbased on the observation that
OF PRODUCTS BLOOD
BACTERIAL CONTAMINATION
most TAS episodes had occurred with
167
platelet concentrate that had been stored
for at least 4 or 5 days (8). In the second study, Gram-staining and microbiologic culturing of concentrates was done prospectively, just prior
platelet
to each planned transfusion of a
pooled platelet concentrate to a recipient. Over a 12 month period, 3,141 random donor platelet concentrate poolswere prepared from 14,481 units. Six of the 3,141, or 19 per 10,000 random donor platelet concentrate pools were found
becontaminated.
The isolatesincluded
to
StaphyZococcusepidermidis (4 isolates),
Bacillus cereus (I isolate) and Staphylococcus aureus (1isolate). The contamination
rate was estimated to be 4.1 per 10,000 platelet concentrate units,with incidencedirectlyproportional
to storageage.
the
These authors alsosuggesteda
shorter platelet concentrate outdatingperiod (12). Recently,aprospectivestudy,usingautomateddetectionequipment, determine the frequency of bacterialcontamination concentrates wasbegunin
to
of random donor platelet
our Centre. The results of the first 6 months of the
study are summarized in Table 1. Using the BacT Alert System(Organon Teknika Corp, Durham,
NC) the overall culture positivity rate was found to be
20.8 per 10,000 platelet concentrate units (25).
contaminationwere
Evidence for bacterial
seen in 32 of 15,838 random donor platelet concentrates
tested. Of these, seven were classified as "confirmed" positives, organismwasagain
that is the same
cultured from the original platelet concentrate whenre-
sampled. Ten were classified as "unconfirmed" positives, that is the blood product unit from which the positive culture was obtained was not available for
re-culture
as they had been issued for patient use prior to the positive result being available; and 12 instances were classified as "non-confirmed" positives, that is the presence of bacteria in the original platelet concentrate unit could not be confirmed even though that unitwasavailable technicalcontaminations.
for re-culture. Three isolateswereclassified Allof
the isolateswere
as
due to organisms that are
usuallyclassified as skin or environmentalcontaminants.Thus,
the "confirmed"
positivity rate of seven per 15,838 platelet concentrate units indicates a bacterial contamination rate of 4.4 per 10,000. This indicates that,
at the very minimum,
approximately one per 2,000 platelet concentrate unitshasasignificantnumber
168
BLAJCHMAN
TABLE 1 Prospective six monthbacteriologicsurveillance of 15,838 random donor platelet concentrate units prepared at the Hamilton Centre of the Canadian Red Cross Society.
Culture Interpretation positivesConfirmed positives Unconfirmed Non-confirmed positives contaminants Technical
Number
Incidence per 10,000 units
7
4.4
10
6.3
12
7.6
3
"_ 1.9
" "
32
TOTAL,
20.2
of bacterial organisms that could be detected and therefore could cause a septic
transfusion reaction in a recipient.
It is very likely that the 'true" contamination
rate issomewhathigherassomeof
the unconfirmed positives are also likely to
have been positives had re-culture.Thus,
the original platelet concentrate units been available for
the "true"positive rate of bacterialconformation
of platelet
concentrates in our Centre is between 4.4 and 10.7 per 10,000. This perspective surveillance study is continuing for a further six months using a second automated system (Bactec 9240, BectonDickinsonDiagnostic
Instrument System,Towson,
MD). BACTERIOLOGIC SCREENING OF CELLULAR BLOOD PRODUCTS
The above study was done using a 1.5 mL sample, taken within
24 hours of
the preparation of each platelet concentrate unit. These data thus indicate that it is possible to determine the sterility of platelet concentrate units when a small aliquot of a blood product is taken soon after collection. It is our view, until such time that it will be possible to sterilize cellular blood products,
that all platelet
concentrate unitsshould be monitoredforbacteriologicsterilityprior
to issue.
OF PRODUCTS BLOOD
BACTERIAL CONTAMINATION
169
The cost of doing such screening would beof the same order of magnitude as that for doingeach of the variousvirologicscreeningtests
that are currently being
done to screen donors for transfusion-associated diseases. More importantly,
the
institution ofsuch a bacteriologicscreeningprogramwouldsignificantly
reduce
the incidence of blood product associated sepsis and would contribute
to a safer
bloodsupply,as
the risk of transfusionassociatedsepsiswould
eliminated.Such removalof supplyprior
be virtually
a bacterialmonitoringsurveillancesystemwouldallow
bacterialcontaminatedcellularbloodproductunitsfrom to issue.Mostimportantly,inaddition
enable the institution of
the prolongedstorage
components. The fullimplementation
the blood
to further improving the
safety of the bloodsupply,suchanapproachwould variousscientificadvancestoallow
the
of cellularblood
of availablescientificknowledgewould
increase the availabilityof safer blood products
for patient care.
ACKNOWLEDGEMENTS
The author would like to thank Leslie Bardossy, Pamela Lyn, Luba Klama, Tammy Tassone and Tim Christmas for their expert technical assistance; and Dr.
A. Aliof the Hamilton Centre of the Canadian Red Cross Society (CRCS) and Dr. H. Richardson of the Department of Pathology,McMasterUniversity;for their important input to this work. The support of the Miles/CRCS R&D Fund
is also gratefully acknowledged.
REFERENCES
1.
C.W. Bordenand W.H. Hall. NewEngl. J. Med. 245, 760-765(1951).
2.B.A.Myhre.
JAMA
244,
1333-1335(1980).
3.
B.A.Myhre.Arch.Pathol.Lab.Med.
4.
KC. Anderson, M.A.Lew,B.C. (1986).
5.
M. Goldmanand
m,982-983(1985). Gorgone et
al.
Am. J. Med. &l,405-411
M.A.Blajchman.Transfus.Med.Rev.73-83(1991).
170
BLAJCHMAN
6.
M.A.Blajchman and A.M.Ali,inBloodSafetv: Current Challenges, S.J. Nance, ed, AABB, Bethesda, MD (1992) pp 213-228.
7.
R.Y. Dodd. NewEngl. J. Med.
8.
J.F. Morrow,J.G.Braine,T.S.Kickler
9.
G. Marduchowicz, S.D. Pitlik,D. Huminer et al. Rev. Infect. Dis. 314 (1991).
l3,307-
10.
R.Yomtovian,H.M.Lazarus,L.T.Goodnough 909 (1993).
33. 902-
327. 419-421,(1992). et al. JAMA
266,
555-558(1991).
et al.Transfusion
11. 0.Heltberg, F. Skov, P. Gerner-Smidt.Transfusion
2,221-227(1993).
33. 189-191(1993).
12.
C.F. Hogman, H. Fritz and L. Sandberg.Transfusion
13.
M.A.E. Stenhouseand L.V. Milner.Transfusion
14.
M.A. Tipple, L.A. Bland, J.J. Murphy et al. Transfusion 30.207-213 (1990).
15.
R.C. Aber.Transfusion
16.
M.J.Arduino, LA. Bland, M.A. Tipple et al. J. Clin.Microbiol. 27. 14831485 (1989).
17.
S.L. Nightingale, JAMA
22. 396-398(1982).
3,193-194(1990).
266,
18. J.M.Sire,C.Michelet,R.Mesnard 954-955(1993). 19. N.M. Heddle, L.N.Klama, (abstr).
190 (1991). et al.ClinicalInfectiousDiseases
L. Griffith et al.Transfusion
20.
J.O.Bordin,N.M.
21.
H.G.Braine,T.S.Kickler,P. (1986).
22.
J.M. Heal, M.E. Jones, J. Forey et al. Transfusion
23.
C.L. Honigand J.R. Bove. Transfusion
24.
K Sazama. Transfusion -03 583-590,(1990).
25.
T.C. Thorpe, M.L.Wilson,J.S. 1612 (1990).
17,
3, 794(1993)
Heddle and M.A.Blajchman.Blood,inDress(1994). Characke et al.Transfusion 27,
26. 391-393
2-5(1987).
a,653-661(1980).
Turner et al. J. Clin.Microbiol. -8 2
1608-
PART III: ALLOTYPES
This Page Intentionally Left Blank
WHAT IS IMPORTANT ON THE RED BLOOD CELL SURFACE Patricia Tippett Medical Research Council Blood Group Unit Wolfson House, University College London 4, Stephenson Way, London NW1 2HE. UK
ABSTRACT Blood group antigens have provided tools for investigation of red the cell surface and been very useful as genetic markers in family, population and forensic studies. Precise definition of phenotypes is veryimportant.ApplicationofMAIEA(monoclonalantibody-specific immobilisation of erythrocyte antigen), a recently reported technique, to identify antigens and to assign red cell antigens to a particular membrane component is described: location of Knops system antigens onCRI is confirmed and provisional assignment of Cromer system antigens to the different short consensus regions of decay accelerating factor (DAF) is described.Variabilityofredcellantigenexpressionisconsidered.Thepossibilityis discussed that factors other than alterations inRh genes may be responsible forsome Rh two ofwhichareassociatedwithlowincidence variantphenotypes.SomeCvariants, 12E7of antigens, are described. The relationship of Xga with the quantitative polymorphism antigen is reconsidered in light of some recent immunochemical studies. INTRODUCTION
The basic function of red cells is delivery of oxygen to tissues round removal of carbon-dioxide. The extracellular components
the body and the
do not affect this function but
structural aberrations may affect the deformabilitycells of and hence their integrity and ability
to fulfil their proper role. The successred of cells depends on correct association of integral proteins and peripheral proteins of the cytoskelton. Although integral membrane proteins other than those that carry blood group antigens are vital components red cell on the surface, only red cell antigens will.be described in this review. Many bloodgroupantigensaremarkers
of integralmembraneproteinsandthishas
contributed to our understanding of biochemical structures of the antigens and biological 173
TIPPETT
174
roles of carrier proteins. The identification and usefulness of blood group antigens as markers will be described and possible explanations for their variation in expression will be discussed. Most red cell antigens have been investigated because they are clinically important [l]. The antibodies to some antigens have caused haemolytic disease of the newborn andlor transfusion reactions. Other antigens are involved in haemolytic anaemia and some are important in transplantation. Red cell antigens provided a tool for investigation of the red cell surface and for use as genetic, immunological and biochemical markers. More than 500 red cell antigens are serologically defined, about half of which have been officially recognised and have been numbered by the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens [2,3]. Antigens are divided into systems (antigens controlled by a locus or closely linked loci) and three holding files: collections (related antigens whose genetic relationship is unknown), antigens of high incidence or antigens of low incidence. THE MAIEA TECHNIQUE Sometimes if an antigen has a very high or a very low incidence it is hard to relate it to other antigensor to assign it to asystem.
Immunochemical studies and in the case of high
incidence antigens, use of cells of rare phenotype can be informative and recently the MAIEA technique, monoclonal antibody specific immobilisation of erythrocyte antigens, has proved useful.MAIEA
was an adaptation ofatechnique,
MAIPA, frequently used for studying
platelets. MAIEA can be used to assign red cells antigens, as recognised by human alloantisera, to particular components of the red cell membrane [4]. Location of antigens on specific red cell membrane components The Knops system consists of4 high incidence antigens Kna,McCa, SIa and Yka with frequencies greater than 90% in populations tested. There is also a low incidence antigen Knb found in Whites [3]. The antibodies to these public antigens are difficult to identify serologically. The antigens show a wide variation of strength on different donor's cells. There is a null phenotype, the Helgeson phenotype, which appears from serological tests to lack all 4 antigens [5]. Cells which lack one Knops antigen may have weakened expression of other Knops antigens. The mists about these serologically difficult antigens were cleared when Moulds et a1 [6] and Rao et al [7] independently showed that these antigens were carried on the CRI (complement receptor 1, CD35) protein. The related antigen Csa was not located on CRI, so Csa and Csb were left in the Cost collection p].
REDBLOODCELLSURFACE
175
The principle of the MAIEA technique depends on the binding of two antibodies made in different species to different determinants on the same membrane component to form of a tri-molecular complex [4]. Briefly, a murine monoclonal antibody (MAb) and human antibody are incubated simultaneously with red cells. Excess antibody is removed, the sensitized cells are solubilised with Triton, so the tri-molecularcomplex
is released into solution. The
complex is detected by an ELISA type assay. The tri-molecular complex is captured by an anti-mouse globulin precoated onto a microtitre plate. The human antibody is then detected by a peroxidase-conjugated anti-human IgG. A positive reaction gives a high absorbance value and a negative reaction gives a low absorbance value. A negative result is obtained when the antibodies used bind to different membrane components, so no tri-molecular complex is formed. A negative result is also obtained when the monoclonal antibody and human antibody compete for the same epitope. Results can be represented as ratios of absorbances for antigen positive to antigen negative cells or as bar charts. In these studies a murine anti-CR1 ( E l l ) and human anti-Kna and other Knops system antibodies were used against antigen positive and antigen negative cells. Absorbances for antigen positive cells with anti-Kna, anti-McCa anti-SIa and anti-Yka were high and results for the antigen-negative cells were low [8].Comparison of chymotrypsin treated Kn(a+) cells with Kn(a-) cells showed that chymotrypsin did indeed destroy Kna antigen; chymotrypsin treated cells, therefore, were suitable cells to use as antigen negative cells when cells of rare phenotype were not available [8]. These reactions gave significantly positive ratios (Table
I). In contrast, low absorbances were recorded for Cs(a+) and Cs(a-) cells with anti-CSa, the 1 : l ratio indicating a negative result (Table l).
Serologically the Helgeson phenotype cells have a Knops null phenotype, all 4 antigens are negative but the antigens could be detected by flow cytometry and in immune precipitation
[6,7].Moulds and colleagues provided an explanation for this when they found that such cells did not completely lack CRI but had a low copy number of CRI molecules per cell [g]. Had it not been known already, the presence of Knops system antigens on Helgeson phenotype cells could have been deduced from the MAIEA results. The absorbance values for Helgeson phenotype cells were significantlyhigher than for antigen negative cells for Kna, McCa and Yka [8]. MAIEA has confirmed that Kna, McCa, SIa and Yka but not Csa are associated with the CRI molecule in the red cell membrane and can detect weak expression of CRI antigens on Helgeson phenotype cells [8].
MAIEA is useful for investigating problem antibodies
suspected to be Knops system antibodies and can also be used to Knops phenotype cells with poor expression of Knops system antigens.
TIPPETT
176
TABLE I Reactions of human antibodies withCRI immobilised by mouse monoclonal antibody E l 1 Red cell phenotype Murine MAb Human antiAbsorbance Ratio Kn(a+) Kn(a-) McC(a+) McC(a-) Yk(a+) Yk(a-) Sl(a+) Sl(a-) Cs(a+) Cs(a-)
}
1 } } }
0.755 Ell
Kna 0.538
Ell
McCa 0.31 5
Ell
Y ka
0.120
1 }
l
4:l 0.195 4:l 0.136 2.6:l
0.342 Ell
SIa
4.6:l 0.074 0.139
El1
CSa
}
1:l 0.108
Mapping relative positions of antigens on a specific protein When several murine monoclonal antibodies to different epitopes on the same protein are available, MAIEA can be used to study the relative position of antigensthat on protein. This application of MAIEA depends on mutual inhibition of murine monoclonal antibodies and human antibodies. A negative result is obtained when human and monoclonal antibodies compete for the same epitope, or bind to very closely located epitopes, so no tri-molecular complex is produced. Several monoclonal antibodies to the Kelt protein have been used in MAIEA to study the relationshipsof the Kelt system antigens [IO]. The decay accelerating factor DAF, CD55, is detected by several monoclonal antibodies. Three antibodies BRlC 230, BRlC 110 and BRlC 216 were known from competitive binding assays to bind to different short consensus repeats (SCR) [l l]. So three of the four SCRs
of the DAF molecule were positively identified (Table H). Strong positive reactions were observed with all three BRlC antibodies and anti-CP, antiM S a , and anti-WESb showing that MAIEA is a useful techique for studying this system [12].
The results showed that CP. M S a , and M S b are not on the first three SCRs and must
RED
177
thereforebe on the fourth SCR or onthe serineltheonine richregion.Bysequencing genomic DNA from Cr(a-) people, Telenand colleagues showedthat a mutation in the fourth SCR was responsiblefor C? [13]. Considering the MAIEA results,the fourth SCR would be a good place to start looking for difference responsible for the WES polymorphism too. Other Cromer system antigens showed someinhibitionwith one of the BRlC antibodies [12]. MAIEA provided biochemical evidence that Esais indeed a Cromer system antigen [12]. Esa was thought to be a Cromer related antigen because of the failureof anti-Esa to react with Cromer-null cells and from its behaviour with proteinaese treated cells [14]. These findings were supported by the observation that Esa was carried by a glycosyl phosphatidylinositol linked protein [15]. However, only a small amount of anti-Esa was available andjherefore, immunoblotting experiments couldnot be done. Strong positive results with BRlC 216 and 110 but a negative result with BRlC 230 suggested that Esa is located on DAF,possibly on the first SCR. Similarly, a negative result with BRlC 230 and Tca suggests that it too is on the first SCR (Table 11) [12]. The resultsof the MAIEA tests for Cromer antigens are summarised in Table 11. They agree with those known fromDNA studies, DP on SCR Ill [15,16,17] and C? on SCR IV [13], and suggest the best places to look for those as yet undetermined.
This demonstrates how
MAIEA may be used to help narrow the field of study to determine the molecular basis of antigens. VARIATION IN EXPRESSION OF SOME Rh ANTIGENS We had hoped to apply MAIEAto Rh but to date the only antibodies to the D protein are of human origin, so MAIEA cannot yet be used to study the relationship of the D antigen to some of the low incidence antigens which appearto be markers of partial D antigens. The Rh antigen D is, after ABO, the most important antigen clinically because it is highly immunogenic. Until the introductionof Rh immunoprophylaxis, anti-D was the most frequent cause of haemolytic diseaseof the newborn and neonatal death [ l ] . Many Rh antigens are good immunogens. Since its
initial recognition in the nineteen-forties, the Rh system has
becomeverycomplex.Thereare48numberedantigens,
that is serologically defined
determinants, the numbers have reached 50 because two numbers have been declared obsolete[2,3,18,19].Someantigensarepolymorphic incidence.
and others are of high or low
TIPPETT
178
TABLE II Possible locations of Cromer system antigens on DAF molecule deduced from MAIEA tests DAF region Reactive monoclonal Cromer antigens antibody SCR Esa I Tca, 230 BRlC SCR II 110 BRlC SCR Ill Ora 216 BRlC SCR IV CP, serinekheonine rich region
UMC
wsa,M S b
Understanding ofthe biochemical structures and molecular basis of Rh antigens is emerging rapidly.AbsenceofRhantigens,asoccurs
in the Rh,
Rh, integrity of red cells and cells from people with an
phenotype,compromisesthe phenotype have been extensively
studied. These studies contributed to the recognition of Rh polypeptides and some related glycoproteins [see 20,21,22]. Partial amino acid sequencing of the proteins in Bristol, Pans and Baltimore [23,24,25] led to recognition of involvement of
two genes and isolation of
cDNA by the Paris and Bristol workers [26,27] and cloning of the D gene [28]. One gene is responsible for the D polypeptide and another for the C and E series of antigens. However, although encoded by the same gene thereis evidence that the C andE series of antigens are carried by different proteins. The molecular genetic basis of Rh antigens is discussed in another presentation. Immune precipitation using anti-D, -c, -E or R6A antibodies demonstrated the proteins which Mr carried the Rh antigens. Two bands are co-precipitated by anti-D: one with an apparent
30,000 called ,D ,
polypeptide by the Bristol group and the other a diffuseband of 50-100
kD called the D ,
polypeptide. Similar bands were observed when immune precipitation
weredone
usinganti-c,-EorR6A[see
20-221.The
D ,,
polypeptide wasan unusual
membrane protein because itwas not glycosylated, the gene producingthis protein and the other Rh protein were subsequently cloned. Assignment of the genes to chromosome 1p34p36 confirmed that they are responsible for the Rh polymorphism [see 221. The role of the Rh glycoproteins, the diffuse band of 50-100kD, is not yet understood: the gene encoding the Rh glycoprotein when cloned was assigned to chromosome 6p2lqter [29].
RED
SURFACE
Both types of Rh,,,
179
cells (amorph and regulator types) lacked the Rh proteins and other
proteins,andshowedsomefunctionalabnormalities. precipitatedbyanti-D
No Rhglycoprotein
but it is present in membranesfromsome
Rh,,,,,,s
is immune
asshownby
immunoblottingstudieswiththemonoclonalantibodyMB-2D10[30].TheLWproteinis missing and some other antibodies only react weakly: anti-U, Duclos, anti-FY5, BRlC 125 (anti-CD47) and the monoclonal antibody 1D8. That
so many determinants encoded by
genetically independent loci are not fully expressed in Rhflull cells has led to the idea of a protein complexorclusterwhichinvolvestheRhproteins,Rh
glycoproteinsandother
proteins[31,32,33].SinceDandCEproteinsareintegralproteinswithabout12 transmembrane domains, is it hypothesised that they and the other proteins interact, perhaps affecting insertion into the membrane. Some of the variation observed in Rh antigens may not depend on mutations
in the Rh
genes but may reflect alterationsin other proteins of the Rh protein complex. D , the most important antigen, has been exhaustively studied. Quantitative and qualitative variation of D
is well documented [see 341. Several other Rh antigens show quantitative and qualitative variation. We
have observed variation in C, E, c, e, G, V, VS, Rh17 and Rh29 antigens.
Possibly it is a common finding for Rh antigens. Variation of C antigen Table Ill shows some of the Blood Group Unit‘s results of testing samples with rare Rh phenotypeagainstpolyclonalanti-C.ComCrepresentscommercialanti-Creagents,all others are single donor antibodies. The first three reagents do not contain any anti-D, the
so are not suitable for tests with enzyme treated cells. next three contain incomplete anti-D The different patterns of reaction would make one suspect that these cells carried different variants of C; most variants are also distinguished by their reactions with antibodiesto low incidence Rh antigens (Table 111).
Low incidence antigen JAL (RH481 JAL+ cells have a very weak C antigen, it is most easily detected with commercial reagents (Table 111).
The antibody in Mrs S Allen’s serum was studied for many years
in several
laboratories interestedin low incidence antigens. There were some hints that it might be an Rhantigen,althoughMrsAllen’shusbandhadbeenanunremarkablecDe.Eventually several samples expressing the JAL antigen were identified. However, family studies had not proved that JAL was an Rh antigen, although 3 of the propositi had a depressed c antigen and 4 had a depressed C antigen, 2 of whom also had a depressed e antigen [35]. A second immune example of anti-JAL, which caused haemolytic disease
of Mrs Pas’ third
)
TIPPETT
180
TABLE 111 Variants of C antigen Anti-C
Red Cells CWDe CXDe CDE Ccde' (C)D(e) (C)D(e) (C)(e)
LA ST
++ +++ +++ ++ ++
+
0 0 0 0 0
++ ++ 0 0 0
1108 SLIT
ComC C+D5
++ ++ +++ ++ +/W
++ ++ +++ ++ +++ +++
+++ cw+ ++ cx+ +++
++ W +++ +++
++
+
o
+++ ++
+++ 0
+++
v-vs+ Rh:32 JAL+ FPTT+
baby, was found in Switzerland. This antibody was used in Bern to screen donors. Four newJAL+propositiwerefound
in 90,000 donors,allfourwereFrenchspeaking.The
to be 0.004% overall but0.06% in French-speaking frequency of the antigen was calculated Swiss [36]. The families of the antibody maker and the4 JAL+ donors were studied. The results proved that JAL was part of the Rh system or encoded by a very closely linked locus 1361. JAL in Whites is associated with depression
of C antigen but
in Blacks JAL is probably
associated with a depressed c [35]. We wondered if there were any difference between the JAL antigen associated with depressed C and that associated with depressed c. Titrations of two anti-JAL with JAL+ cells of weak C and weak c phenotypes showed that there was no significant difference between the two samples [35]. is noteworthy It that the anti-JAL Mrs of S Allen was stimulated by pregnancy and her husband was cDe JAL+ but J Pas's anti-JAL,
also stimulated by pregnancy, was madein response to a depressed C JAL+ complex. So the expression of JAL does not depend on the C or c withit is which associated. Both these anti-JAL were responsible for haemolytic disease of the newborn [35].
Low incidence antigenFPTT (RH50) In contrast to JAL, the FPTT+ sample was more easily detected by the polyclonal anti-C (the ones with incomplete anti-D) than by commercial anti-C (Table 111). FPTT presentsa much more difficult problemthan JAL. Adsorption/elution tests are needed to identify the antigen. 5 different depressed antigenic Family studies showed that FPTT is associated with at least
complexes(TableW).
One complex, that of propositus
1 [37], had depressed C and e
antigens, the complex of propositus 2had depressedC antigen and depressed e antigen 1371
RED BLOOD CELL SURFACE
181
TABLE IV Variation of expression of FPTT antigen FPTT+ samples serum Eluate from Mol IAT papain
1 or 4 3
DFR
We)
Prop 1
+
+
+
+
+
(C)D(eS)
Prop 2
-
+/-
+
-
nt
Prop 3
-
W/-
+
+ +
+ + +
+
+
+ + +
+ + + +
(e)
(D)(e)Rh:33Prop
4
+
partial D
CDDFRe
partial D
C D ~ ~ +~ E
(and expressed VS antigen) and the complex of propositus 3 [37] has depressed e antigen but a normal C antigen but others involve depression of D antigen, one a partial D antigen and the other, that of propositus 4 [37], associated withthe weak D antigen characteristicof RoHar and a depressed e (Table IV) [19,37]. The expression of FPTT was variable. The reactions of the cells of propositus
1 [37], all
examples of Rh:33 and all examples of DFR were uniformly strong but those for propositi 2 and 3 [37] were much weaker (Table IV). The partialDantigenof
DFRpeople
differedfrompartialDantigens
of thereported
categories [19]. In 24 DFR propositi, the partial Dwas associated with Ce and in only two was it associated with cE: the partial D associated with Ce was indistinguishable from that associated with cE
.
We did not observe any weakness of Rh antigens, other than D, on
DFRcells.FPTTbehavesas
amarkerforthemissingpart
of D and, therefore, we
wondered if the weakD associated with Rh33 was a weakened form of the D partial of DFR. The results of testing monoclonal anti-D against untreated cells are shown in Table V. Four patterns of reaction were observed. Thirty-seven monoclonal anti-D reacted with DFR cells, the majority of anti-D which reacted with DFR cells failed to react with RoHarr cells and one anti-D reacted with RoHarr cells but not with DFR cells (Table V). Twenty-one monoclonal anti-D were negative with both types of FPTT+ cells. From these results we concluded that the weak D of Rh:33 cells was different from the partial Dof DFR.
182
TIPPETT TABLE V Comparison of D antigen of FPTT+ samples Monoclonal Number anti-D MAb-D Samples
reactions
o + + o o + + o + o + o
24 2 Number MAb-D
1 21 35
2
So FPTT is associated withtwo different types of D antigen and three different typesCe of antigens (Table IV). These results suggest that similar a amino acid sequence corresponding to the FPlT antigen is encoded by D genes and by CE genes. Since the genes are highly homologous and proteins very similar, it is possible that similar changes mayhaveoccurred.
Several mechanisms could be involved: mutation, recombination or
geneconversionhavebeeninvoked
in otherbloodgroupsystems
to explainrare
phenotypes. The large number of Rh antigens and their quantitative and qualitative variantswill not be easy to explain. Variation in the Rh genes may explain some variants but we know that Rh
RH,homozygosity of oneunlinked
expressionisaffectedbysuppressorsunlinkedto suppressor causes the regulator type of
Rh,.
Mutation in one of the genes encoding a
non-Rh protein required for formation of the Rh protein complex may affect the presentation ofsomeRhantigensat
thecellsurface.Rhgroupswillcontinue
to beclinicallyand
immunologically important until their genetic control is fully understood. Xga AND THE RELATED 12E7 ANTIGEN Unlike Rh antigens, Xga is not clinically significant but was a very valuable marker for studies of the X chromosome. Our interest
in Xga and the related 12E7 antigen was rekindled
recently by a report ofPBDX,a candidate gene forXG [38], and by speculation of the role of 12E7 antigen as an adhesion molecule[39,40]. Xga is red cell specific; in contrast, 12E7 antigen is almost ubiquitous.
12E7 antigen, the
M C 2 gene product, has been numbered CD99 at the fifth Leucocyte Workshop and this
183
RED designationwillbeusedforthe12E7antigen.CD99wasfirstdetectedby12E7,a
monoclonal antibody made in response to a T-cell line, and was initially thought to be a ‘thymus-leukaemia’ marker antigen [41]. Many similar antibodies were made which reacted with different epitopes of the same molecule [see421. Independently, CD99 was identified as E2, a T-cell adhesion molecule, and as a marker antigenfor Ewing’s tumours [see 401. CD99 is expressed on many tissues including red cells. By somatic cell hybridization and biochemical studies, Goodfellow and his colleagues have shown thatMIC2, the structural locus encoding the 12E7 antigen, is located on the short arm of the X chromosome and on the short arm ofYthe chromosome within the pairing regions [43]. MICZ hasbeencloned[44].
XG is X-borne.On
redcells,CD99expression
is a
quantitative polymorphism [45]. Family studies proved that this polymorphism is also caused by regulator genes on X and Y chromosomes. XG appears to be the regulator on the X [46]. There is variation in CD99 expression on cells other than red cells. In a recent publication, CD99 was found on all haemopoeitic cells but was variably expressed during leucocyte differentiation[40].Use
of differentmonoclonalantibodiesandvariabilityofexpression
during maturation offered an explanation for the previous apparently contradictory findings by different laboratories. Both Xga and CD99 are sialoglycoproteins [47,48,49]. These glycoproteins differ in M, and in their sialic acid content [49]. lmmunostaining of separated membrane components with 12E7 and similar antibodies had demonstated that the MIC2 gene product was a 30-32 kD protein. 12E7 also bound to an intracellular bandof 28 kD which was found in mouse cell lines in addition to human cell lines, platelets, lymphocytes and encodedbythe
red cells but it was not
MICZ gene[47].lmmunoblottingassayshaveshownthatXgawas
associated with two diffuse bands of 22-25 kD and 26.5-29 kD [49]. ThesefindingssupportedtheevidencethatXgaandCD99wereproductsofdifferent structural loci. However, XG appears to regulate CD99 expression on red cells and Latron and colleagues foundthat purified CD99 protein inhibited binding of 12E7 and of anti-Xga to red cells [48]. We have studied the immunochemical relationship of Xga and CD99 [50]. One approach was immunoprecipitation of membrane components from biotin labelled cells. Bands are detected by chemiluminescence via peroxidase-conjugated avidin. The 32 kD protein of CD99 was visualised by this technique and the quantitative polymorphism was also demonstrated since the 32 kD bandis seen on X-ray film after 2 minutes in membranes
184
TIPPETT
from CD99 high expressors but membranes from CD99 low expressors required exposure of 5 minutes before the 32 kD band was apparent(501. Unfortunately, these tests gave no information about the Xga protein because the position of the Xga band was maskedby the antibody light chain which became labelled. However, a 32 kD band was seen in the Xga-immunoprecipitate from Xg(a+) but not from Xg(a-) cells
[50]. It has not yet been proved that this is the CD99 protein because this band was not stainedbyimmunoblottingXga-immunoprecipitateswith12E7.Theluciferin-enhanced luminescent proceedure to detect the avidin-biotin label isvery much more sensitive than immunoblotting. Our results support the theory that Xga and CD99 may be associated in the membrane. Cloning of the XG gene will increase our understandingof this relationship. The important blood group genes have been clonedtwo butbig problems remain, regulation on antigen expression and the function of blood group polymorphisms. Rare phenotypes should still be studied because they will contribute to unravelling the mechanisms responsible for the polymorphisms. The wealth of serological information which continues to increase includes many examples of variable expression of red cell antigens. Some antigens do not show the same variation on other cells suggesting that some modes of regulation may be limited to redcells. Association of blood group antigens with proteins of known function and identification of red cell antigens on cells other than red cells will contibute to understanding the functions of the blood group polymorphisms. REFERENCES 1.
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FUNCTIONAL FACTORS IN THE RED CELL MEMBRAm. INTERACTIONS BETWEEN THE MEMBRANE AND ITS UNDERLYING SKELETON
D J . Anstee, N.J. Hemming, M.J.A. Tanner* International Blood Group Reference Laboratory, Bristol BSlO SND, UK and *Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 ITD, UK
ABSTRACT Recent studies involving two abnormal red cell phenotypes (South-east Asian ovalocytosis andLeach phenotype) provide novel informationconcerningthenatureandsignificanceof interactions of both the anion transport protein AE-1 (syn. band 3) and Glycophorins C and D with the underlying skeleton. The location of Wra and Dia blood group antigens to mutationson AE-l at residues 658 and 854 respectively, together with the availabilityof monoclonal antibodies recognising epitopes dependent upon the integrity of the third extracellular loop of AE-l, have allowed us to study the mutantAE-1foundinSouth-eastAsian theorganisation of the membranedomainof ovalocytes(AE-1 SAO). The resultssuggestthat the organisation of the wholemembrane of other integral membrane proteins domain of AE-l SA0 is abnormal and that the organisation like those involved in expression of Rh blood group antigens may also be affected. Increased homo- and hetero-associations involving AE-1 SA0 and other integral proteinsmay in turn result in reduced membrane flexibility. Purified protein4.1 binds with 50-fold higher affinity to protein 4.1 depleted normal red cell membranes than to protein 4.1 depleted red cell membranes of Leach phenotype which lack Glycophorin C (GPC) and Glycophorin D (GPD). Experiments using purified protein 4.1 and p55together with syntheticpeptidescorresponding to differentregions of the cytoplasmic domain of Glycophorins C and D (GPCID) demonstrate that protein 4.1 interacts directly with GPC through residues 82-98. They alsoshow that p55 binds to GPC through residues 112-128. Since p55 also binds directly to protein 4.1 itis clear that protein 4.1 can bind to GPC through two different sites either directly through residues 82-98or indirectly through p55. These results show that GPC and GPD provide major attachment sitesfor the red cell skeleton via protein4.1 and that p55 is part of this complex.
INTRODUCTION The human erythrocyte relies on a networkof proteins underlying the membraneto maintain its structural integrity and deformability. This network, known as the skeleton, consists predominantly, of spectrin, actin and protein 4.1, along with several other minor components [protein 4.9 (syn. dematin), protein 4.2 (syn. pallidin), p55, ankyrin, adducin, 187
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ANSTEE, HEMMING, AND TANNER
tropomyosin, myosin, tropomodulin, caldesmon]. The major feature of the skeleton is defined by long filamentous spectrin heterodimers, aboutlOOnm in length, which associate (head to head) into tetramers and higher order oligomers. These spectrin tetramers (and oligomers) interact with short actin filamentsat so-called junctional complexes (1). Protein 4.1, dematin and adducinare located at the site of junctional complexes, whereasankyrin is attached to the skeleton near themid region of the spectrin molecules (2). The skeleton is anchored to the membrane through at least two protein interactions which involve ankyrin and protein 4.1 respectively. Ankyrin binds to the N-terminal cytoplasmic domain of the erythrocyte anion transport protein, AE-1, (syn. band 3) and the nature of this interaction has been the subjectof recent reviews (3,4). The site of interaction between protein 4.1 and the membrane is lessclear, although available evidence favours the cytoplasmic domainsof Glycophorins C (GPC) and D (GPD)(5,6). In this paper,we describe recent studies utilising two abnormal red cell phenotypes [Southeast Asian ovalocytosis (SAO) and Leach phenotype] which throw further light on the nature and significanceof interactions of both AE-1 and GPC/D with the underlying skeleton. STUDIES ON SOUTH-EAST ASIAN OVALOCYTOSIS (SAO)
Several recent studiesof SA0 red cells have shown that the phenotype resultsfrom inheritance of an abnormal formof AE-l which lacks nine amino acids (400-408) at the junction of the N-terminal cytoplasmic domain and the first transmembrane domain (Figure 1) (7,8). The occurrence of this abnormal form of AE-1 (AE-1 SAO) confers a selective advantage in the heterozygote (resistance to malaria) but is probably lethal in the homozygote because it does not functionas an anion transport protein (9). S A 0 membranes are 10-20 fold more rigid than normal membranes but this does not appear to be a consequenceof altered interaction between the mutant band 3 and ankyrin (8). Schofield et aL(8) concluded thatthe deleted segment inSA0 cells controls membrane elasticity either directly or through a structural perturbationof the membrane domainof the band 3 as a whole. Three recent observations suggested an immunological approach to investigate the consequencesof the AE-1 SA0 mutation on the membrane domain of band 3 as a whole. Rodent monoclonal antibodies to theextracellular face of AE-1 We have recently produced and characterised a series (11 in all) of murine and rat monoclonal antibodies which recognise epitopes on the extracellular face of AE-1 (10,ll). The antibodies were produced in response to immunisation with intact humanerythrocytes and were characterisedby immunoprecipitation from intact erythrocytes followed by of the immune precipitates immunoblotting of the electrophoretically separated components with monoclonal antibodies specificfor epitopes on theN- and C-terminal cytoplasmic
RED CELL MEMBRANE
189
-r
\l/
Segment
deleted in AE-1
SA0
FIGURE 1 Schematic illustration of AE-1 (Band 3) showing various extracellular sites and the position of the mutation found in Southeast Asian ovalocytes
domains of AE-l. All of the antibodies failed to react with pronase-treatederythrocytes. Pronase cleaves AE-1 in the regionof the third extracellular loop. Chymotrypsin, which also cleaves AE-1 at this loop (Tyr 553 and Tyr558), reduced the reactivity of 7 of the 11 antibodies. The remaining 4 antibodies failed to react with red cells treated sequentially with chymotrypsin and trypsin (under low ionic strength conditions). Trypsin under low ionic strength conditions cleaves after Lys-562(12). These results indicate thatall of the antibodies see epitopes whichare dependent upon the integrity of the thirdextracellular loop of AE-1 (Figure 1).
Location of the Wra antigen to AE-1 Sequence analysis of AE-1 cDNA from an individual (MF) of phenotype Wr(a+b-) revealed a single base change resulting in conversion of Glu 658 + Lys (13). The
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ANSTEE, TANNER HEMMING, AND
correlation of this change with the occurrenceof the Wra antigen was confirmed by analysis of genomic DNA from 7 unrelated individualsof phenotype Wr(a+b+). This amino acid sequence change is located on the fourth extracellular loop of AE-1 (Figure 1). Evidence has been presented which suggests that the Wra and Wrb antigens are encoded by alleles of the same gene (14). These results are consistent with Glu 658 as the primary determinant of the Wrb antigen. Considerable evidence exists which implies arole for Glycophorin A (GPA) in the expression of the Wrb antigen nevertheless, the amino acid sequence of GPA from Wr(a+b-) red cells is normal (13,15). Together, these results suggest that an interaction between Glu 658 AE-1 on and the region of Glycophorin A corresponding to residues 61-70 is requiredfor expression of the Wrb antigen.
Location of the Dia antigen to AE-1 Recently, we have shown that the AE-1 variant known as Memphis variantII is associated with the expressionof the Diego (D?) blood group antigen (16). Further studies have revealed that the Occurrenceof the Dia antigen is dependent upon the AE-l mutation Pro854 + Leu (17). Sequence analysis of cDNA from a MexicadAmerican individual (ESC) of phenotype Di(a+b-) revealed the Memphis polymorphism (Lys56 + Glu) and the additional change Pro854+ Leu. The same sequence changes were found on analysis b-) and heterozygous of genomic DNA from three Japanese individuals of phenotype Di(a expression of these changes was found in two individuals (one Brazilian, one Japanese) of phenotype Di(a+b+). These results confirm the extracellular locationof the loop connecting transmembrane segments 13 and 14 (Figure 1) and suggest thatthe Dib antigen is dependent uponPro854 of AE-l.
+
The observations described above define antigenic sites on three different extracellular regions of AE-1 and have proved useful for the analysisof the membrane domain ofAE-l in SA0 red cells. AE-1 SA0 is expressed at the plasma membrane of Xenopus oocytesin both the presence and absence of GPA, but monoclonal antibody BRIC 6, which reacts with a chymotrypsin-sensitive extracellular epitope on normal AE-1, does not immunoprecipitate AE-1 SA0 from oocytes (18). This result suggested that the BRIC 6 epitope is concealed or misfolded in native AE-l SA0 and prompted us to examine the expression of the BRIC 6 epitope on intactSA0 erythrocytes. Quantitative binding assays using purified radioiodinatedBRIC 6 IgG demonstrated aclear reduction (54% of normal) in the number of BRIC 6 epitopeson SA0 red cells when compared with normal redcells. BRIC 6 IgG gave 286,000 f 30,000 sites on normal red cells and 156,000 f 16,000 on SA0 cells (11). Similar quantitative binding assays were performed using a murine monoclonal anti-Wrb (BRIC 14) and these revealed a significant reduction(79% of normal) in the number of BRIC 14 epitopes recognised by radioiodinated Fab fragments onSA0 cells (842,000 & 18,000 for normal red cells and 563,000 f 68,000 for SA0 cells). Since the BRIC 6 epitope is dependent on the third extracellular loopof AE-1 and the BRIC 14 epitope is
RED CELL MEMBRANE
dependent uponthe fourth extracellular loop in association with GPA (Figure l), these results suggest a substantial alteration in the organisation of the membrane domainof AE-1 SA0 in SA0 erythrocytes. Selective depressionof blood group antigens on SA0 cells has been reported previously by Booth and colleagues (19). Amongst the antigens shown to be depressed were Wrb and Dib (Table I). Since the Dib antigen is now knownto be defined by mutation at yet another extracellular loop on AE-1 (Figure1, vide supra) these results provide further support for the contention that the mutation which gives rise to AE-1 SA0 has a dramatic affect on the membrane domain of band 3 as a whole. SA0 mutation affects The results of Booth et d ( 1 9 ) also raise the possibility that the AE-1 the organisation of several other red cell membrane proteins. In particular, they report depression of Rh, S, S, U and LW antigens. There is now considerable evidence that under certain circumstances bothAE-l and AE-1 SA0 associate with GPA (18,20,21,22). Since GPA forms heterodimers with Glycophorin B (GPB) and there is evidence that GPB is associated withthe Rh 50Kd glycoprotein (23,24) a large complex involving AE-1, GPA, GPB, Rh 50Kd glycoprotein, Rh 30Kd polypeptides and LW glycoprotein seems possible (Figure 2). The depressed Rh, S, S, U and LW antigens noted by Booth er a1 (19) may be a consequenceof an organisational change in this complex resulting from the AE-1 SA0 mutation. Preliminary quantitative binding studies suggest thatSA0 cells have a reduced number of epitopes for theRh monoclonal antibody BFUC 69 (11).
THE INTERACTION BETWEEN GLYCOPHORINS CID AND PROTEIN 4.1 The first evidence of an interaction between Glycophorins CID (GPC/D) and protein4.1 was provided by Mueller & Morrison (25) who observed that while Glycophorin C (syn. Glyconnectin) is normally present inTriton shells, it was not presentin Triton shells prepared from an individual with total protein 4.1 deficiency. Subsequent studies demonstrated reduced levels of GPCID in the membranes o f protein 4.1 deficient individuals (26). Recently, Pinder er a1 (5) have shown that protein4.1 can be more readily extracted from the membranes of individuals with GPC/D deficiency (Leach phenotype) than from normal membranes. Nevertheless, other candidates for protein 4.1 membrane interaction havebeen proposed including AE-1 (27-29), Glycophorin A(30) and the lipid bilayer itself (31,32). We have examined the protein 4.1 interaction with GPClD in an in vitro ELISA system by comparing the bindingof purified protein4.1 to normal and Leach phenotype membranes which have been depletedo f endogenous protein 4.1 either by treatment with 0.1M NaOH or lMKCl (6). The results clearly demonstrated a dramatic difference between normal and Leach phenotype membranes. Protein 4.1 bound with high affinity to normal membranes (Kd 6.25 x 10-8M) while the binding to Leach phenotype membranes had Q a 50 times lower.
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ANSTEE, HEMMING, AND TANNER
Table I Selective depression of blood group antigens associated with Hereditary Ovalocytosis among Melanesiansfrom ~ 0 0 t het d , (19) Depressed antigens
mI Ena, f LW, D, C,
IT IF,
e, S,
Antigens not depressed A,, ID, i, P,, M, N, Lub, k, Fya, CO*V-
Spectrin
S,
U, Kpb, Jka, ab, Xga, Scl
RED CELL MEMBRANE These results led us to examine the region ofGPC/D involved in the binding. Three synthetic peptideswere prepared corresponding to theentire cytoplasmic domainof GPC/D. One peptide (GPC-l) corresponds to GPC residues 112-128, GPC-2to residues 99-111 and GPC-3 to residues 82-98. Each peptide was tested for its ability to inhibit the binding of purified protein 4.1 to protein 4.1 depleted normal membranes. GPC-3 was by far the most potent inhibitor (up to 80% inhibition of protein 4.1 binding). Furthermore, GPC-3 was shown to bind directly to purified protein 4.1. GPC-3 also bound directly to Leach phenotype membranesprior to protein 4.1 depletion but not to normal membranes or Leach membranes depleted of protein4.1, suggesting that the GPC-3 binding site on protein 4.1 is not occupied in Leach phenotype membranes. These results led us to conclude thatdirect interaction between protein 4.1 andGPC/D is mediated by the protein sequence on GPC/D located within residues 82-98 (6). Recently Alloisio et a2 (33) showed that red cell membranesfrom individuals deficientin protein 4.1 and also those deficient in GPC/D lack an additional protein, p55. This protein (p55) has been cloned and sequenced (34). In order to investigate the possible significance of p55 in GPC/D interactions with protein4.1, we have purified p55 from normal membranes and examined the ability of peptides GPC-1, -2 and-3 to bind to it. The results show that GPC-1 binds strongly to p55 in a concentration dependent manner whilst binding of GPC-2 and GPC-3 is much less (35). These results are in contrast to GPC binding to protein 4.1 where GPC-3 binds most strongly. Protein 4.1 purified from normal membranes may contain small amounts of p55 andso in order to study the bindingof GPC peptides to protein 4.1 in the complete absenceof p55 we repeated our earlier experiments using protein 4.1 purified from Leach phenotype membranes. The results clearly demonstrated that GPC-3 binds directlyto protein 4.1 and suggested that low levels of GPC-1 and GPC-2 binding in protein4.1 preparations prepared from normalred cell membranes may be due to low level p55 contamination. These results clearly show that protein 4.1 and p55 bind to GPC at different sites. Next, we turned our attention to the binding siteson protein 4.1 for GPC and p55. Purified protein 4.1 was digested with chymotrypsin and the 30Kd N-terminal fragment purified from other fragments by immunoaffinity chromatography using a rabbit anti-30Kd domain. The purified 30Kd fragment bound to both GPCand p55 whilst protein 4.1 fragments depleted of the 30Kd fragment did not bind (35). These results are in agreement with those of Marfatiaer aL(36). These results show that the 30Kd domainof protein 4.1 can bind directly to the region of GPC-3 and also indirectly (residues 112-138), through p55. In order to determine if the 4.1 binding site for GPC is distinct from thatfor p55 the isolated 30Kd fragment ofprotein 4.1 was incubated with GPC-3 under saturating conditions and the effect on p55 binding determined. The results showed that p55 binding is not inhibitedby bound GPC-3 and that therefore the 30Kd domain of 4.1 contains two distinct binding sites, one for GPC and one for p55 (35).
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The location of p55 binding site to the extreme C terminus of GPClD is entirely consistent with the resultsof Pinder et aZ.(5) who showed that after extensive proteolysis of normal red cell membranes, GPC/D is cleaved between the transmembrane and cytoplasmic regions but that the fragment containing the C terminus remains bound to the membrane. This fragment was identifiedby immunoblotting using a monoclonal antibody (BGRL 100) to the synthetic peptide GPC-1 corresponding to residues 112-128 of GPC (37). In order to explain the retentionof this fragmentin the membrane Pinderer aZ(5) postulated binding to p55. Both the p55:GPC/D interaction and protein 4.1:GPCID interactions are of high affinity (6,36). Recent quantitative binding assays using Fab fragments of monoclonal antibodies to extracellular epitopes on GPCand GPD suggests thatthere are about 225,000 GPC and GPD molecules on normal red cells(38) and so protein 4.1 and GPC/D are present in equal amounts in normal red cell membranes. In contrast, available evidence suggests that thereare only approximately 80,000 p55 monomerslnormal red cell membrane (33). At the present time it is not clear whether a singleprotein 4.1 molecule can bind simultaneously to GPC directly (through the GPCS region) and via p55, or whether two moleculesof protein 4.1 can simultaneously bind a single GPC molecule via amino acids 82-98 of GPC and p55 respectively. A proportion of Leach phenotype redcells are elliptocytic (39) and this has beenattributed to a reduced levelof protein 4.1 (ca 80% o f normal) in these cells (33). Recent studies by Discher er aL(40) have shown that additionof the spectrin binding lOKd domain ofprotein 4.1 can normalise the mechanical abnormalities of Leach phenotype red cells. The function of GPC/D/4. Up55 interaction, therefore, remains unknown.
CONCLUDING REMARKS In this paperwe have considered how the study of red cell alloantigens can give useful information about functional propertiesof the red cell membrane and particularly about the relationship between red cell membrane proteins and the underlying skeleton. In 1977 Booth and colleagues (19) described the selective depression of several genetically unrelated blood group antigens in individuals with ovalocytic redcells found in the coastal regions of Papua New Guinea (Table I). They sought to explain their findings by proposing that "a membrane anomalyexists, and that the series of depressed determinants all depend, for their full expression, upon the same membrane components(s), theproper synthesis of which is being genetically affected". It is now clear that a mutationin the AE1 gene is the underlying causeof these changes and thedirect demonstration that the Diego and Wright antigens are defined by AE-l provides a clear link between the band3 mutation and the diverse array of surface changes documentedby Booth et a2 (19). These results are not simply a satisfying confirmationof the power o f red cell serology in skilled hands, they provide new insights into the organisationof red cell proteins withinthe membrane by pointing to those proteins which may exhibit some functional interdependence [for example, AE-l, GPA, GPB, Rh(SOKd), Rh(30Kd), Figure 21.
RED CELL MEMBRANE
195
About 35% of AE-1 in normal red cells is immobile and this contrasts with65% in SA0 cells. Nevertheless, the ankyrin binding region of AE-l SA0 is unaffected and it is the deleted segmentat the membrane interface which is responsiblefor the reduced mobility. AE-l from SA0 cells contains a higher proportionof tetramers (50%)than normal AE-1 (33%) (41). The absence of this deleted segment in AE-l SA0 probably results in abnormal assembly of the membrane domain (as evidenced by abnormal expression of extracellular epitopes) leadingto increased homo- and hetero-associations between integral membrane proteins which in turn result in reduced membrane flexibility. An awareness of this organisational relationship between proteins at the cell surface is likely to be of particular relevance to the interpretationof experiments which show that binding of antibodies to extracellular epitopes on AE-1 andGPA induces membrane rigidity and inhibits red cell invasion by malarial parasites (42-44). By no means all of the blood group antigens examined by Boother aL(19) were found to be depressed on SA0 cells (Table I). The Gerbich antigens whichare located on GFWD were not depressed. It is often the case that rare individuals whose cells lack all of the antigens of a particular blood group system (null phenotypes, reviewedby Issitt (45) provide the most useful information concerning the function of particular membrane proteins. Individuals of the Leach phenotype were first examined because o f their Gerbich negative phenotype and shown to represent an unusual subset of Gerbich negative individuals becauseof the failure of their red cells to react with monoclonal anti-GPC(40). As discussed above, these cells have been extremely valuable in elucidating the nature of GPClDlprotein 4.11~55interactions and establishing that GPClD rather than AE-1 or GPA provide the major attachmentsites for protein 4.1 in the normal red cell membrane(Figure 2). REFERENCES
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D. M. Gilligan and V. Bennett, Semin. Hematol., 3,74-83 (1993).
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S.-C. Liu, L. H. Derick, Semin.Hematol., 29, 231-243(1992).
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P. Jarolim, J. Palek, D. Amato, K. Hassan, P. Sapak, G. T. Nurse, H. L. Rubin, S. Zhai, K. E. Sahr, S.-C. Liu, Proc. Natl. Acad. Sci., B, 11022-11026 (1991).
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2:Recent C 25. T. J. Mueller and M. Morrison in Wthrocvte M-es m e n t a l Advances, W. C. Kruckeberg, J. W. Eaton, G. J. Brewer, eds, Alan R. Liss, New York (1981). 26. N. Alloisio, L. Morle, D. Bachir, D. Guertarni, P. Colonna, J. Delauney, Biochim. Biophys. Acta., Hlh,57-62 (1985). 27. G. R. Pasternack, R. A. Anderson, T. L. Leto, V. T. Marchesi, J. Bid. Chem., 3676-3683 (1985). 28. T. Jons, D. Drenkhahn, EMBO. J., U, 2863-2867
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29. C. R. Lombardo, B. M. Willardson, P. Low, J. Biol. Chem., 30. R. E. Lovrien, R. A. Anderson, J. Cell. Biol.,
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39. D. J. Anstee, S. F. Parsons, K. Ridgwell, M. J. A. Tanner, A. H. Merry, E. E.
Thornsen, p. A. Judson, P. Johnson, S. Bates, I. D. Fraser, Biochem. J.,
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Mohandas, Blood, 8 2 , suppl. 1,p. 309A (1993). 41. V. E. Sarabia, J. R. Casey, R. A. F. Reithmeier, J. Biol. Chem., 2 6 8 , 10676-10680 (1993).
42. G. Pasvol, J. A. Chasis, N. Mohandas, D. J. Anstee, M. J. A. Tanner, A. H. Merry,
Blood, 74, 1836-1843 (1989). 43. K. Rangachari, G. H. Beaven, G. B. Nash, B. Clough, A. R. Dhuzewski, 0. Myint,
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44. J. A. Chasis, S. McGee, D. J. Anstee, N. Mohandas, Blood, 8 2 , suppl. 1, Abstract 683 (1993). 45. P. D. Issitt, Transf. Med. Rev.,
1,139-155(1993).
HOT
SPOTS IN THE
MOLECULAR
RED
CELL
MEMBRANE:
ASPECTS OF SOME RED CELL
ANTIGENS
Geoff Daniels MRC Blood Group Unit, Wolfson House,
4 Stephenson Way, London, NW1 2HE. UK ABSTRACT After decades of studying the human blood groups by serological and, more recently, biochemical techniques, analysis of blood group genes at the molecular level has confirmed that a variety of different genetical events have given rise to the vast complexity of blood group systems. In order to 4 blood group systems have been selected: AB0 and H, illustrate this involving carbohydrate determinants, and MNS and Rh, involving predominantly protein antigens. The molecular basis of the A I , A2. B. and 0 groups, and of the rare H-deficiency phenotypes will be described. The S t a antigen of the MNS system will be discussed in order to illustrate the variety of different genetic mechanisms that can give rise to a single rare antigen. Finally, recent work on the molecular basis of the polymorphic Rh antigens, D , C, c , E, and e, and on some rare Rh phenotypes, Rhnu1l, D--, and r's. wjll be explained briefly in order to emphasize the complexity of blood group genetics. THE
AB0AND H BLOOD
GROUP
Blood groups are nearly a century old. sera of some people agglutinated
SYSTEMS
In 1900 Landsteiner noticed that
the red cells of some others and
the
discovery of the AB0 groups followed. AB0 is the most important system in blood
transfusion as
transfusion purposes.
it
is
imperative
that matched
blood
be
used for
A and B antigens may be glycolipid or glycoprotein.
with the antigen expressed within the carbohydrate portion of the molecule. Consequently the A and B genes do not encode the A and B antigens directly, but
code for transferase enzymes which catalyze
the
addition
of
monosaccharides to a specific oligosaccharide chain. The Type
1 H and Type
2 H oligosaccharide chains in Table I represent
precursors of A and B and are abundant antigenwhich
wil I
be
discussed
in group 0 people.
below.
199
The
A
They express H
gene produces
an
200
DANIELS TABLE I H, A. and
B active oligosaccharides
Type l H
Fucal-2Galp1-3GlcNAcpl-R
H
Fucal-2GaIB1-4GlcNAcpl-R
2Type A
Fucal-2Galp1-3/4G1cNAcpl-R 3
I Ga 1NAcal
B
Fucotl-2GaI~l-3/4GlcNAc~l-R 3 I
Ga la1
N-acetylgalactosaminyltransferase
which catalyzes
N-acetylgalactosamine from a nucleotide donor,
which catalyzes
the
transfer
of
UDP-N-acetylgalactosamine,
of an H acceptor,
to the fucosylated galactosyl residue A-active molecule. Likewise,
the
to produce an
the B allele produces a galactosyltransferase to the H
transfer of galactose from UDP-galactose
acceptor to produce the B-active molecule. The 0 allele produces no active enzyme, and so in group 0 people the H-active precursors remain unconverted to A or B. Following the purification of A-transferase from human lung tissue, and the acquisition
of
partial
amino acid sequences, Yamamoto
et
al.
Cl1
isolated A cDNA from a cDNA library constructed from a human stomach cancer cell line expressing high levels of A antigen. Subsequently were also cloned
B and 0 cDNAs
and sequenced. Nucleotide sequences predicted proteins
with a 3 domain structure characteristic of a glycosyltransferase.
Ai and B cDNAs differ by 7 nucleotide substitutions, 4 of which predict amino acid substitutions [ l 1 266, and 268.
(Table 1 1 ) .
These are
at residues 176, 235.
By a process of site-directed mutagenesis and transfection
into group 0 HeLa cells, Yamamoto and Hakomori
C21 demonstrated that the
amino acid substitutions at position 266 and,
to a lesser extent, 268 are
most
important
in
determining whether
N-acetylglactosaminyltransferase
the gene product has predominantly
or galactosyltransferase activity. The
0
sequence was identical to the Ai sequence, apart from a single nucleotide deletion. This causes a reading frame shift at codon 86 and the generation of a stop codon at codon 117. explaining why no active glycosyltransferase is produced by the 0 allele.
20 1
MOLECULAR ASPECTS OF RED CELL ANTIGENS TABLE II Nucleotide differences between Ai, B, and 0 cDNA, and the resulting amino acid changes in the protein products
261 297 526 657 703 796
G
Ai
803
AC
C
G
C
Ars
G Gly
Leu Gly
266235176
G
B
G G
268
T
A
A
C
GlY Ser
"_"
0
C
G
C
G
G
117
A2 cDNA differs from A1 cDNA by (nucleotide 1059,1060,
A Met Ala 268
266235176
nt 261 G deletion & AC
930
G
a
single base deletion near the 3' end
or 1061) C31.
This deletion, like that responsible
for the 0 gene, results in a frameshift; but this time i t abolishes a stop codon so the A2
extraneous 21 amino acid residues.
transferase ha3 an
These changes greatly reduce the N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e activity of
this protein.
There is also a
single base change responsible for
converting Pro-156 in Ai transferase to Leu in Az transferase, but this has little effect on enzyme activity. In group 0 people
the acceptor
substrate of A-
and B-transferases
remains unmodified and this structure expresses H antigen (Table I ) .
The
immunodominant sugar of H is the terminal fucose, and this is added to its precursor by an otl-2fucosyltransferase. There
are
very
rare
red
cell
phenotypes
in which
H
antigen,
and
consequently A and B antigens, are not present on the red cells due to otl-2fucosyltransferase deficiency.
A. B. and H antigens have been called
histo-blood group antigens as they are present in many tissues, and they are also present in soluble form in body secretions. The presence or absence of H in secretions is polymorphic, this polymorphism controlling A and B antigen expression in secretions when the appropriate A and B genes are
present.
In
about
20% of
people,
so
called
non-secretors,
no
otl-2fucosyltransferase is present in the secretory tissues, no H substance
202
DANIELS
is produced, and no A or B can be synthesized
.
People with H deficiency of
their red ce11s may be secretors or non-secre tors of H. The
most
likely
explanation
for
these
two kinds
of
H
deficiency
phenotype involves 2 crl-2fucosyltransferases: one the product of the FUTl gene, active in ectoderm and mesoderm and responsible for H on red cells; the other the product of the FUT2 gene, active in endoderm and responsible for
H in secretions.
(Galp1-4GlcNAc)
The FUTl product is specific for a Type 2 substrate
FUT2
andthe
productis
more
active
onaType
1
I t is probable that these two genes are very
(GaIpl-3GlcNAc) substrate.
closely linked on chromosome 19.
Lowe's group in Ann Arbor C4-61 used a
gene-transfer method to isolate one of the ul-2fucosyltransferase genes, and the expressed product had the catalytic properties expected of the red cell H-transferase ( F W f product). Kelly et al.C71 demonstrated that an individual H deficient on red cells and in secretions (Bombay phenotype), change
within
the
H gene which
termination codon (Table 1 1 1 ) .
was homozygous for a single base
replaced
Tyr-316
with
a
translation
The resulting polypeptide would lack 50
amino acids from its C-terminus.
Tranfection experiments showed that this
mutated gene did not produce an active enzyme.
Two secretors of H with
H-deficient red cells also had point mutations within the FUTl gene, one encoding an amino acid substitution, the other creating a premature stop codon (Table I l l ) . Johnson C 8 1 screened the H fucosyltransferase gene for mutations by PCR with
4 sets of primers covering the single coding exon of the gene,
followed
by
denaturing
gradient
gel
conformational analysis, and direct
electrophoresis,
sequencing.
Only a
single
strand
single synonomous
base change was found in over two hundred donors of normal ABH phenotypes. Nine different coding mutations were found in around 60 propositi and their immediate families with H deficient red cells; some secretors and some nonsecretors.
Mutations
were
concentrated within the 3'
found
end.
throughout
the gene.
the
majority
Five mutations have been determined by
nucleotide sequencing and are shown in Table 1 1 1 .
Some encode amino acid
substitutions, others consist of one and two base deletions and a resulting frame shift.
The one
non-secretor
was
of
European origin, no
coding
mutations were detected in the classical Bombays. the true H deficient nonsecretors of Indian origin.
So what do these data tell us?
Firstly. they strongly the support the
idea of two ul-2fucosyltransferase genes cells, the other in secretions
-
-
one producing H antigens on red
because most of these mutations are in
individuals with normal H in their saliva.
Secondly, they confirm that the
MOLECULAR ASPECTS
OF ANTIGENS RED CELL
203
TABLE I I I Mutations within the H (FU7'1) gene associated with red cell H deficiency
Kelly et a1 C71 Tyr-316
ter
-f
Non-secretor
Leu-l64 + His
Secretor
ter
Secretor
Gln-276
-f
Johnson C81 Trp-267 + Cys
Non-secretor
Ala-l10 + Thr
Secretor
Asn
Secretor
Asp-278
-f
codon 294
Secretor
CTG + CT codon 330
Secretor
TTT
u1-2fucosyltransferase
-f
T
gene
that was
active in red cells (F'LIT1).
cloned and sequenced
is the gene
Thirdly, they demonstrate that single amino
acid substitutions can have quite severe effects on the activity of the al-2fucosyltransferase product of this gene, particularly when close to the N-terminus.
The failure by Johnson 181 to detect any mutation in the true
H-deficiency non-secretors of Indian origin suggests there may be more than one mechanism mutation in the
responsible for
this phenotype.
Perhaps one
involves a
promoter or some other critical non-coding region of the
gene. THE MNS BLOOD GROUP SYSTEM
MNS i s a highly complex system. of them of low
frequency.
I t comprises at least 38 antigens, most
These are located on either or both of two
sialic acid rich red cell membrane glycoproteins called glycophorin A (GPA) and glycophorin B (GPB). GPA and GPB are encoded by discrete, closely linked homologous genes called GYPA and CYPB (Fig. l ) .
There is a third gene in this complex,
GYPE. which may produce low levels of a third glycoprotein glycophorin E. CYPA has 7 exons and
GYPB has 5
exons.
Exons A2 and B2 encode
the
extracellular N-terminal 26 amino acids, which are identical in GPB and the N form of GPA.
Exons A3. A4.
and B3 encode the rest of the extracellular
domains, exons A5 and B4 encode the transmembrane domains, and exons A6. A7, and B5 encode the cytoplasmic domains.
There is a region within the
204
DANIELS A2
A3
A4
A5
A6
r””.
E3
E4
A7 3’
GYPA
El
E2
GYPE
Y
Y
FIGURE l Diagram showing the exons of GYPA, GYPB. and GYPE. and the pseudoexons of GYPB and GYPE.
(v)
second intron of GYPB, called the GYPB pseudoexon, which is homologous to
I t is not expressed because there is
exon A3 of GYPA. S‘spicing signal of
a mutation in the
what would be intron 3. and the exon-like region is
spliced out and not represented in the mRNA or in the resulting protein. There are a number
of different genetic mechanisms responsible
for
altering GYPA and GYPB and explaining how so many red cell antigens arise from these 2 genes, including a number of rare phenotypes in which more than one low incidence antigen is expressed.
In some cases expression of
low incidence antigens may arise from simple point mutation, but in others there is a rearrangement of GYPA and GYPB to produce hybrid glycophorins. These rearrangements may occur as
a result of gene misalignment and pairing
of homologous regions of the 2 genes. followed by unequal crossing-over or gene conversion.
This may also lead to
the activation of the normally
si 1 ent GYPB pseudoexon. The rare MNS antigen St= (MNS15) arises by a number of different genetic mechanisms.
From
the
1960s i t
was known
that St= was
almost
always
associated with an N antigen, but that in one family i t was associated with an unusual M antigen phenotype was
C91.
Anstee et al.
associated with
an abnormal
Cl01 showed that the Stcat)
glycophorin
structure which
appeared to consist of the N-terminal domain of GPB and the C-terminal domain of GPA. crossing-over
They proposed that this arose as a between
homologous
genes.
confirmed by DNA analysis C11-131. occurs between them, 2 fusion genes
This has
result of unequal subsequently
been
If 2 genes misalign and crossing-over result: one which encodes a hybrid
glycophorin with GPA at its N-terminus end GPB at its C-terminus; and one the other way round (Fig. 2 ) . GYPA and GYPB.
The latter fusion gene is flanked by normal
In the example shown in Fig. 2 , the GYPCB-A) fusion gene
encodes the St= hybrid glycophorin.
Huang and Blumenfeld C141 have shown
MOLECULAR ASPECTS OF RED CELL ANTIGENS
205
8
FIGURE 2 Diagrammatic representation of chromosomal misalignment and unequal crossing-over at the third intron of GYPA and the homologous region of GYPB. The black boxes represent GYPA exons, the white boxes GYPB exons, and the hatched boxes the GYPB pseudoexon.
that intron 3 of GYPA and the homologous intronic region of GYPB represents a recombination hot-spot.
They identified 3 types of St. fusion gene, all
producing an identical protein product, but with different recombination sites within
the
intron.
Twoof
these
Americans, the other in Japanese.
St.
types were
found
in African
is almost always associated with N
antigen; no surprise as GPB almost always has N GYPB pseudoexon is not expressed, so the St.
at its N-terminus.
The
fusion gene produces a protein
with the product of exon B2 fused to the product of exon A4. The
amino
acid
responsible for the St.
sequence
-Gln-Thr-Asn-Gly-Glu-Arg-
is
I t does not exist in normal
antigen.
but occurs when the product of the
probably
GPA or GPB.
3' end of exon B2 (-Gln-Thr-Asn-) is
fused to the product of the 5' end of exon A4 (-Gly-Glu-Arg-). One family (MZ), first studied in the 1960s. had St. associated with an abnormal, trypsin resistant M N-terminus, so Dahr et al.
antigen
C151
C91.
GPB
never has M
at
its
suggested that the abnormal St.-active
glycophorin jn this family, instead of being a GP(B-A) hybrid, may be a GPA molecule in which the product of exon A3 is missing. exons
2
of
associated
GYPA
amino
and
GYPB are
acid
sequence.
identical,
As the 3' ends of
this would
This proposal
was
create
the St.
confirmed
byDNA
analysis when Huang et al. C161 identified the mechanism responsible for lack of representation of exon A3 in the abnormal glycophorin.
The variant
gene is GYPA in which the whole of exon A3 plus the 5' end of intron A3 has been replaced by the homologous region from a
GYPB gene (Fig. 3).
Once
206
DANIELS
GY P(A-B-A) FIGURE 3 Diagram to demonstrate proposed mechanism responsible for the abnormal glycophorin gene in M.Z. which produces an St. and M active GPA molecule of reduced size. As a result of gene conversion exon A3 and the flanking intronic regions of GYPA are replaced by the homologous region from GYPB, including the GYPB pseudoexon. No exon A3 is expressed by the resulting GYP(A-B-A) gene and an St.-active glycophorin molecule is produced.
again the recombination hot-spot in intron 3 is involved.
This means that
the functional splice site at the 5' end of intron A3 is replaced by the mutated splice site of GYPB. just as
So the third exon has become a pseudoexon,
i t is in a normal GYPB gene.
A2-A4 junction. and hence the St.
The resulting protein has the exon
antigen.
The M antigen is derived from
exon A2; i t is trypsin resistant because the trypsin cleavage site lies within the absent exon A3 encoded domain. Huang et al. C161 suggest that the
exchange of genetic material which
has created the abnormal GYPCA-B-A> gene in the result of gene conversion.
MZ family occurred as a
Although the mechanism for gene conversion is
unknown, i t involves the non-reciprocal exchange of genetic material from one gene to a homologous region of another. genes become misaligned breaks (Fig. 4).
I t may occur when homologous
at meiosis and one strand of the heteroduplex
The broken strand invades and pairs with a strand of the
opposite gene, and then acts as a template for repair of the region which is now unpaired.
The broken strand is repaired normally and the invading
strand degraded.
Other mechanisms, involving double-strand breaks, have
also been proposed C 1 7 1 . We have recently identified a new MNS low incidence red cell antigen called ERIK
C181.
In the four families with ERIK+ members, all ERIK+
individuals were St(a+) and all ERIK- individuals were Stca-). St(a-)
No ERIK+
person has been found, although most Stcat) pepople are ERIK-.
-
MOLECULAR ASPECTS OF RED CELL ANTIGENS
207
3"
5'
3' 5' 3'///,,/////////////////////////////////,/~ 5' 5'7,/////////////////////////////////////,3'
FIGURE 4 A possible mechanism to explain the non-reciprocal exchange that occurs as a result of gene conversion. Homologous regions oftwo genes become misaligned during meiosis, one strand breaks and pairs with the opposite gene, the broken strand is repaired normally, but the unpaired strand is repaired using the invading strand as a template.
lmnunoblotting of red cell membranes provided unexpected results. anti-St=
imnunostained
components
normally associated with St.. molecule.
characteristic
of
the GPCB-A)
Whereas hybrid
anti-ERIK bound to an apparently normal GPA
So one rare MNS haplotype
located on two different proteins.
was producing
two rare antigens
Once again analysis of the
how this had occurred [l91 (Fig. 5). In
DNA showed
ERIK+ people a GYPA gene contains
a point mutation, a G to A change in the most 3' nucleotide of exon A3. This results
in a Gly-59
responsible for molecule.
to Arg substitution, which is presumed to be
the ERIK antigen on an
otherwise normal
glycophorin A
However, the mutation also causes partial disruption of the 5'
splice site of intron A3, resulting in some degree of exon skipping: some
of the mRNA lacks exon A3 and so some of the resulting protein lacks the product of exon A3.
This produces a GPA molecule of reduced size with the
exon 2-4 junction responsible for expression of the St. antigen. Description of St. has shown howone rare antigen of the MNS system can arise from a variety of genetic mechanisms: unequal crossing-over, gene conversion, point mutation, and exon skipping. been shown to be
responsible for many of
Similar mechanisms, have
the other variant phenotypes
within this system and for their associated low frequency antigens.
208
DANIELS G I y+A rg ERIK
t GY PA
FIGURE 5 Diagram to show how a point mutation in exon 3 of GYPA results in the production of at least 2 abnormal glycophorin products: ( 1 ) an apparently normal GPA molecule apart from Arg-59 which is probably responsible for expression of the ERIK antigen (above); (2) a GPA molecule lacking the product of exon A3 and expressing St. antigen (below), due to exon skipping as a result of the mutation disrupting the consensus spice site.
THE Rh BLOOD
GROUPSYSTEM
L i k e MNS, there are at least two genes encoding the red cell antigens of
the Rh system: one encoding D antigen, the other the C and E antigens.
In
D-positive people both genes are present, but in D-negative people only one of the genes is present, explaining the absence of any antigen allelic to D
C201.
The rare D-- phenotype, in which D antigen. but no C, c, E. or e
antigens are detected on the red genetical background.
cells, appears to have more than one
Six unrelated individuals with the D-- phenotype
were analyzed by Blunt et al. C21,221: in 5 a D gene was present and most of the C€ gene is deleted; but in one D-- individual and her D-- brother apparently normal D and CE genes were present. Colin et al.
In one example of D".
1201 also found that both genes were present.
The Rhaull
phenotype, where no Rh antigens are expressed, was shown by family studies to have 2
genetical backgrounds: one resulting from homozygosity for a
silent or amorph gene at the RH locus; the other due to homozygosity for a rare gene at a regulator locus unlinked to
RH.
In the amorph type of
Rhnu11 a CE gene. presumably inactive, is present and may have a C or c
MOLECULAR ASPECTS OF RED CELL ANTIGENS
209
background in different families: no D gene is present C23,241. regulator type of Rhnull
In the
just the C€ genes were
either both genes or
present C231. The Rh proteins are highly hydrophobic and span the red cell membrane 12 times, with cytoplasmic N- and C-termini and up to 6 extracellular domains C251.
They are fatty acylated, but not glycosylated.
amino
acid
sequence
at
any part of the
Minor changes in the
molecule
may
result
in
conformational changes elsewhere and this may explain, in part, the extreme serological complexity of the Rh system. Mouro et al. C261 found that a single base substitution in exon 5 of the C€ gene, resulting in an amino acid substitution at residue 226, determines
the E/e polymorphism and that 6 nucleotide substitutions in exons l and 2 are associated with C/c polymorphism, 4 of them resulting in amino acid substitutions (Table IV). Mouro et al.
Only residues 226 and 103 are extracellular.
C261 believe that Cc and Ee
antigens reside on different
proteins, despite being encoded by the same gene.
They explain this by
postulating that at least 3 different transcripts arise from the C€ gene. in 2 of which exon skipping has occurred.
They propose that the product of
the transcript containing all 10 exons of the C€ gene expresses E or e, but although exons 1 and 2 are both present the protein does not have the When exon 5 is missing. as i t
correct conformation for C or c expression.
is in the other 2 transcripts, the resulting protein cannot express E or e, Although mRNA
but the conformation is then suited to C or c expression.
molecules of reduced size have been detected, no proteins representing these transcripts have been found and so this model remains speculative. The red cells of some D-positive people fail to react with some anti-D These cel Is appear to lack part of the D antigen and have been
reagents.
called partial capable
of
D.
making
The individuals with antibodies
therefore, behave as anti-D.
which
these unusual D antigens are
react
with
normal
D
antigen
and,
There are numerous varieties of these partial
D antigens, the least rare of which is a heterogeneous collection called category V I . Mouro et al.
associated with a D gene
C271 have found that DV1 is
lacking exons 4 , 5 , and 6. This occurs in two ways: ( 1 ) a deletion of the 3 exons; or (2) the replacement of the 3 exons with the equivalent 3 exons of
the
C€
gene, probably
intergenic crossing-over. apparent
molecular
weight
as
a
result
of
gene
conversion
or
double
Strangely, both produce a protein of identical as
determined
by
SDS
polyacrylamide
gel
electrophoresis, and of the same apparent molecular weight as the normal D protein.
The deletion type is always associated with c and E (DvlcE): the
210
DANIELS TABLE IV Amino acid substitutions inthe CE protein associated with the E and e, and C and c polymorphisms C261
Amino acid cC
Encoding e E exon
16
l
60
Cys I le
Trp Leu
2
68
Ser
Asn
2
103.
Ser
Pro
2
226.
Ala Pro
5
= extracellular
conversion type with C and e ( D v l C e ) .
Lomas and Mougey C281 identified an
antibody, called anti-BARC, which generally reacted with the Dv'Ce
complex,
but
exceptions. Presumably
not
a
DvrcE
product of a
complex,althoughthereweresome
i t was detecting part of the C E g e n e insert, or the
product of one of the junctions of the insert and the D exons. Finally, an unusual gene complex often called r'Caucasians,
but
relativelycommon
in people
or dCces. very rare in
of Africanorigin.
In
a
simplified form, r's produces no D, both c and a form ofC. and an unusual e called e.
(or V S ) .
Dr Ben Carritt of the MRC Human Biochemical Genetics
Unit and his colleagues
C291 made the following observations. Firstly,
that the D gene is not entirely absent; sequences from the 5' and 3' ends of the gene, probably involving exons 1 and 2 and 8 to 10. are present. appears that the D gene has a large internal deletion. We do
It
not know if
the deleted segment is replaced by the equivalent segment from the C€ gene as
a
result
Secondly,
a
of
doubleintergeniccrossing-over
restriction fragment
length
this
Perhaps the C is produced by the rearranged D
gene. Thirdly, a mutation within exon responsible for e.
geneconversion.
that the C€ gene of
correlates with C and c expression suggests complex does not encode C.
or
polymorphism which always
5 of the CE gene which is probably
(VS) expression. CONCLUSION
The collection
of phenotypes and antigens belonging
systems described here illustate some mechanisms available for
to 4 blood group
of the variety of different genetic
the diversification of cell
surface proteins and
MOLECULAR ASPECTS
OF ANTIGENS RED CELL
211
The final example, r’s of the Rh system, demonstrates the
glycoproteins.
complexity of blood group genetics. Our extremely detailed serological knowledge of invaluable
the complexities
in
the
of blood group antigens
understanding
of
the
molecular
is now proving biology
of
the
diversification of cell surface antigens. ACKNOWLEDGEMENTS I am extremely grateful to Dr Ben Carritt of the MRC Human Biochemical
Genetics Unit and to Dr Philip Johnson and Ms Fiona Steers of the MRC Blood Group Unit for permitting me to quote their unpublished work. REFERENCES 1.
2. 3.
F. Yamamoto, H. Clausen, T . White, J. Marken and S. Hakomori. Nature, (1990).
345. 229-233
F. Yamamoto and
S. Hakomori. J Biol Chem,
265,
19257-19262 (1990).
F. Yamamoto, P.D. McNeill and S. Hakomori. Biochem Biophys Res C o m , 366-374 (1992).
187, 4.
L.K. Ernst. V.P. Ra jan, R.D. Larsen, M.M. Chem, 2 6 4 , 3436-3447 (1989).
5.
V. P. Ra jan, R. D. Larsen, S. Aimera, L.K. Erns t and J.B. Lowe. J Biol Chem. 2 6 4 , 11158-11167 (1989).
6.
R.D.Larsen, L.K. Ernst, R.P. USA, E, 6674-6678 (1990).
7.
R.J. Kelly, L.K. Ernst, R.D. Larsen. J.G. Bryant, J.S. Robinson and J.B. Lowe. Cited by Lowe, in Innnunobiology of Transfusion Medicine, G. Garratty, ed, Dekker. New York, (1994) pp. 3-36.
8.
P.H. Johnson, personal comunication (1994).
9.
M.N. Metaxas. M. Metaxas-Buhler and E.W. (1968).
Ruff and J.B. Lowe. J Biol
Nair and J.B.
Lowe. Proc Not1 Acad Sci
Ikin. Vox Sang, l5, 102-117
10. D.J. Anstee, W.J. Mawby, S.F. Parsons, M.J.A. Tanner and C.M. Giles. J Imnunogenct, 9 . 51-55 (1982). 1 1 . C.H. Huang, M.L. Guizzo, 836-843 (1989).
M. Kikuchi and 0.0. Blumenfeld. Blood, 7 4 ,
12. A. Rearden. H. Phon, T. Dubnicoff. S. Kudo and M. Fukuda. J Biol Chem, 2 6 5 , 9259-9263 (1990). 13. C.H. Huang and 0.0. Blumenfeld. Blood, 77, 1813-1820 (1991). 14. C.H. Huang and 0.0. Blumenfcld. J Biol Chem.
266,
23306-23314 (1991).
15. W. Dshr. D. Blanchard. C. Chevalier, J.P. Cartron, K. Beyrcuther and B. Fournet. Biol Chem Hoppe-Seyler, 3 7 1 , 403-410 (1990). 16. C.H. Huang, M.E. Reid and 0.0. Blumenfeld. J Biol Chem.
268,
4945-4952
(1993).
17. P. Kourilsky. Trends Genet, 2. 60-63 (1986). 18. G.L. Daniels, C.A. Green, J. Poole. D. J c m e . E. Smart, D. Wi lcox and S. Young. Transfusion Med, 3 , 129-135 (1993).
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19. C.H. Huang, M. Reid, G. Danielsand 0.0. Blumenfeld. J Biol Chem, 25902-25908 (1993).
268.
20. Y. Colin, B. Ch6rif-Zahar. C. Le Van Kim, V. Ranynal, V. Van Huffel and J.P. Cartron. Blood. 7 8 . 2747-2752 (1991). 21. T. Blunt, F. Steers, G. Danielsand B. Carritt. Ann HumGenet, 24, (1994).
58,
19-
23. B. Cherif-Zahar. V. Raynal. C. Le Van Kim, A.M. D'Ambrosio. P. Bailly.
J.P. Cartron andY. Colin. Blood, 8 2 , 656-662 (1993).
271, 25. N.D. Avent, K . Ridgwell. M.J.A. Tanner and D.J. Anstee. Biochem J. 821-825 (1990).
26. I . Mouro. Y . Colin, B. ChCrif-Zahar, J.P. Cartron and C . Le Van Kim. Nature Genet, 5 , 62-65 (1993).
P.Y. Le Pennec, 27. I . Mouro, C. Le Van Kim, C. Roui 1 lac, D. J. van Rhenen. P. Bailly, J.P. Cartron andY. Colin. Blood, 8 3 , 1129-1135 (1994). 28. C. Lomas C, and R. Mougey. Abstract. Transfusion,29 supvl. 14s (1989). 29. T. Blunt, G . Daniels. B. Carritt. Serotype switching
deleted RHD gene. Vox Sang, in press.
in a partially
BLOOD GROUP ANTIGENS AS TUMOR MARKERS, PARASITIC/BACTERIAL/VIRALRECEPTORS, AND THEIR ASSOCIATION WITH IMMUNOLOGICALLY IMPORTANT PROTEINS G. Garratty Research Department American Red Cross Blood Services Southern California Region Los Angeles, CA 90006 ABSTRACT Blood group antigens (BGAs) are chemical moieties on the red blood cell (RBC) membrane. Some BGAs (e.g., A, B, H, Lewis, P, I) are widely distributed throughout the body and may not be primarily erythroid antigens. Statistical correlations with AB0 blood groups and disease have been made for years and have been highly controversial. It is not known if BGAs have a biological function. There are increasing reports of BGAs [e.g., L e x (an isomer of Lea), Ley (an isomer of Leb), T, Tn, "A-like"] appearing as "new" antigens on malignant tissue. Their presence and membrane density appears to correlate with the metastatic potential of the tunlor. This often parallels loss of normal BGAs (e.g., ABH) from the tissue. Some of these antigens have been shown to influence the humoral and cellular response and have been used in assays to determine preclinical cancer, and in tumor immunotherapy. Interactions of some parasites and bacteria with human cells have been shown to depend on the presence of certain BGAs. P. vivux malarial parasites only enter human RBCs when the Fy6 Duffy blood group protein is present on the RBCs. Certain E. coli will only attach to the epithelial cells of the urinary tract if P or Dr BGAs are present in the epithelial cells. The P antigen is also the RBC receptor for Parvovirus B19. Leb has recently been found to be the receptor for H. pylori in the gastric tissue. The high frequency BGA, AnWj, is the RBC receptor for H . i~flucnzuc. BGAs have been shown to be associated closely with some important complement proteins. Ch/Rg BGAs have been found not to be true BGAs but are RBC-bound C4 (C4d). Knops/McCoy/York BGAs have been located on the C3blC4b receptor (CRI). The high frequency BGAs of the Cromer (Cr) system are located on decay accelerating factor O A F or CD55). Cartwright (Yt) BGAs are located on RBC acetylcholinesterase molecules. DAF and acetylcholinesterase are on phosphatidylinositol-glycan (PIG) linked proteins. When the PIG anchor is missing from RBCs, as in paroxysmal nocturnal hemoglobinuria, the affected RBCs lack all Cr, Yt, JMH, Hy/Gy, Do and Emm BGAs. The most important ligand for P, E and L selectins is sialyl-LeX. This interaction is the tethering stage that start the leukocytes' journey from the circulation into the tissue. It appears that malignant cells may move through tissue in a similar way and may explain the close association of L e x with metastasis. Thus, there are increasing data suggesting a biological role for BGAs unrelated to the RBC. 213
2 14
GARRATTY INTRODUCTION There have been numerous reports of associations of blood groups with disease (1-4).
Many of these were statistical differences between the number of group A and group 0 individuals with a certain disease. Such reports were highly controversial and led to vitriolic attacks in the literature, by such well-known figures as Alexander Wiener, who regarded the subject as one of the myths of blood transfusion medicine (5). It is true that the reports before 1950 could be criticized because of the lack of appreciation of the large number of tests and controls required for valid interpretations. In addition, it was not generally appreciated at that time that one could see a wide variation in AB0 groups over quite short geographical distances, even within populations appearing homogeneous. The results by Aird et a1.(6,7), in 1953 and 1954, regarding an association of AB0 groups with cancer of the stomach and peptic ulceration respectively, seem to have stood the test of time, having been confirmed by literally hundreds of studies in different countries. The statistical associations tend to fall into three major groups: malignancy; 2) associations withcoagulation(bleedingandthrombosis); associations with infection.Othermorespecificassociationshave include: associations of blood groups with hematological
1) associations with
and, 3)
been described. These
disorders [e.g., leukemia,
congenital dyserythropoietic anemia type 2 (HEMPAS); abnormal RBC shape]; blood group antigens (BGAs) as markers for malignant tumors; BGAs as receptors for parasites and bacteria; and, associations of BGAs with immunologically important proteins. These associations pose a very fundamental question. Do blood
groups have a
biological function, or are BGAs coincidentally present on other functional molecules? In contrast to Wiener ( 3 , Ibelieve that evidence is emerging to show that both are true. I believe that some BGAs do have a biological role, and their presence on RBCs and their presence on other functional structures is sometimes only coincidental. It is worthwhile to emphasize that some blood group antigens are widely distributed throughout the body. Clausen and Hakomori (8) and Oriol (9) point out that ABH antigens are not only present on RBCs; they constitute the major allogeneic antigens on most epithelial cell types, and are also found on primary sensory neurons, and in many body
secretions. They appear earlier in
evolution in ectodermal tissue than in mesenchymal hematopoietic tissue and cells, including RBCs. Other related antigens such as I, i, P, Lewis are also widely distributed throughout the body.
BLOOD GROUPS AS MARKERS ON MALIGNANT TUMORS Aird et a1.(6) reported a 20% increase of group A over group 0 in cancer of the stomach. Many
other investigators have confirmed this since then. When
one reviews the
literature, there is an overwhelming trend towards an increase of group A over group 0 in
BLOOD GROUP ANTIGENS
215
TABLE I. STATISTICALLY SIGNIFICANT INCREASE OF GROUP A COMPARED TO GROUP 0 IN VARIOUS MALIGNANCIES
Type of Cancer
Relative Increase of A : O
Salivary Ovaries Stomach Uterus Cervix Colon/Rectum
1.64 1.28 1.22 1.15 1.13 1.11
many malignancies (10,ll). Table I shows some of the most impressive associations that were either the result of very large studies andlor the studies have been repeated by several investigators with similar results. When cells become malignant, some antigens that are normally present tend to decrease and new, so-called tumor antigens tend to appear. Such antigens
may be truly new
antigens, or more often perhaps, precursors of the antigens normally present. We
see these
same phenomena occurring with BGAs and malignancy. LOSS OF ABH ANTIGENS FROM MALIGNANT CELLS Oh-Huti (12) seems to be the first to have described a loss of ABH antigens from malignant cells, and this was confirmed by Masamune et
al,(13), Kawasaki (14), Kay and
Wallace (15), Nairn et al.(16), and Davidsohn (17). In a series of papers, Davidsohn and his co-workers (reviewed in reference 18) showed that A, B and H, present in epithelial cells of some normal tissues, could not be detected when carcinoma developed in these tissues. The specific red cell adherence test, a modification of Coombs’ mixed cell agglutination test, was used for the detection of ABH antigens. The test was highly sensitive and specific.
The age of paraffin-embedded tissues
and hematoxylin-and-eosin stained slides did not affect the sensitivity of the test. The loss of the antigens was increasingly progressive from carcinoma in situ to anaplastic, invasive, and metastatic carcinoma, and was interpreted as evidence of immunologic dedifferentiation analogous to morphologic dedifferentiation of anaplasia. With few exceptions, the loss of ABH antigens preceded the formation of distant metastases. test held promise to:1)
The authors suggested that the
diagnose early carcinoma in tissues that normally contain ABH
antigens and in the prognosis of advanced carcinoma; 2) reduce the need for radical surgery in carcinoma in situ of the cervix. Since Davidsohn’s studies (17), there have been hundreds
216
GARRATTY
of publications studying ABH antigens and many types of malignancy. In general, the results agree with and extend the earlier observations. Hakomori, in particular, has helped us considerably to understand the etiology of these changes (18-24). The A, B, H genes are responsible for governing the synthesis of glycosyl transferases that are responsible for the blood group antigens; other genes such as the Lewis and secretor genes also play a role. Singhai and Hakomori (24) recently suggested that many of the carbohydrate changes associated with cancer take place at various stages of development and differentiation, and that the changes, associated with cancer, may result from activation of increased synthesis of different glycosyltransferases. APPEARANCE OF "NEW" BLOOD GROUP ANTIGENS ON MALIGNANT CELLS "A-Like Antigens":
As early as 1929and1930,Hirzfeld
et aL(25) andWitebsky
(26) reported the presence of "A-like" antigens in cancer patients who were not group A. Hakomori and Jeanloz (21) isolated a glycolipid of unusual carbohydrate composition from a human adenocarcinoma; attempts to isolate a similar component from normal tissues were unsuccessfill. Glycolipids extracted from different adenocarcinomas were shown to possess weak Leb and H activity and moderate Lea activity regardless of the Lewis type of the tissue donor (22). Neither blood group A nor B activity was detected in glycolipid
fractions from any adenocarcinomas, even though some tumor donors were group A or B. In contrast, A and B activity was detected regularly in normal glandular tissue (21,22).
Although the tumor glycolipid had no A activity, there appeared to be a special relationship between it and blood group substance A. Rabbit antisera against tumor glycolipid agglutinated type A red cells more readily than group B, AB or 0 red cells. This and other experiments suggested that blood group A substance and group A red cells may contain a larger concentration of a hapten structure similar to, or identical with, the carbohydrate moiety of the tumor glycolipid, than substances or red cells of group B or Om). Hakomori et al.(23) suggested that it may be more
difficult for the immunological system of the group
A patient to recognize tumor cells, possessing "A-like'' glycolipid, as foreign, because of the
latter's similarity to the host's own A substance and thus fail to reject them. It was suggested that this may relate to the increase of group A compared to 0 in cancer patients. Hiikkinen and co-workers (27,28) demonstrated clearly that "A-like'' antigen could be detected in gastric juice and mucosa from group 0 and B patients with gastric cancer but was not detected in mucosa from patients with peptic ulcers. More recent studies suggest
that the
"A-like'' antigen may be a true A antigen, or an antigen cross-reacting with Forssman or Tn antigen (19,29-34). Glycolipids with clear A activity have been demonstrated in some cases of group 0 cancer patients (30,31,33). About 10-15percent of group 0 or B patients with colonic cancer express mono- or di-fucosyl Type 1 chain A antigen; A transferase activity
S
BLOOD GROUP
217
was also detected in the A-expressing 0 tumors (33). The Forssman antigen is usually present on malignant cells independently from the "A-like'' antigen
(30,31). It seems that
most of the "A-like'' antigens appearing on malignant cells may be Tn (32).
Lewis Antigens: 0rntoft (35) reported that Le(a-b-) individuals who did not have Lea or Leb in their saliva or 0114 fucosyltransferase (the primary Lewis gene product) in saliva or normal tissues, showed expression of Lea, Leb and a 1 4 fucosyltransferase in their tumors. Lacto-series type 1 chain is a normal component of gastrointestinal cells. It appears that type 2 chain-based structures [e.g., Lex, sialosyl Lex @Lex),dimeric and trimeric Lex] are major oncofetal antigens in human gastrointestinal colorectal and lung cancers (36). Large concentrations of Lex antigen, an isomer of Lea [fucosylated type 2 chains, Gal/314(Fuc 011+3)+GIcNAc], first known as SSEA-I (37), have been demonstrated in various tumors (38). Levels of another type 2 antigen, L$, an isomer of Leb,
[Fuc011+2Gal~l4(Fuc011+3)GlcNAc]are also significantly increased in malignant cells (39). It appears that when malignant cells lose A and B antigens due to blocked activity of A and B transferases, the type 2 chain is fucosylated by enhanced
011+3 fucosyltransferase and
accumulates Lex hapten glycolipid. Type 1 chainisfucosylatedbyenhanced
0114
fucosyltransferase as well as 011+2 fucosyltransferase to accumulateboth Lea and L e b hapten, regardless of the Lewis status of the host (18,19). In the last five years there have been a huge number of papers published on the association of Lex, Ley and sLex with malignancy; many of these references (up to 1992) are listed in the reference list of reference 4. Antigens of the P Blood Group System: Anti-PPIPk was originally called anti-Tja because
it was found in a patient called Jay with a gastric adenoma, and the lyophilized tumor inhibited the activity of the RBC antibody (40). Thus, the new specificity was called Tja (T for tumor, j for Jay). The patient was given a small amount of incompatible blood which resulted in a severe hemolytic transfusion reaction, and boosted the titer of the antibody. The patient had a subtotal gastrectomy and survived for 22 years following surgery. At no time was there any evidence of tumor recurrence or metastasis. The patient's sister, who
also was of the rare p phenotype and was not given incompatible blood, died of adenocarcinoma of the uterus.
Levine suggested that the adenocarcinomas in these p patients
had produced "illegitimate" P antigens, and the first sister's hyperimmune response to the incompatible blood had led to the destruction of the P+ malignant cells (41). In 1987, Levine found some frozen tumor from his original patient with anti-Tja and had it examined by Hakomori's group. Hakomori's group found that the major glycolipid isolated from the tumor had the same mobility on thin-layer chromatography and antigenic reactivity as a new glycolipid of human RBCs that cross-reacts with antigloboside and the structure was identified as G a l N A c ~ 1 ~ 3 G a l ~ l 4 G l c N A c ~ l + 3 G a l ~ 1 4 G(42). lc~~er
218
GARRATTY
Although the purified glycolipid fraction displayed a clear inhibition of anti-Pl agglutinins, only a minor component had the same mobility on thin-layer chromatography as P1 glycolipid, which is a ceramide pentasaccharide susceptible to cy-galactosidase. These results indicated that the tumor activated the synthesis of the globo-series glycolipid; it also showed an enhanced synthesis of the lacto-series glycolipid with the same terminal structure as
globoside (42). The biochemical results supported the 1951 findings of a p individual having made illegitimate P antigen on malignant cells (40). Levine coined the term illegitimate (or
incompatible) antigens for new antigens that were genetically foreign to the host (e.g., P antigens appearing in a p host). The P blood group antigen (globoside) is a precursor of Forssman antigen (43,44). Forssman antigen was found to be present in many animals but until relatively recently humans were thought to be Forssman negative. Most human sera
were known to contain
naturally occurring Forssman antibodies that would react strongly
(i.e., agglutinate and
hemolyze) with Forssman positive cells (e.g., sheep RBCs). It has also been known for many years that Forssman antigen cross-reacts with the A blood group antigen, and later a cross-reaction with P antigenwasdescribed (45,46). Hakomori (18) considered the possibility that the "A-like'' antigen appearing in malignant tissue might be Forssman antigen. Forssman antigen was not detected in the gastrointestinal mucosa of most normal individuals, but it was detected in about 30% of the population studied (47). Tumors derived from Forssman-negative tissue contained Forssman antigen whereas tumors from Forssmanpositive tissue did not contain Forssman glycolipid
(47). Other workers have also detected
Forssman antigen in malignant tissue (48-51). Sera from patients with cancer showed decreased Forssman antibody activity (52,53). It was suggested that the decrease in Forssman antibody activity in cancer was due to the appearance of "illegitimate" Forssman antigen on the malignant tissue (53). Mori et a1.(54) also reported that the sera of cancer patients contained less Forssman antibody than normal sera. Although Forssman antigen does appear as an "illegitimate" antigen in malignant tissue and can appear to be "A-like", Hakomori now believes that this only accounts for 10-15%of the "A-like'' antigens appearing in malignant tissue (19). Some "illegitimate"
A antigens appear to be true A
antigens, but the majority of them are probably Tn antigens (see later) that cross-react with A antigens (32). T, Tn, and Sialyl-Tn Antigens: In 1975, Springer et al (55) showed that malignant cells,
but not normal cells or benign tumors, had T antigen, a cryptantigen known to be present on human RBCs, and Tn antigen, a precursor of T, on their surface. T is formed when neuraminic acid is cleaved from the normal
RBC sialoglycoprotein. Tn is formed when the
terminal galactose is removed from the T structure. Springer et a1.(56,57) also showed that naturally occurring anti-T was severely depressed in the sera of many cancer patients. T and
BLOOD GROUP ANTIGENS
219
Tn antigens were first described as blood group antigens that were acquired by RBCs in association with certain conditions. T and Tn antigens
are not usually detected on the RBCs
of healthy individuals. T antigen can appear in vivo as a transitory antigen on RBCs as a result of the action of bacterial sialidases; the acquisition of Tn by RBCs is a rare event and is often persistent; it is found usually in association with thrombocytopenia, leukopenia and/or hemolytic anemia, or leukemia (58-65). Tn expression
on RBCs is thought to result
from somatic mutation within stem cells of the bone marrow, resulting
in lack of the a3-8-
D-galactosyltransferase needed to convert Tn to T. Since Springer's first observations, there have been many publications confirming the association of T, Tn and sialyl-Tn with malignancy (see reference 4 for many of these). There appears to be a correlation with the appearance of T and Tn and increasing metastases. It has been suggested that T and Tn
are important for adhesion of cancer cells to
their preferred target (e.g., hepatocytes) in metastasis. (57,68-70). The attachment of malignant cells (Esb T-lymphoma cells), which expressed T and Tn on their membranes, to hepatocytes was competitively inhibited by minute quantities of T and Tn antigens (57). Springer (57) suggested that T and Tn may be involved in
specific cell-cell adhesions
required for invasion and metastasis by cancer cells. T and Tn also appear to be differentiation antigens. Barr et a1.(71) studied fetal tissue, 45-117 days after ovulation, for T and Tn reactivity. Most fetal tissues during normal morphogenesis appear to contain some T and Tn specific structures during gestational days 45 through 117; their highest density is in fetal epithelial and mesothelia. One monoclonal anti-T reacted with
all elements of
erythropoiesis but with none of the epithelia (i.e., hepatocytes and bile duct epithelia). The authors suggested that as ABH antigens appear around the third month of gestation, that this probably is when T and Tn are no longer detectable in fetal tissue. This work would suggest that "illegitimate" T and Tn are examples of carcinoembryonic antigens. Springer's group has shown that T/Tn antigens can stimulate both a humoral and cellular immune response (57,72). Delayed-typed skin hypersensitivity reactions (DTHRs) were observed following skin tests, using T antigen. Of 951 patients and
controls, only one
of 127 healthy controls gave a positive reaction; over 80% of the malignancies, most of which were adenocarcinomas, yielded a positive result (88.2% of lung cancer, 83.4% of breast cancer, 88.5% of pancreatic cancer, 86.7% of colon cancer, 85.3% of bladder cancer) (72). Positive DTHRs were observed
in less than 10% of patients with benign tumors of the
lung, breast, pancreas, colon and genital tract.
No positive DTHRs were observed in 45
patients with other benign tumors or diseases (72). The results of these studies has led to the development of tests for detecting preclinical stages of cancer and vaccines used in tumor immunotherapy. Using a combination of measuring anti-T levels in the serum and DTHRs, Springer's group have
GARRATTY
220
reported remarkably successful results in predicting cancer. They have evaluated longitudinally 34 patients (32 with breast and lung cancer) who yielded repeatedly positive assays, but were free of cancer, according to biopsy and radiographic results at that time. As of May 1990, 32 of 34 (94%) of patients who showed positive results more than once developed biopsy-verified cancer within 3 months to 10 years of follow up. BGAS AS RECEPTORS FOR
PARASITES/BACTERIA/VIRUSES
Table I1 lists BGAs that have been described to be receptors for, or interact with, parasites, bacteria, and viruses. ASSOCIATIONS WITH MALARIA In 1975, Miller et a1.(73) showed an association between malaria and Duffy blood
group antigens. RBCs lacking Fya and Fyb [Fy(a-b-)]
were shown to be resistant to
invasion by a monkey malaria, P. knowlcsi. P. knowksi is genetically related to the human malaria P. vivux. It had been known
since the 1930’s that many black people are resistant to
infection by P. vivux. It had also been known that about 70% of the American black population and nearly 100% of the West African black population are Fy(a-b-). Epidemiologic studies in endemic areas showed that P. vivax infections do not occur in individuals who are Fy(a-b-).
P. vivux does not occur in West Africa where nearly 100%
of the population is Fy(a-b-). Studies using
P. vivux in volunteers demonstrated that the
resistance inblack people correlated with the Fy(a-b-) that P. knowlcsi cannot invade Fy(a-b-) RBCs
phenotype (74). It has been shown
(75). It is important to note that Fya and Fyb
epitopes per se do not appear to constitute the actual binding site recognized by the parasite for the following reasons:
1. P. knowksi parasites invade Fy(a+b-) and Fy(a-b+) human
kra monkeys, which express Fyb,
erythrocytes equally well. 2. Erythrocytes of rhesus and can still be invaded after Fyb is removed by chymotrypsin.
It is likely the actual binding site
recognized by P. knowlcsi and P. vivax is on the same protein as the Fya and Fyb determinants (76). It is of interest that P. vivux requires two ligands for invasion. One ligand may be associated with Duffy BGAs, which appears to
be Fy6 (72), and the other may
be expressed on reticulocytes but not on mature RBCs (78). The ligand for P. fulcipurum is different from that of P. vivax. P. falcipurum invades Fy(a-b-) RBCs
equally to Fy(a+) and Fy(b+) RBCs. Miller et a1.(77) tested RBCs of
various null phenotypes and found that none resisted invasion by noted with Fy(a-b-) and
P. falcipurum to the extent
P. vivux. RBCs of the En(a-) phenotype, neuraminidase-treated
RBCs of common phenotype, showed a 50% reduction in invasion by P. fakiparum. Subsequent studies in other laboratories using different strains of P. fulciparum showed a 95% reduction in invasion with sialidase-treated RBCs.
All the phenotypes that showed
221
BLOOD GROUP ANTIGENS
TABLE 11. ASSOCIATION OF BLOOD GROUP ANTIGENS WITH PARASITIC/BACTERIAL/VIRALRECEPTORS Blood Group
ParasiteslBacterialViruses
P:vivux
FY6 I,sialyl I (FIISia-bl) P, Dr, "MN" AnWj
M. pncumoniuc E. coli H. influcnzclc H . pylori Parvovirus B19
Leb
P
resistance to invasion by P. ,fulciparum had abnormalities of glycophorin A or B. Pavsol et al. (79) suggested that the initial attachment of the merozoite surface coat to the red cell may
reflect a lectin-ligand like interaction in which the parasite binds in a specific manner to a cluster of oligosaccharides present on glycophorin A or B (or both). Once attachment has occurred and the apical end of the merozoite with its specialized organelles has oriented to the membrane, further specific conformational alterations may occur which trigger the process of red cell deformation and parasite entry. The above results suggest that as glycophorin A deficient E@-)
RBCs are resistant
to invasion, that glycophorin A is an important ligand. Glycophorin B also appears to play a role as glycophorin B deficient RBCs (S-S-U-)
are less susceptible to invasion. Trypsin-
treated RBCs are also resistant to invasion; trypsin treatment cleaves glycophorins A and C, whereas glycophorin B remains unchanged. Although trypsin-treated En(a-) RBCs
are more
resistant to invasion than untreated En(a-) RBCs, which would suggest that glycophorin
C
may also be involved, sialidase-treated Gerbich null RBCs of the Leach type, which lack glycophorin C, are invaded by P. fulcipurum comparably to normal neuraminidase-treated RBCs (80). Sialic acid is required for all strains of P. .fulciparum to bind to human RBCs. MkMk RBCs, that lack glycophorin
A and B, showed resistance to invasion by some strains
of P. fulcipcrrum but were invaded by other strains. Genes for RBC-binding proteins for P. knowkcxi @l), P. vivux (82), and P. ,fulcipurum (83,84) have also been cloned and
sequenced.Hadley (76) suggestedtwohypotheses.Hypothesis
1: There is a proven
receptor heterogeneity among P. .fulcipurum parasites. Parasites that depend heavily on sialic acid for invasion also depend heavily on the presence of glycophorin A; parasites that are less dependent on sialic acid are less dependent on glycophorin A. HvDothesis 2: The importance of glycophorin A resides in the fact that it provides sialic acid for a sialic aciddependent site, rather than a peptide domain, for parasite binding.
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In 1993, Horuk et a1.(85) described a further fascinating finding concerning the relationship of the Duffy blood group antigen protein to malaria. The Duffy blood group antigen was found to be on the RBC receptor for the chemokines interleukin-8 (IL-8) and melanoma growth stimulating activity (MGSA). IL-8 bound minimally to Fy(a-b-) RBCs. A monoclonal antibody to Fy6 blocked binding of IL-8 and other cytokines to Duffy-positive RBCs. Both IL-8 and MGSA blocked binding of
the parasite ligand and the invasion of
human RBCs by P. knowlec.i, suggesting the possibility of receptor blockade for antimalarial therapy. ASSOCIATIONS OF BGAs WITH BACTERIA In the early 1980’s a series of papers were published showing that E. coli
hemagglutinins reacted specifically with RBCs having P blood group antigens (86-91). Over 90% of E. coli isolated from 30 children with pyelonephritis showed mannose-resistant hemagglutination. The reactive target on human RBCs was found to be the P blood group antigen. The P glycolipids were shown to be the receptors for bacterial fimbriae.
The P
glycolipids are also present on uroepithelial cells and renal tissue where they mediate the adhesion of E. coli and their subsequent ascent to the kidney (89-92). This adhesion is necessary for the E. coli to resist the rinsing effect of urine and for efficient colonization (92). Some other blood group antigens whose specificity is not determined by carbohydrates have been shown to be associated with the receptors for bacteria. Vaidnen et aL(93) reported that one strain of E. coli (018:Kl:H7), associated with sepsis and meningitis of the newborn, had hemagglutinating specificity associated with the MN sialoglycoprotein
(SGP),
glycophorin A. Their results suggested that the E. coli binds to the NeuNAccr2-3Galpl3GalNAc sequence of the 0-linked saccharides of the MNSGP (glycophorin A). The specificity of about 20% of pyelonephritis-associatedE. coli that are mannoseresistant hemagglutinins has not been identified and has been termed
X. Nowicki et al (95)
used a systematic approach with RBCs of different phenotypes, including very rare types, to identify the receptor for an X hemagglutinin associated with the serotype 075 E. coli The receptor was identified as the high frequency Dr blood group antigen. The Dr antigen is a component of the Cromer-related blood group complex. The molecule recognized by the Dr hemagglutinin is a chloramphenicol-like structure; tyrosine seems important to the specificity. Nowicki et al (95,96) isolated the Dr hemagglutinin from a recombinant bacterial E. coli strain (BN406). A rabbit anti-Dr hemagglutinin was prepared and used by indirect immunofluorescence to study different tissues. The Dr antigen was expressed in different parts of the digestive, respiratory, urinary, and genital tracts and skin. Intense staining by the Dr hemagglutinin was shown in colonic, bronchial, and endometrial glands. The
BLOOD
223
strongest fluorescence was observed in the luminal domains o f glands, but focal weak staining was also present in cell membranes. Renal tubulzr basement membrane and Bowman’s capsule were strongly stained. P-fimbriated E. coli have been shown to adhere to glomeruli and to lumens of proximal and distal tubules but not to collecting ducts and peritubdar sites. Dr hemagglutinin positive strains show adherence to Bowman’s capsule and renal interstitium. The authors (95,96) suggested that high density of the Dr-rich structures in the colon and urinary tract may permit E. coli to colonize the colon. Colonization of the lower urinary tract may occur due to attachment of E. coli to Dr-rich transitional epithelium in the ureter. High density of Dr in Bowman’s capsule may facilitate colonization of the glomerulus. Rosenstein et aL(97) described a new type of adhesive specificity, revealed by oligosaccharide probes, in E. coli from patients with urinary tract infection. This adhesive specificity was unrelated to the presence of fimbriae. The new oligosaccharide receptor was affected by the presence of blood group genetic markers. It involved sequence linked to the membrane-associated lipid moiety of
the disaccharide
the host-cell glycolipids. It was
proposed that this type of adhesive specificity may have had an
important role in the invasion
of damaged epithelial membrane where the saccharide-lipid function
may be exposed. When
the lactose-containing sequence is modified by additional monosaccharides (including the blood group monosaccharides), binding of E. coli is greatly impaired. It was predicted that the secretor gene and the genes which code for blood group enzymes, or for other glycosyltransferases whose levels change in epithelial cells during differentiation, proliferation, and maturation, would strongly influence binding and hence susceptibility to invasion. It was pointed out that an association has already been suggested between nonsecretion of blood group B and AB antigens and susceptibility to recurrent urinary tract infections (98). Some strains of the Huemophilus influanzae also have fimbriae and cause in vitro hemagglutination (99). The degree of hemagglutination was found to correlate with adherence to buccal epithelial cells (99). The receptor for the
fimbriae of H. influenzue has
been found to be the AnWj blood group antigen (100,101). RBCs from the rare individuals of the Lu(a-b-)
dominant phenotype lack, or have very weak, AnWj and H. itfluenzae
adherence receptors. The receptor molecule on the surfaces of epithelial cells andRBCs are different, but the binding sites for the fimbriae are similar (102).
Bor6n et a1.(103) reported in 1993 that
the Leb blood group antigen was the receptor
for Halicobucm pylori. H. pylori is associated with gastritis, adenocarcinoma, and is thought to be a major cause of gastric ulcers. When terminal N-acetylgalactosamine, the A determinant sugar, was substituted to form A-Leb, H . pylori did not bind, suggesting that H.
pylori receptors might be reduced in groups A and B compared to group 0. This finding
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224
provides some scientific rationale for the report of Aird et al.in 1954 since, that gastric ulcers are much more common in
(7), and many others
group 0 than in group A (4).
ASSOCIATIONS OF BGAs WITH VIRUSES Brown et aL(104) showed that the receptor for Parvovirus B19, a virus that replicates only in erythroid progenitor cells, was the P blood group antigen. In a further publication, the authors showed that individuals with the rare p phenotype were resistant to Parvovirus
B19 infection (105). Of 17 subjects of the phenotype, none had serologic evidence of previous Parvovirus B19 infection (compared with 47 and 71
% of two control groups).
In
vitro, bone marrow from donors with the p phenotype maintained normal erythropoiesis despite very high concentrations o f virus, with no infection of erythroid progenitor cells by parvovirus B19. Three papers have reported a possible association of blood group antigens with acquired immune deficiency syndrome (AIDS). Adachi et aL(106) found that human T cell lines infected with human immunodeficiency virus (HIV) and
T cells from AIDS patients
expressed Ley antigen, whereas normal T cells did not. Arendrup carbohydrate epitopes on HIV may be
et aL(107) reported that
targets for monoclonal antibody (Mab)-mediated virus
inhibition. Specificity of the Mab-mediated inhibition was shown using A antigen (tetrasaccharide). The same workers had previously 'shown that a monoclonal antibody, directed against blood group A antigen, precipitated the major envelope glycoprotein gp120 and that A-specific Mabs were able to inhibit cell-free HIV infection even though the A antigen is not normally expressed by the cells used for the production of HIV or as target cells for infection (108). Arendrup et a1.(107) suggested that HIV infection of mononuclear cells from donors with blood group A appears to induce expression of host-cell-encoded carbohydrate blood group A epitopes on HIV which can be
targets for Mab-mediated virus
neutralization. The authors (108) are trying to identify the possible A transferase, mRNA, in HIV-infected cells. Glinsky (109) has postulated that blood group antigen-related glycoproteins may be key structural determinants in the immunogenicity and pathogenicity of AIDS. ASSOCIATIONS OF BLOOD GROUP ANTIGENS WITH IMMUNOLOGICALLY IMPORTANT PROTEINS
Table 111 lists some of the associations of blood group antigens with immunologically important proteins. The associations of the Bg antigens with HLA and the Ch/Rg antigens with the complement C4 molecule were reported 15-20 years ago, and have been discussed (e.g., 4,110,11 l). They will not be discussed
in many publications
here as Bgand Ch/Rg are not true blood
BLOOD GROUP ANTIGENS
225
TABLE 111. ASSOCIATION OF BLOOD GROUP ANTIGENS WITH IMMUNOLOGICALLY IMPORTANT PROTEINS Protein (C4d)
Blood Group
(B7, HLA B17, Bga,A28) C4 C3blC4b receptor (CRI) Cromer accelerating (DAF) Decay factor CD44 Selectins
Bgb, BgC Ch, Rg Kn/McC/Yk Inb sialyl-hX
group antigens but rather remnants of HLA, and C4, molecules respectively, present on normal human RBCs. ASSOCIATION OF BLOOD GROUP ANTIGENS WITH THE COMPLEMENT SYSTEM Apart from the association of Ch/Rg with RBC-bound
C4, two other interesting
associations with the complement system have been described. The Knops (McC), and York (Yk) blood
(Kn), McCoy
group antigens have been shown to be present on complement
receptor 1 (CRl), the receptor for C3b and C4b (1 10,112,113). The known variation in the density of CRI receptors on the RBC membrane probably accounts for the serological characteristics of Kn/McC/Yk antibodies (e.g., high titer, low avidity reactions).
CRl,
which is present on RBCs, lymphocytes, monocytes, granulocytes, and dendritic cells, has an important role in the clearance of immune complexes. Low numbers of
CRI sites on RBCs
have been described in patients with AIDS, systemic lupus erythematosus, some tumors and autoimmune disease (1IO). Some important proteins involved in the control of complement activation are attached
to the cell membrane via a complex phosphatidylinositol-glycan (PIG) linkage (known fondly as a "PIGtail") (1 14). These complement proteins are decay accelerating factor (DAF or CD%), membrane inhibitor of reactive lysis (MIRL or CD59), and C8-binding protein (C8BP). A number of blood group antigens are reported to be on "PIG-linked'' proteins. Cromer BGAs are found on DAF; Cartwright (Yt) BGAs are found on acetylcholinesterase; JMH and Hy/Gy BGAs are found on 76kD and 47-58 kD glycoproteins respectively, and Dombrock and Emm BGAs
are on as yet uncharacterized proteins.
RBCs, neutrophils and platelets derived from affected clones in patients with paroxysmal nocturnal hemoglobinuria (PNH) lack PIG-linked proteins. Patients with PNH have variable proportions of RBCs that are highly susceptible to complement-mediated lysis.
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226
This is highly associated with the absence of the regulatory PIG-linked complement molecules, particularly MIRL (CD59). The populations of RBCs most susceptible to lysis lack all blood group antigens in the Cromer system, and Yta, JMH, Hy/Gy/Do and Emm blood group antigens (I14). ASSOCIATION OF BLOOD GROUP ANTIGENS WITH ADHESION MOLECULES Homing-associated cell adhesion molecule (HCAM or CD44): CD44 is a widely
distributed cell surface proteoglycan that has been implicated in a wide range of biologic functions. The molecule has been found to mediate recirculation of lymphocytes between blood and lymphoid organs, from which i t derived its original name of lymphocyte homing receptor. CD44 has
also beenimplicated in T-cell activation (121), hematopoietic
development (1 16,117) and tumor metastasis (118). There is an increasing stream of publications implicating CD44 in tumor progression; studies of non-Hodgkins lymphoma are particularly impressive (1 19-122). CD44 has been found to be suppressed (21-61 .
Lu(a-b-)
%) on RBCs from individuals with the
In Lu form of inheritance(123-126). Inb,
phenotypeassociatedwiththedominant
a high frequency blood group antigen present on RBCs, granulocytes and lymphocytes, is present on CD44 (127). Parsons et a1.(128) recently described a fascinating form of congenital dyserythropoietic anemia associatedwith a deficiency of RBC CD44. The patient’s RBCs had the unique phenotype o f h(a-b-),
Co(a-b-), and lacked
the high incidence
antigen AnWj. Selectins: There are three major selectins:
E. selectin (or ELAMI), is present on
endothelium following activation by endotoxin or inflammatory cytokines (e.g., tumor necrosis factor or [L-l); neutrophils, monocytes and CD4 lymphocytes can bind to Eselectin. P selectin (or GMP-140) is localized in platelet
(Y
granules and appears during
platelet activation; it is also present on endothelial Weibel-Palade bodies. L-selectin (LAM1) is a glycoprotein present onmost leukocytes (118,129,130). The selectins recognize
terminal sialic acid and fucose in appropriate linkage; their most potent ligand is sLex (129). To get to a site o f injury, leukocytes must get from the circulation into the tissue. To do this they must first adhere to the endothelium. The selectins are responsible for the initial tethering of the leukocytes to the endothelium. Selectins are ideally suited for this role because they have a long molecular structure that extends above the surrounding glycocalyx and allows them to capture passing leukocytes that express the appropriate receptor (130). The tethering is a loose bond as the leukocytes must next roll along the endothelium. This rolling is essential for the leukocyte to search the endothelium for appropriate trigger factors (e.g., IL-8) that activate leukocyte integrins (130). The integrins mediate strong adhesion of
S
BLOOD GROUP
227
the leukocyte to the endothelium. After this stage,
the leukocytes change shape and migrate
through the endothelium. There is increasing evidence that malignant cells may migrate through the tissues in a
for leukocytes (131). As discussed previously, L e x
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THE ROLE OF THE LYMPHOCYTE IN A N IMMUNE RESPONSE Kamala Balakrishnan and
Louis E. Adams
HLA-Laboratory, Hoxworth Blood Center University of Cincinnati Cincinnati, Ohio 45267
ABSTRACT
The immune system has evolved in the human being as an elaborate mechanism to distinguish itself fromall else thatis not self. This process serves in the defence against invaders. The cells of the immune system learn to tolerateall tissues, cells and proteins of the body. Failure to control the state of tolerance results in autoimmunity. The understanding of the role of T-cell receptors (TCR), the Major Histocompatibility Complex(MHC), adhesion molecules and growth factors in antigen recognition has lead to the exploration of various means to modulate the immune response. Safety measures exist to prevent the immune system from attacking its host. The antigen has to be recognized by the T-cell. This involves the TCR and the MHC. In addition it must receive a second signal to become activated. The second signal involves a protein such as B7 binding with CD28. Certain specialized cells, macrophages, dendritic cells and activated B-cells can deliver this second signal for activation; receipt of only one signal can prevent activation. The elucidation of the role of cell-to-cell interactions, the adhesion molecules involved and the accessory growth factors provides modalities for selectively modifying the immune response. This would be of great relevance in autoimmunity and transplantation. INTRODUC!L'ION
An immune response in man is believed to be highly
individualistic, a process which defines "self" and also defends the organism. This is evident by the response toselect environmental allergens or antigens and the specific rejection of allografts. Such restriction in the immune response is known to be determined by genetic variations in the class I and I1 major histocompatibility complex (MHC) (1-5). The totality of
233
234
the response encompasses and external.
BALAKRISHNAN AND ADAMS
the
array
of
experiences both internal
Initially, the ability of the immune system to differentiate from "non-self" is an educational process. During the maturation process, the system must ignore an infinite variety of self-molecules and yet be primed and ready to respond to an array of exogenous antigens. This immunomodulatory control mechanism leads to immune tolerance of T-cells encounter selfself (6-11). In the thymus immature antigens and in the process they are deleted. Since all of "self" is not encountered in the thymus, additional modalities are required to be present to maintain tolerance to peripheral self-antigens. Therefore, control of reactivity to self is part of an individual's acquiredimmunesystem, whereby the development of autoimmunity is precluded. However, T-cells responsible for autologous mixed lymphocytereactions appear to be a consquence of positive selection in the thymus since they
.
are specific forMHC antigen (4) Experimental data have shown that T-cells are more easily tolerized than B-cells (7,11,12); this has also been suggested for self-antigens. Both T-and B-cell tolerance can be induced by deletion of specific cells bearing the antigen receptors that recognize self (7) or the cells can be inactivated by changes in surface phenotype (13). Tolerance at the T-cell level is an effective way for maintaining long-lived unresponsiveness toselfantigens. Deletion of self-reactive T-cells in the thymus is a major mechanism of tolerance preservation to self-proteins (9,14). A critical threshold of antigen concentration is necessary in the thymus for deletion of self-reactive T-cells to occur ( 1 5 ) . However, the mechanisms of tolerance for self-proteins that are rare or not present in the thymus are not well understood. Furthermore, select self-antigens are thought to be hidden from immune recognition, thereby avoiding the initiation or the provoking of an autoimmune response. The clinical expression of an autoimmune disease is rare, even though low titer autoantibodies are frequently found in sera from patients with a chronic disease or after an infectious illness; these include antinuclear antibodies, antibodies directed to lymphocytes, collagen type 11, and anti-immunoglobulin idiotype antibodies
235
LYMPHOCYTES AND IMMUNE RESPONSE
(16-20). Although the B-cells are the antibody producers, Tcells specificfor autoantigens have been isolated from peripheral blood in healthy individuals (20) The loss of
.
tolerance to self may occur by random mutation of a receptor resulting in the failure to delete a forbidden clone as suggested by Burnet (6) or it may occur as proposed recently by Cohen ( 7 ) , in a more organized and cognitive way by using information already built into the immune system. Such temporal physiologic responses may be an important form of immunomodulation that occurs to preclude the development of an autoimmune disease.However, T-cells specific for type I1 collagen have been reported in healthy, elderly individuals (20), as well as patients with osteoarthritis and rheumatoid
.
arthritis ( 4 , 1 4 1 In the process of maintaining immunologic hemostasis, the T-cell is known to play a major role in coordinating the immune response. Under the influence of the antigen presenting cell (APC), MHC complex and the various cytokines produced by accessory cells or T-cell subsets, the T-cell controls the immune response. For example, Talcells are known to produce IFNCY,which suppresses TE2 cells, while the TE2 cells synthesizes IL-10, which in turn, suppresses thefunction of TE1 cells. ROLE OF HLA MOLECULES IN T-CELL ANTIGKN RECOGNITION
In contrast to the Ig molecules on B-cell that bind directly to antigen, the T-cell will only recognize antigen fragments after the fragments are bound to class I or class 11 trimolecular complex MHC molecules (9) The requirement for this - including antigen, MHC molecule and T-cell receptor - by the
.
restricted allelic forms ofthe HLA molecule limits the recognition properties ofthe immune system. First, the restricted differences in amino acid sequence of allelic forms of MHC molecules control the binding of the amino acid side chains of an antigen fragment. The second way MHC molecules influence the immune response is by regulating the T-cell receptor. It is postulated that T-cell the repertoire is formed prior to birth in a process of "positive and negative clonal selection" (12); this process would helpexplain the restriction of immune response to se1f"HC antigens andthe establishment of innate tolerance.
236
BALAKRISHNAN AND ADAMS
T-CELL ACTIVATION Dendritic cells have the abilityto present antigen to Tcells by virtue of their class 11 molecules and cell surface ligands. Complete cellular activation requires co-stimulation by a second receptor-ligand signal; if this signal is &sent then further proliferation and cytokine production does not occur. This results in an inactive and anergic cell. The co-stimulatory molecule CD28, presenton T-cells during activation, is required for T-cell proliferation and IL-2 production. The cell-surface molecule- B-7, expressed on dendritic cells and B-lymphocytes acts as a ligand for CD28.The co-stimulatory molecule B7-2 has been shown to be important in transplantation. Recent studies suggest that CTLA4Ig, a CD28 antagonist, may inhibit T-cell proliferation and suppressimmune responses (47). This may be a new avenue to explore for donor-specific tolerance induction. (See Table la and lb) Class I molecules are presenton most nucleated cells. The nature of antigen bound iscytoplasmic and an example would be a viral capsid. The location of this MHC antigen would be in the endoplasmic reticulum (See Table 2 A ) . In contrast, class 11 antigen would be bound to a soluble protein (eg. tetanus toxoid or bacterial exoenzyme) and the
.
antigen processing is cathepsins 2b)
.
located
in
the endosome (Table
Recognition of antigen fragments is effected by receptors on CD4+ cells involved in the induction TA of cell function for antibody synthesis ordelayed type hypersensitivity (DTH) reaction. The class I1 molecules are composed of01 and p chains that encode in the MHC to form a superdomain for binding antigenic fragments. Activated but not resting T-cells are MHC class 11-positive (1,22) while B-cells and dendritic cells express class I1 antigens (13,14) The significance of expression on T-cells is unknown, but evidence suggest that
.
class 11-positive T-cell8 canpresent peptide antigens and they may function as antigen presenting cells in anmixed lymphocyte reaction (MLR) (221, however, their ability to present soluble antigens may be restricted by their lack of an proficient mechanism of antigen acquisition (13,22,23). in the The class I1 molecules thatareimportant immunogenetics of diseaseassociations and transplantation
237
LYMPHOCYTES AND IMMUNE RESPONSE
TABLE 1A INTERACTION MOLECULES T-CELL I. T-cell Receptor Antigen
Ag. peptide in MHC Cleft
T-cell receptor ( TCR
11. Adhesion Molecules
MHC LFA-1 B-7
CD4+, CD8+ ICA" 1 CD2 8 CTLA4 CD2 Other
LFA Others 111. Growth Factors
Growth Factors
Growth Factor Receptor
Interleukins 1-12 Interferons Tumor Necrosis Factor Other
TABLE 1B ACTIVATION Non-self
Tolerance
I. Antigen Presentation
11. Growth factors
MHC -Ag/TCR CD4 CD28/B7
MHC - TCR CD4 CD28 only
IFN gamma IL-2 Other inflammatory factors
IL-4 IL-10 TGF Beta Non
genes (1,4,9). A major include HLA-DR, -DQ and -DP 01 and function of these class I1 molecules is to provide processed antigen-derived peptides and to assist in determining if a apecific autoantigen can be efficiently presented to CD4+ T, cells. During secondary responses, antigen-primed animals produce increased numbers of T-cells into the efferent lymph; they also express the MHC class I1 DR and DQ locus products (1,10,14). These changes in the expression of cellphenotypes raises some important questions concerning activation, function and
238
BALAKRISHNAN AND ADAMS
TABLE 2A MHC CLASS I MOLECULE ANTIGEN PRESENTATION CYTOPLASMIC PROTEIN
----------- >
PEPTIDES
-"""""
>
Nine amino acids (Could be multiple).
__""""-
>
Delivered to TAP Transporter in the Endoplasmic Reticulum. Heterodimer of TAP1 & TAPS.
DEGRADED INTO PEPTIDES (by LMP Complex) Subset of proteosomes.
Transported into ER Lumen Bind to class I molecules Conformational Change Export to cell surface.
TABLE 2B MEC CLASS I1 MOLECULE ANTIGEN PRESENTATION Assembled from a,/3 and y chain in ER. Transported as nine subunit complex from ER through Golgi to Golgi reticulum. Sorted by y chain to the endocytic pathway. Gamma chain degraded.
CLASS I1 MOLECULES - - - - - - - - - >
ANTIGEN
> Internalized via coated pits,
"""""
endosomes, MHC and lysosomes. Antigen degraded, fragments associated with Class 11. Peptide-Class I1 complex transport to surface.
expression of these class 11-positive T-cell8 as theyrelate to autoimmunity and organ transplantation in man. HLA-DR, expressed by activated human CD4+ - T-cell clones, has been shown to play a major role in signal transduction ( 4 , 9 ) . Antibody cross-linking of DR but not DQ results in an increase in tyrosine phosphorylation and T-cell activation (9).
LYMPHOCYTES AND IMMUNE RESPONSE
239
These and other data suggest that DR and DQ may perform different roles; implying that DQ molecules when expressed on activated T-cells may be more committed to the presentation of suppressor rather than helper epitopes (24). MHC-IN BONE MARROWTRANSPLANTATION Both Host-versus-graft-disease (HVGD) and Graft-versushost-disease (GVHD) remain major problems following transplantation. In bone marrow transplantation (BMT), the utilization of tissuefrommatched, unrelated donors or allogeneic bone marrow is limited by the incidence of sideeffects (25). Research into the pathogenesis of GVHD has been primarily in murine models with limited observations in humans (22,25) Such evaluation of patients with acute versus chronic CVHD has revealed a functional dichotomybetween CD4+ and CD8+
.
T-cells. Functional collaboration between CD4+ and CD8+ T-cells in allograft immunity isconjectured to be more complex in vivo than the observed in v i t r o IL-2 production by responsive Tcells. BMT has become an important treatment for a variety of hematologic malignancies and immunologic disorders (26-28). Post transplant, nearly every immunologic element from T-cells to Bcells to monocytes is alteredin function. I m u n e reconstitution proceeds from the development of "immature" CD8+ and NK-cells, to the more mature specialized CD4+ T-cells that produce and respond to a variety of cytokines in a correlative manner. However, the signals that regulate the synthesis of these cytokines as well as of their surface receptors remain largely unknown.Furthermore,the mechanisms involved in membrane signaling events in T-cells during the establishment of a tolerant state in the hostremain unclear. Alternatively,T-cell to T-cell collaboration leading allograft immunity or tolerance may also require the active participation of the APC and cytokines in controlling the final outcome of the graft. The CD4+ T,-cell would be involvedin the regulation of APC function via MHC class I1 signaling or by controlling the APC and/or CD8+ T-cell function by local cytokine production (23,29,30). Chimerism analysis followingallogeneic BMT permits documentation of importantclinicalevents such asearly engraftment, graft rejection and leukemia relapse (31) Originally, a complete conversion to donor hematopoiesis had
.
240
BALAKNSHNAN AND ADAMS
.
been believed to occur following allogeneic BMT (32-36) More recently, however, there has been increasing evidence that persistence or reappearance of recipient cells is not an unusual occurrence (37). The reappearance of malignant host cells may indicate relapse, while normal recipient cells may coexist with donor cells for various periods of time resulting in stable mixed hematopoietic chimerism (37-41). Graft rejection may occur prior to detection of host cells after BMT (37,40,41). Various other factors such as infection, drugs and GVHD have been implicated in the development of late graft failure in marrow transplant recipients (42). The residual hostimmune state plays a minor role in immune reconstitution; however, persistence of residual host lymphocytes is very important because it is associated with a high risk of graft rejection (43). Many nonspecific immunsuppressor factors such as IFN-a, TGF-8, IL-4, -6 and -10 have been described in the control of GVHD, but the mechanism of control remains unresolved ( 8 ) . It therefore becomes imperative in allograft transplantation to ensure that the best available matched donor is utilized. To help accomplish this, the selection should be made following class I1 compatibility testing using molecular techniques. Functional tests such as the MLC test would also be useful in choosing the best donor. ROLE OF MEC IN S O L I D ORGAN TRANSPLANTS
In solid organ transplantation, the detection of pre-formed antibody produced by B-cells is extremely important to avoid hyperacute rejection. In the past, the mechanism involved in the development of chronic rejectionwere not well defined. Newer evidence suggest that T-cells, cytokines and the adhesion cell molecules, may be directly involved in the acute and chronic rejection phases. Ithas also been proposed that the recognition of foreign MHC antigens by T-cells after transplantation may occur by two different routes; directly as intact molecules or indirectly as peptides after antigen processsing (21). Other mechanisms whereby T-cells are involved in allograft response may include collaborative interaction between CD4+ and CD8+ Tcells with or without the influence from the APC (29). A detour in the direct pathway of allorecognition can frequently be accomplished by immunosuppressive therapy to
LYMPHOCYTES AND IMMUNE RESPONSE
241
prevent the effectsof acute rejection. Conversely, exposure to an alloantigen prior to transplantation has been reported to induce a degree of unresponsiveness (21). The effectiveness of indirect exposure to these antigens depends on the route of antigen presentation,thephysical state of the antigen administered and the genotype of the recipient. An example of indirect antigen presentation that resultedin unresponsiveness in clinical transplantation is from work by Lagaaij et al. whereby they found thatif a recipient was transfused with the donor's blood that shared at least one HLA-DR antigen prior to transplantation, the renal graft survival was prolonged (44)
.
This suggests that intravenous exposure toalloantigen prior to renal transplant can induce a state of "tolerance" in the recipient. Additional studies by van der Mast et al. suggest transfusion of leukocyte-containing blood pre-transplant may induce a GVHD in immunocompetent patients that may ultimately be a self-protective mechanismagainst rejection oftissue. However, factors that must be considered are the immune status of the patient, the HLA match between the host and the donor, and the dose and viability of the leukocytes to be injected (45).
MICROCHIMERISM IN SOLID ORGAN TRANSPLANTATION The ultimate goal of organ transplantation is the induction of specific graft tolerance. Current practice of organ transplantation fails to achieve this objective in the vast majority of organ transplant recipients. The mechanism(s) involved in the development of solid organ graft tolerance is unclear. Microchimerism between donor and recipient may play a major role in a successful long-term graft outcome. In liver transplant recipients, microchimerism has been documented in the transplanted organs as well as other tissues such as skin, lymph The migratory phenomenon of nodes, heart and intestines ( 4 6 ) . donor cells into other organs makes the liver allograft recipient more tolerogeneic. This may account for an
.
immunological advantage over other solid organ transplants This may be due to the fact that organs such as heart and kidney have a lower leukocyte content which make them less tolerogeneic after transplantation. Augmentation of posttransplant cell migration leading totolerance has been achieved with donor specific transfusion and bone marrow infusion. These
BALAKRISHNAN AND ADAMS
242
methods need futher study in solid-organ transplantation and long-term graft outcome. The role and outcome of microchimerism after renal transplantation is not well understood and has not been adequately studied. Every exposure to histoincompatible lymphoid cells has the potential for sensitizing andinitiating an immune response in the recipient. This can occur after transfusion of blood, BMT and solid-organ transplantation. Transfusion associated GVHD is a severe illness which often has a fatal outcome - if the recipient's immune system is compromised. However, transfusion The ultimate related GVHD is rare in immunocompetent recipients. outcome depends on the immune status of the recipient, the number of leukocytesinfused and the disparity in the histocompatibility antigens between the donor and the recipient. REFERENCES 1.
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D.C. Roy, R. Tantravahi, C. Murray, K.C. Anderson, A.S. Freedman, L.M. Blood 7 5 , 296-304 (1990).
z,
K. Dear, B. Corgone, Nadler and J. Ritz.
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K.
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S. Nakao, T. Nakatsumi, T. Chuhjo, O.H. Tsuchiya, T. Niki, S. Shiobara, T. Mori and T. Matsuda. Transplantation, 9 , 107-111 (1992).
43.
A. J. Jef freys, A.MacLeod, R. Neumann, S. Povey and N. J. Royle. Genomics, 2, 449-452 (1990).
44.
E.L. Lagaaij, I.P.J. Henneman, M.B. Med., 3 2 1 , 701-705 (1989).
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B. J.van Der Mast, N.Hornstra, M.B. Ruigrok, F.H. J. Claas, J.J. van Rood, E.L. Lagaaij. Lancet, 3 4 3 , 753-757 (1994).
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T.E. Starzl, A.J. Demetris, N. Murase, A.W. Thomson, M. T ~ U C C O ,C. Ricordi. Imunology Today, l4, 326-332 (1993).
47.
M. Isobe, Y.K. Yagita, O.A. Ihara. Science, (1992).
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1125-1128
NEUTROPHIL ANTIGENS, FROM BENCH TO
Albert E.G.
Kr. von dem Borne*, MD PhD; Masja
BEDSIDE
de Haas, MD; Dirk Roos,
PhD; Christa H.E. Homburg, C.Ellen van der Schoot, MD PhD Department of Hematology and Centre for Blood Cell Research, Academic Medical Centre, and Department of Experimental Immunological Hematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands
INTRODUCTION Neutrophil antigens are of great interest, because of their involvement in a number of important immune mediated diseases of the blood as well as in transfusion reactions. But they are also of interest as markers of certain neutrophil membrane glycoproteins. In the past two decades these antigens have been intensively studied. New methods to detect, measure and characterise antigens and antibodies have been devised. This has led to the recognition of new antigen systems
and to new insights in the
pathophysiology of immune mediated disorders of the blood in which theyare involved. Studies at the molecular level have added a new dimension. Moreover,
*Correspondence: Dr. A.E.G. Kr. von dem Borne, Department of Hematology, Academic Medical Centre, University of Amsterdam, P.O.Box 22700, 1l00DE Amsterdam, Netherlands. Phone: (20)5665785, Fax: (20)6968833, E-mail
[email protected] or
[email protected] 245
246
BORNE
DEM
ET AL.
VON
soluble products of antigen-carrying glycoproteins have been detected in the blood, which can be used in the evaluation of neutrophil kinetics in health and disease. In this chapter various clinical, immunologicaland biochemical aspects of neutrophil antigens will be discussed. NEUTROPHIL ALLOANTIGENS
.
On neutrophils different types of antigensare present Common antigens shared with other blood cells and/or tissue cells are glycoconjugate antigens of the blood group I and P system and glycoprotein HLA class I (ABC) antigens. Blood group ABH and Le antigens are not present on neutrophils 19. On their surface membranes they carry another type of complex sugars (unbranched and fucosylated in another way) different from those of red cells and platelets, which can not act as substrates for A,B,H or Le transferases. These complex sugars are also present on monocytes (mainly in a sialated form). They react with murine monoclonal antibodies of the cluster CD15 or its subcluster CDlSs, which react with the L e x structure or with sialyl-Lex ,respectively ’. Recently, the reason for this marked difference in surface membrane carbohydrate make-up has become clear. Complex carbohydrates on leukocytes play an important role in adhesion processes, while those of red cells (and platelets?) appear to have an anti-adhesive function. Inorder to migrate to the tissues leukocytes must adhere to and pass through the endothelium. In this process there
are four different steps: tethering, triggering, strong adhesion and migration
3.
Tethering, which is the first step promotes under flow conditions rolling of leukocyte on the vessel-walls. It is mediated by a family of three lectin-like molecules called selectins
(L-,E-, and P-selectin) L-selectin
is present on
leukocytes, E-selectin and P-selectin on activated endothelial cells, and P-selectin also on activated platelets. The ligands of the selectin molecules are specific carbohydrate structures, on leukocytes of the CD15 cluster. A rare hereditary disorder has been described, Leukocyte Adhesion Deficiency typeI1 (LADII), with recurrent infections, marked neutrophilia and a neutrophil aggregation defect
NEUTROPHIL 22.
247
There is a generalised fucose deficiency, leading to a deficiency of sialy1-k".
HLA class I1 @P,DQ,DR) are not present on neutrophils either. However, in
some donors they are expressed on neutrophils under the influence of cytokines such as GM-CSF and/or G-CSF both in vitro 26 as well as in vivo (unpublished observations). Neutrophils carry also antigens, that have been detected onlyon these cells so far. These "neutrophil specific antigens" are the antigens of the NA-, NB-, NC-, NDand NE-system. The first three systems were all described by Lalezari and coworkers 41 in a series of classical studies with sera from mothers who gave birth to a neutropenic child. The last two systems were discovered with sera from patients with autoimmune neutropenia, and are less well defined
I2*".
A new system, LAN,
has quit recently been described and found to be responsible for neonatal alloimmune neutropenia in Australian arboriginals
".
Finally, neutrophils express antigens which they share with other white cells (monocytes and lymphocytes). These antigens were defined in studies with selected sera from individuals alloimmunised by blood transfusion and/or pregnancy and are the 9-system antigens, Onda=E27 and Mart"36*52*66~68. The 9system antigens werelater found to be identical with the antigens of the human monocyte system HMA-I
35.
In table 1 the phenotype and gene frequencies ofthe different neutrophil-specific
and leukocyte-associated antigen systems are shown. All appear to be biallelec systems. A possible exception is the NA-system. In studies in various laboratories
a very strong association @>0.001) has been found between positivity for the NA2- and the NC1-antigen. It might indicate the existence of triallelic system, with the alleles NA(Z+)NC(I +), NA( 1+)NC(l +) and NA(1+)NC( I-). A interesting observation is that Japanese and Chinese havea much higher
frequency of the NAl-genotype than Europeans and Northern-Americans (table 2) 6142*49.
Divergent phenotype and gene frequencies between Mongolian and
Caucasian people have also been found for platelet and red cell antigens.
248
BORNE VON DEM
ET AL.
Methods to detect neutrophil antibodiesand tot y p e and characterise neutrophil antigens Tests most commonly used to date are agglutination and immunofluorescence, but cytotoxicity and antibody dependent cellular cytotoxicityare being applied as well 21,45
The neutrophil agglutination test (NAT) and the neutrophil immunofluorescence test (NIFT) are most suitable for routine use. Both have an acceptable sensitivity and specificity, although the NIFT scores higher in this respect. Both tests detect IgM and IgG antibodies 69. Agglutination with IgG antibodies is an active process that requires both F(ab) binding and Fc-receptor binding, and for which viable cells, energy and an intact cytoskeleton are necessary. As a consequence it is inhibited by a low temperature, metabolic inhibitors, inhibitors of microfilament and/or micrutubule formation, etc. n. However agglutination by IgM antibodies is purely an immunological process, occurring most optimally at low temperatures. The neutrophil cytotoxic test (NCT) is not suitable for routine use. Only
complement binding antibodies are detected in this assay. Moreover, aspecific positive test results often occur. The antibody dependent cellular cytotoxicity assay 43 is the most sensitive and specific test for neutrophil antibodies available at present. It measures lysis of "Cr-tagged neutrophils by killer lymphocytes (NK-cells) upon sensitisation with antibodies. Because this is also an Fc-receptor dependent process only IgGantibodies (of the IgG1, IgG2 and IgG3 subclass) are being detected. Unfortunately, the test it is too complicated for routine use. For scientific studies methods suchas immunoprecipitation, immunoblotting and monoclonalantibodyimmobilisation(MAINA)
are in use
MAINAmaygain
a place in the routine laboratory.
A new method for neutrophil antibody detection that was recently developed is the
neutrophil chemiluminescence test (NCLT). It is based on the activation of neutrophils by antibodies and the subsequent generation of luminescence-inducing superoxides. The test appears to be as applicable as the G I P .
NEUTROPHIL ANTIGENS
249
CLINICAL IMPORTANCE OF NEUTROPHIL ALLOANTIGENS Antibodies against neutrophil antigens are involved in neonatal alloimmune neutropenia, autoimmune neutropenia, notably when occurring in infants, and in transfusion reactions. Neonatal alloimmune neutropenia (NAINP)40 NAINP is a rare disease but important disease, occumngin less than 1 per 2000 new-borns. It may lead to severe and sometimes (S%?) lethal infections in the newborn. However, it may also be an entirely asymptomatic disease. Infections are often recurrent and caused by gram positive bacteria (streptococci, staphylococci). They preferentially affect the skin and mucosal membranes of the airway tract (ENT, respiratory tract), but sepsis may also occur. Detection of neutrophil specific alloantibodies in the blood of the mother is of great diagnostic value (sensitivity 96%). The blood of the newborn shows selective neutropenia and (often) compensatory monocytosis, while the bone marrow is normal or shows a maturation arrest of neutrophils. If untreated, alloimmune neutropeniamay persist for 2-4 month. Treatment modalities are antibiotics (also given prophylactically), plasma exchange, high dose intravenous gammaglobulin and G-CSF. For review see elsewhere
37,45.
Autoimmune Neutropenia (AINP) of infancy38 Chronic idiopathic and secondary neutropenia is quite a rare haematological disorder. Serological analysis of patients with this disease shows that often neutrophil-bound immunoglobulinsare present and that the serum may contain neutrophil (aut0)antibodies. In adults these antibodies show only rarely antigenspecificity 70. However, in primary autoimmune neutropenia of infancy, neutrophil autoantibodies of the IgG and/or IgM class with antigen specificity are found in about 50% of the cases. This is a peculiar disease, in that it occurs at a very early age (a few months to less then two years) without any apparent cause, and
250
VON DEM BORNE ET AL.
spontaneously resolves with disappearance of the antibodies in 6 months to 3 years. Blood and bone marrow show the same abnormalities as found in NAINP patients. Recurrent infections may occur. These infections are mostly of mild to moderate severity, and usually affect of the skin (boils, cellulitis), the middle ear (otitis media), the oropharynx and upper respiratory tract (stomatitis, tonsillitis, pharyngitis) and the digestive tract (gastroenteritis). Few affected children develop pneumonia. Sepsis occurs only rarely. Usually, infections can be managed with routine antibiotic therapy, but in severe cases corticosteroids, high dose intravenous IgG, G-CSF and plasmapheresis are therapeutical options. For more detailed information see elsewhere 15.39*45,46. Transfusion reactions
In most cases a transfusion reaction due to neutrophil reactive alloantibodies is an uncomplicated febrile reaction. Treatment with an antipyretic drug is then an adequate measure. Leukocyte removal by filtration of the blood
will usually
prevent the occurrence of further episodes. The responsible antibodies are usually HLA-antibodies, but .sometimes neutrophil specific antibodiesare involved 17. In some patients a life threatening acute respiratory distress syndrome may develop, also referred to as transfusion induced acute lung injury (TRALI)
53.
Transfusion induced acute lung injury (TRALI)
TRALI has always been considered to be a rare complication of transfusion. However, analysis of the causes of transfusion associated deaths in the USA reported to the FDA from 1976 to 1985 57suggest that this is not an entirely correct view. Although haemolysis (acute, delayed) is the leading cause
with
71.9% (184 of 256) of transfusion associated deaths, TRALI is the second cause
with 12.1% (31 of 256), occurring more often than death due to bacterial contamination of blood or blood products (26 of 256 i.e. (8 of 256 i.e. 3.1%).
10.2% ) or anaphylaxis
NEUTROPHIL ANTIGENS
25 1
TRALI is an acutely occurring dramatic complication of transfusion therapy. It may develop within minutes after starting a transfusion. Signs are nausea and vomiting, a persistent cough with production of sero-sanguineous sputum
,
dyspnea, cyanosis, development of mental confusion,and even coma with progression to death. Symptoms are a noisy respiration, rales over the lungs, tachycardia, hypotension, hypoxia and respiratory acidosis. A lung X-ray shows diffuse, mottled infiltrates. Treatment is difficult, and consists of intensive respiratory and circulatory support, corticosteroids, prophylactic antibiotics. Plasma(leuka)pheresis may have a beneficial effect TRALI has occurred during the administration of leukocytes to individuals sensitised against leukocyte antigens. However, most cases reported in the literature were due to alloantibody infusion i.e. infusion of whole blood or plasma from donors, containing alloantibodies reactive with the recipient’s leukocytes. TRALI is supposed to result from an intravascular reaction between neutrophils (or leucocytes in general) and alloantibodies, leading to massive sequestration of these cells in the lung. Damage to the lung tissues may be caused by substances released from activated neutrophils such as neutral proteases and superoxides, but perhaps also by various cytokines. Detailed studies are still necessary to obtain more exact information about the incidence of TRALI,and the role of leukocyte antibodies in its pathogenesis. Neutrophil alloantibody specificity in different clinical settings
The neutrophil alloantibodies that are most commonly involved in NAINP are anti-NA1 and anti-NB1. Anti-NA2 and anti-NI32 are found less often, and antiNC1 rarely. In many cases (40%) specificity is not clear
37*45.
Some may be due to
panreactive antibodies in cases of neonatal isoimmune neutropenia (see later). As discussed, autoantibodies show only rarely antigen-specificity in adults with AINP. Auto-anti-NAl,-NDl and - N E 1 have been found in a few cases 70. However, in primary AINP of infancy neutrophil autoantibodies with antigen specificity are found in about 50% of the cases (mostly anti-NA1, sometimes antiNA2) 39.45.46.70
VON DEM BORNE ET AL.
252
TRALI has occurred after infusion of whole blood or plasma from donors, containing neutrophil specific antibodies (anti-NA1, -NA2, -N€31,-Sb) and HLA class I antibodies
Recently, we observed a case in which TRALI
439*20348*64*75.
occurred upon infusion of a gammaglobulin preparation containing high titers of both HLA class I and class I1 antibodies Is. GLYCOPROTEIN LOCALIZATION AND OTHER CHARACTERISTICS OF NEUTROPHIL ANTIGENS The study of the biochemical nature of neutrophil antigens has made significant progress lately; see Table 1. NB-system antigens With the help of immunoprecipitation and immunoblotting it was established that NB1 is present on a membrane glycoprotein of 56-62kDa under reducing conditions (Mr 49-55 kDa non-reduced). It was shown that this glycoprotein is a glycosyl-phosphatidylinositol (GP1)-anchored N-glycosylated protein(see also later). The allotypic NBl-epitope is alsorecognised by a mouse monoclonal antibody (1B5) 14*25*58@. NB1 antigen staining of neutrophils varies greatly among different donors (range0-100%), but is mostly constant in individual donors. Blood cells other than neutrophils do not stain n. NB1 antigen is expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. Cross linking of NB1 antigen on the plasma membrane resulted in internalisation of the complex, while in-vitro stimulation of neutrophils caused an increase in intensity of plasma membrane staining with antiNB1, but only of those cells that were positive already prior to stimulation25@’.
N B 1 glycoprotein thus appears to identify a distinct subset of neutrophils. Its function remains unclear, but its behaviour upon cross linking and stimulation suggests a possible role as receptor molecule. Recently, a role in the margination of neutrophils was suggested
59.
253
NEUTROPHIL ANTIGENS TABLE I. NEUTROPHIL SPECIFIC AND LEUKOCYTE "SPECIFIC" ANTIGENS
Antigen
System
Phenotype
Gene frequency
frequency in 9%
NA1
46
0.38
NA2
88
0.63
NB1
97
0.83
NB2
32
0.17
NC
NC1
91
0.72
ND
NDl
98.5
0.88
NE
NE1
23
0.12
9=HMA
9'= HMAl
69
0.44
9b = HMA2
81
0.56
Mart
Malt?
99
0.91
Ond
Ond' (E27
> 99
> 0.91
NA
NB = S?
NA-system antigens (see Tables I and II) Immunoprecipitation, immunoblot and monoclonal antibody immobilisation, failed to localise the antigens of the NA-system. However, a different approach solved this problem.
TWO
murine McAb's against the human neutrophil Fc -receptor
(FcGranl and Granl 1) were produced 74. Later, these antibodies were found to be directed against the major Fc-receptor of neutrophils, FcRIII . In Monoclonal Antibody Workshops the antibodies were clustered in the CD16 McAb cluster. One antibody (FcGranl) was panreactive i.e. it reacted with the neutrophils of all donors tested. The other antibody (Granl1) reacted with only about 50% of the neutrophils of normal donors and appeared to be a murine anti-NA1. Such antibodies are now clustered in the subcluster CD16b. In precipitation
254
VON DEM BORNE ET AL.
NA2
NA1
Netherlands 0.625
0.374
Fl7UVX
0.332
USA 0.663
0.337
0.302 Japan
0.651
0.309 China (Taiwan)
0.680
0.640
studies the panreactive antibody reactedwith the whole neutrophil FcRIIIstructure, a typical smear-like precipitate of50-80 &a upon SDS-polyacrylamide gelelectrophoresis. This broad smear is due to heavy, but variable glycosylation of the molecule. Monoclonal anti-NA1 Gran11 precipitated only the lower half of the FcRIII smear, indicating that NA1-FcRIII has a faster electrophoretic mobility than NA2-FcRIII. This was confirmed when precipitations were performed with the neutrophils of different NA-typed donors. Thus, the NA-allotypes of FcRIII are also reflected in electrophoretic mobility differences
30750.
Neutrophils also carry another type of FcR for IgG, FcRII. Other blood leukocytes that carry FcR for IgG are monocytes, with FcRI (the high affinity Fcreceptor) and FcRII, and natural killer (NK) cells with only FcRIII. However, NA-antigens have only been detected on neutrophils 29. It indicated that FcRIII from neutrophils and NK cells are different structures, as was later confirmed in molecular genetic studies 32*33. They are called FcRIIIb and FcRIIIa, respectively. Monoclonals have been produced that react only with FcRIIIb, while others react both with NA2-FcRIIIb and FcRIIIaMcAb’sspecificforNA2-FcRIIIb FcRIIIa have not yet been produced.
or
NEUTROPHIL ANTIGENS
255
"NA-NULL" PHENOTYPE In two clinical situations neutrophils have the phenotype NA(1-2-)(NA-null). This is in paroxysmal nocturnal hemoglobinuria and in hereditary neutrophil FcRIII deficiency. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) This is an acquired clonal defect of the bone marrow stem cell. In PNH the red cells (and also other blood cells) miss multiple membrane glycoproteins, including the important complement regulatory proteins decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL) and C8 binding protein. This makes these cells very sensitive to lysis by activated complement components. Because some complement activation always occurs in vivo chronic intravascular haemolysis results, in some patients (for unknown reasons) occurring most strongly during the night. Recently, it was found that the missing membrane proteins all belong to a new class of membrane glycoproteins, so called PIG-linked proteins, which are anchored in the outer lipid layer of the cell membrane via phosphatidyl-inositol glycan
PIG^.
In PNH a mutation has occurred in the hematopoietic stem cell which leads to defective anchoring of such proteins. The nature of the mutation has been clarified. The proteins are synthesised normally in blood precursor cells of PNH patients. But, PIG-anchoring is abnormal because of an acquired mutation in the gene of the enzyme PIG-A. This enzyme is necessary for the first step in the biosynthesis of the anchor. The gene is on the short arm of the X-chromosome When studying PNH neutrophils we found that they do not, or in
62.
a much lower
concentration, express FcRIIIb and NA-antigens, while PNH NK cells had normal expression of NA-antigen negative FcRIIIa
30p34-67.
It has been established that the
FcRIIIa of NK cells (and of macrophages) is a transmembrane and not a PIGanchored molecule.
VON DEM BORNE ET AL.
256
HEREDITARY NETUROPHIL FcRIII DEFICIENCY AND NEONATAL ISOlMMUNE NEUTROPENIA In studying a case of neonatal neutropenia, we found that the cause was isoimmunization of the mother against neutrophil FcRIIIb of her child 31. She appeared to have a hereditary deficiency of this receptor. Clinically, it was a classical case of neonatal immune neutropenia. The affected baby (the third child of the family) recovered after intravenous gammaglobulin treatment without any severe complications. The mother, who was healthy, had panreactive neutrophil antibodies in her blood not reacting with her own cells. Her neutrophils were typed as NA(1-2-) and lacked expression of FcRIIIb. The antibodies were isoantibodies directed against neutrophil FcRIIIb, strongly reactive in the immunoblot with the whole FcRIIIb smear. Also a healthy male blood donor was found who had the same abnormality. His neutrophils were unreactive with the mother’s antibodies. He had not produced neutrophil antibodies himself. Hence, the isoantibodies in the mother were probably the result of transplacental immunisation during pregnancy. The defective neutrophil FcRIIIb expression in these two individuals was studied in detail at the RNA/DNA level. In both a deletion of the neutrophil FcRIIIB gene was found, but not of the FcRIIIA gene. Recently, other cases of neonatal isoimmune neutropenia due to neutrophil FcFUIIb deficiency have been described. Also more deficient but healthy individuals and a patient with systemic lupus and the defect have been discovered
11913924.61.
All
individuals studied at the genomic level so far appear to have the same deletion of the FcRIIIB gene. Thus, hereditary neutrophil FcRIIIb deficiency due to gene
deletion does not seems to be an extremely rare genetic defect. Whether it will have any clinical effect, other than being a cause of neonatal immune neutropenia, has still to be studied.
SOLUBLE FcRIII AND NA-ANTIGENS IN PLASMA
In vitro, neutrophil-FcRIIIb is released in a soluble form into the fluid phase upon stimulation of neutrophils with various agonists. FcRIIIb is cleaved off, apparently
NEUTROPHIL
257
by a latent membrane protease that becomes activated by the agonists. In vivo, cleaved soluble(s) FcRIII is present in body fluids 2B; see Table I. We found it in plasma, ascites and lymph fluid in quite high amounts and in traces also in the urine. Immunochemical studies have shown in most individuals and patients sFcRIII stems mainly from neutrophils i.e. that it is mainly sFcRIIIb. Plasma sFcRIIIb also carries the NA-antigens. Its concentration is markedly decreased in the plasma of PNH patients and it is undetectable in plasma from most individuals with neutrophil-FcRIII deficiency. The level of sFcRIII in plasma is not clearly related to the blood neutrophil count and not markedly increased in patients with infectious or inflammatory disorders. Thus, it seems that the level of plasma sFcRIII is determined mainly by constitutive shedding or excretion from neutrophils. It may be a good measure for the overall neutrophil mass in the body. Indeed, its level is decreased in patients with neutropenia due to cytostatic treatment and normalises upon recovery. Recently, we found that under certain circumstances also sFcRIIIa may
be present
in plasma. It was detected in plasma from one individual with neutrophil-FcRIIIb deficiency, who had a chronic arthritis. Later it was also found in plasma from some other arthritic patientsand in patients with NK-lymphocytosis 16. It appeared to originate from NK-cells. Whether sometimes also macrophage-derived sFcRIIIa may be present in plasma in measurable amounts is still under study. Mechanism($ of in vivo release of soluble FcRIII
Neutrophils kept in maintenance culture in vitro loose FcRIIIb without loosing viability. Also this loss is due to a proteolytic event. It can be inhibited by the addition to the culture of cytokines such as G-CSF, GM-CSF
and interferon
. In
recent studies in our laboratory we obtained strong evidence that it is apoptosis (programmed cell death), switched on in the cultured cells, that initiates sFcRIIIb release. Apoptosis is accompanied by the externalisation of charged inner membrane phospholipids (phosphatidyl-serine (PS) and phosphatidyl-ethanolamine
DEM
258
VON
BORNE ET AL.
(PE)), so called flip-flop. It can be detected by the acquisition of binding sites for (fluorescent) annexin 5 27. Annexin V is a PS binding protein. In vitro, all annexin
V positive neutrophils had completely lost membrane FcRIIIb. These findings indicates that also in vivo sFcRIIIb might originate from naturally occurring apoptosis. I t might even be a more general mechanism for the release of cell membrane glycoproteins. NC- AND ND-ANTIGENS PNH neutrophils are not only NAl- and NAZantigen negative, but also NC1-, ND1-and NB1-antigen negative, indicating that all these antigens
are on one or
more PIG-linked membrane proteins. PIG-linkage of the NBI-antigen has been discussed already. PIG-linked structures can be removed by the enzyme GPI-PLC (glycophosphoinositol specific phospholipase C). Indeed, neutrophils treatedwith GPI-PLC loose all the antigens mentioned above. All FcRIII deficient individuals, studied in our laboratory so far, were typed negative for NAI, NA2, NCI and N D 1 , but positive for NB1. Thus, it appears that not only NA-, but also NC- en ND-antigens are located on PIG-linked FcRIIIb, and that NB-antigens are carried by another structure, as was known already from the studies discussed above. The newly described LAN-antigen was also found to be present on FcRIIIb
47.
OND- A N D MART-ANTIGENS The antigens Ond' and Mart" (see Table I), are expressed on neutrophils, monocytes and lymphocytes. This suggests the presence of these antigens on
a
more common glycoprotein type. Likely candidates are leukocyte integrins i.e. molecules of the Leu-CAM or ,-integrin family.
To date three members of this family have been identified: LFA-I ( L 2), complement receptor 3 (CR3)(
M
), and complement receptor 4 (CR4)( x
2).
They are detected by McAb's of the cluster groups CD1 1 ( -chain antibodies),
NEUTROPHIL
259
including CDlla, CDllb, CDllc, and CD18 ( -chain antibodies). Definite localisation of the Ond and Mart alloantigens on Leu-CAM structures was achieved by applying immunoprecipitation and monoclonal antibody immobilisation. It was further evidenced by immunofluorescence on cells from patients with Hereditary Leukocyte Adhesion Deficiency (LAD) type I. These patients do not have Leu-CAM molecule expression because ofa genetic defect in the synthesis of the ,-chain. Ond" -antigen was located on the on the
L
M
-chain (CD1 lb molecule) and Mart' -antigen
-chain (CD1la molecule) of the Leu-CAM family, respectively
In 1987 Pischel et al.
52
described an alloantiserum (E23 also reacting with a
polymorphous structure on LFA-1 -chain. Serologically E27-antigen
and
0nd"-antigen were found to be identical. Many platelet alloantigens (HPA-1, -3, -4 and -5 system antigens) are located on integrin structures as well, notably the
- or-chainof
the cytoadhesin or
3-
integrin family (GPIIIa or GPIIb). Thus, the general message is that integrins tend to be polymorphic, which may lead to antigenicity and immune mediated diseases. THE MOLECULAR NATURE OF NEUTROPHIL ANTIGENS So far, only the molecular nature of the NA-system antigens has been elucidated;
(seeTable 111). This was made possible by the findings mentioned above, and by the cloning and sequencing of FcRIII-cDNA 50*51*54*63. From cloning it became again clear that there are two types of FcRIII: FcRIIIb, which is a shorter structure of 233 aminoacidsand FcRIIIa, which is a somewhat longer structure of 254 aminoacids. FcRIIIb, the neutrophil form, becomes PIGlinked (possibly via a serine on position 234). FcRIIIa, the NK cell and macrophages form, becomes a transmembraneous structure. FcRIIIa and FcRIIIb are the products of two different genes present on chromosome 1, denoted FcRIIIA and FcRIIIB, which are very closely linked. The coding sequences are identical for more than 95%.
260
VON DEM BORNE ET AL. TABLE m. DIFFERENCES IN DEDUCED AMINOACID SEQUENCE
FcRIIX Type
A
A Aminoacid positions
number 36
65
82
NA1-FcRIIIb
233
arg
asn
NA2-FcRIIIb
233
ser
m**m
FcRIIIa
254
ile arg asp ser
158 176 234 203
147 106
asp val
ile
asp
his
val
ser*
-
asp
his
val
ser*
-
gly phe tyr
Phe asp
* PIG binding site? ** Extra glycosylation site underlined
In the aminoacid sequences of FcRIIIb and of FcRIIIa from NA1 or NA2 donors only very subtle differences have been found (table 3). Some are specific for the FcRIII type. Others are probably related to theNA polymorphism of the FcFUIIb. These are the aminoacids arginine, asparagine, aspartate and valine (at positions 36, 65, 82 and 106) in NAl-FcRIIIb and serine, serine, asparagine and isoleucine
in NA2-FcRIIIb. The aminoacid differences are all based on one nucleotide difference in the coding triplet. The serine and asparagine substitution in NA2FcRIIIb (at positions 65 and 82) lead to two extra glycosylation sites. This explains the slower mobility of NA2-FcRIIIb compared to NA1-FcRIIIb in SDSpolyacrylamide gelelectrophoresis.
NEUTROPHIL ANTIGENS
26 1
Thus, the molecular basis ofthe NA-antigens differs from that of the platelet antigens in that multiple aminoacid substitutions are found. The precise location of the NA epitopes on the FcRIIIb molecule is not yet clear.
Other FcR polymorphism’s Two allelic forms of FcRIIa have been identified, the other Fc-receptor present on neutrophils (as well as on monocytes and macrophages). Originally it was discovered as a functional polymorphism, based on the binding capacityof mouse IgGl, the low-responder (LR) and high-responder (HR) allotype. It is caused by a single aminoacid difference, a histidine (H) or an arginine (R) at position 131 of the mature polypeptide chain. The two alleles are therefore designated as FcRIIa-
H131 (LR) and FcRIIa-R131 (HR), also because it was found that the LR-form was in fact more avidly binding human IgG2 than the HR-form
65*73.
A McAb
(41H16) is available that is specific for IIa-R131, however, human alloantibodies against these allotypes have not (yet) been found. Recently, we discovered a polymorphism of FcRIIIa (article in preparation). Size differences were detected inthe deglycosylated FcRIIIa from different individuals (upon SDS-polyacrylamide gelelectrophoresis). In fact two forms were encountered, a slower (S) and a faster (F) moving form, and heterozygotes with both forms (SF)were found as well. By analysing 28 individuals we could (tentatively) calculate a phenotype frequency of 96 % for S-FcRIIIa and 25% for F-FcRIIIa, and a genotype frequency of about 0.83 and 0.17, respectively. Further studies showed that the polymorphism is caused by a single aminoacid difference, a leucine (L) or an arginine (R) at position 49 of the mature FcRIIIa polypeptide. It results from a single base substitution (T G), which can be detected at the genomic level by the acquisition of a site for the restriction enzymeAci I. Whether this polymorphism has any functional consequences is under study. McAb’s or human alloantibodies which recognise FcRIIIa-WR49 allotypes have still to be found.
262
VON DEM BORNE ET AL.
FcR-polymorphism’s and disease susceptibility Recent studies have shown that Fc-receptor allotypes may influence susceptibility for certain infectious diseases, notably meningococcal disease. In 25 children who survived a fulminant meningococcal septicaemia the FcRIIa-R/R313 allotype appeared to be over represented
’. Moreover, in 15 individuals with complement
factor C6 or C8 deficiency the occurrence of meningococcal disease (in 8 ) was significantly associatedwith the FcRIIa-WR131/FcRIIIb-NA2/NA2 allotype 23. This finding is explained by the fact that humoral defence mechanisms (specific antibodies, complementand phagocytes with their Fc- and complement receptors) play an important role in the defence and protection against meningococci. Human IgG2 and IgG3 seems to interact more readily with the FcRIIa-H131 allotype than with the FcRIIa-L131 allotype, and IgGl and IgG3 more readily with FcRIIIbNAl than FcRIIIb-NA2 allotype *.Thus, individuals with the FcRIIa-R131and/or NA2-gene seem to be less able to defend themselves against meningococci, especially at young age or when an immune deficiency exist, such as a complement deficiency. It remains to be studied whether these polymorphism play also a role in the susceptibility for other infectious diseases. Moreover, the involvement of other polymorphism’s of Fc-receptors and of other membrane glycoproteins of phagocytes (such as integrins and lectins) should be studied as well. CONCLUSION Much has been achieved in the field of neutrophil immunology in the past decades. The insight in the various immune mediated disorders and the nature of the antigens involved has markedly increased. Important is the finding that (antigenic) polymorphism’s of neutrophil glycoproteins may be associated with susceptibility to certain infectious diseases. Nevertheless, much has still to be learned. Still we do not understand how and why an immune response which leads to disease is generated. A more detailed knowledge of the process of presentation
of antigens or antigenic peptides via HLA-molecules, and of selection and activation of T-cells and B-cells is necessary for further progress in this field.
263
NEUTROPHIL ANTIGENS
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anchored via a glycosyl-phosphatidylinositol linkage. J Leukocyte Bioi 49: 163,
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123:247, 1994
60. Stroncek DF, Skubitz KM, McCullough JJ: Biochemical characterization of the neutrophil-specific antigen NB1. Blood 75:744, 1990
61. Stroncek DF, Skubitz KM, Plachta LB, Shankar RA, Clay ME, Herman J, Fleit H B , McCullough J: Alloimmune neonatal neutropenia due to an antibody to the neutrophil Fc-gamma receptor I11 with maternal deficiency of CD16 antigen.
Blood 77: 1572, 1991 62. Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T: Deficiency of the GP1 anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell
73:703, 1993
63. Trounstine M, Peltz G, Yssel H, Huizinga TWJ, von dem Borne AEGK, Spits H, Moore K: Reactivity of cloned, expressed human FcgammaRIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of FcgammaRIII.InternationalImmunology 2:303, 1990 64.
van Buren NL, Stroncek DF, Clay ME, McCullough J, Dalmasso AP:
Transfusion-related acute lung injury causedby an NB2 granulocyte-specific antibody in a patient with thrombotic thrombocytopenic purpura. Transfusion
30:42, 1990 65. Van de Winkel JGJ, Capel PJA: Human IgG Fc receptor heterogeneity: Molecular aspects and clinical implications. Immunol Today
14:215, 1993
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66. van der Schoot CE, Daams M, Huiskes E, Clay M, McCullough J, van Dalen C, von dem Borne AEGK: Antigenic polymorphism of
the Leu-CAM family
recognized by human leukocyte alloantisera. 1994 (in preparation) 67. van der Schoot CE, Huizinga TWJ, van 't Veer-Korthof ET, Wijmans R, Pinkster J, von dem Borne AEGK: Deficiency of glycosyl-phosphatidylinositollinked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay. Blood 76: 1853, 1990 68. van Rood JJ, van Leeuwen A, Schippers AMJ: Leukocyte groups, the normal lymphocyte transfer test and homograft sensitivity. Histocompatility Testing 37, 1965 69. Verheugt FWA, von dem Borne AEGK, Decary
F, Engelfriet CP: The
detection of granulocyte alloantibodies with an indirect immunofluorescence test. Br J Haematol 36533, 1977 70. Verheugt FWA, von dem Borne AEGK, van Noord-Bokhorst
JC, Engelfriet
CP: Autoimmune granulocytopenia: the detection of granulocyte autoantibodies with the immunofluoresence test. Br J Haematol 39:339, 1978
71. Verheugt FWA, von dem Borne AEGK, van Noord-Bokhorst JC, Nijenhuis LE, Engelfriet CP: ND1, a new granulocyte antigen. Vox Sang 35:13, 1978 72. Verheugt FWA, von dem Borne AEGK, van Noord-Bokhorst
JC, van Elven
EH: Serological, immunochemical and immunocytological properties of granulocyteantibodies.VoxSang35:294,1978 73. Warmerdam PAM, Parren PWHI, Vlug A, Aarden LA, Van de Winkel JGJ,
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Cape1 PJA: Polymorphism of the human Fcgamma receptor I1 (CD32): Molecular basis and functional aspects. Immunobiology 185:175, 1992 74. Werner G,von dem Borne AEGK, Bos UTE, Tromp JF, van der Plas-van Dalen CM, Visser FJ, Engelfriet CP, Tetteroo PAT: Localization of the Human NA1 Alloantigen on Neutrophil Fc-gamma-Receptors, in Reinherz EL, Haynes BF, Nadler LM, Bernstein ID (4s): Leukocyte Typing 11. Volume 3. Human Myeloid and Hematopoietic Cells, New York, Berlin, Heidelberg, Tokyo, Springer-Verlag, 1986, p 109 75. Yomtovian R, Kline W, Press C, Clay M, Engman H, Hammerschmidt D,
McCullough J: Severe pulmonary hypersensitivity associated with passive transfusion of a neutrophil-specific antibody. Lancet 1:244, 1984
ANTI-IDIOTYPES
TO HLA AND T H E I R ROLE I N TRANSPLANTATION
E . Reed Departmentof Pathology College of Physicians and Surgeons of Columbia U n i v e r s i t y New York, New York 10032
ABSTRACT The network theory proposed by J e r n e i s based on t h e f i n d i n g thatvariableregions of T and B c e l l a n t i g e n r e c e p t o r s a r e s t r u c t u r a l l y d i v e r s e and expressuniquevariableregion determinants. W e have p o s t u l a t e dt h a ti d i o t y p e sp r e s e n t on t h e V region of t h e anti-HLA antibodymolecule (Abl) can e l i c i t t h e productionofanti-anti-HLA a n t i b o d i e s o r Ab2 and t h a t such Ab2 may play a r o l e i n t h e s u p p r e s s i o n ofanti-HLA antibodyresponses. W e first tested the validity of t h i s concept i n pregnancy, n a t u r e s most p e r f e c t model of a l l o g e n e i ct o l e r a n c e . O u r s t u d i e sr e v e a l e d that anti-Id antibodies were p r e s e n t d u r i n g pregnancy a t times when HLA a n t i b o d i e s had disappeared from t h e c i r c u l a t i o n ( 1 , 2 ) . T h i s i n v e r s e c o r r e l a t i o n between Ab1 and Ab2 suggested that Ab2 suppressestheproduction ofAbl. Once t h e v a l i d i t y of t h i s concept was s u b s t a n t i a t e d i n t h e modelof pregnancy, we t r i e d t o determinewhether a n t i - I d a n t i b o d i e s t o HLA a l s o p l a y a r o l e i n t h e down r e g u l a t i o n of t h e alloimmune response t o t r a n s f u s i o n s and t r a n s p l a n t a t i o n . For t h i s , we monitored p a t i e n t sf o rt h e development of anti-anti-HLA antibodies following donor s p e c i f i c blood t r a n s f u s i o n ( 3 ) . W e found t h a t most p a t i e n t s developed Ab2 t o t h e mismatched HLA c l a s s I1 a n t i g e n s of the blood donor two weeks f o l l o w i n g t r a n s f u s i o n and were a s s o c i a t e d w i t h s u c c e s s f u l t r a n s p l a n t a t i o n . Furthermore, we found a p o s i t i v ec o r r e l a t i o n between the presence of anti-anti-HLA a n t i b o d i e s and t o l e r a n c e t o t h e g r a f t i n p a t i e n t s w i t h a h i s t o r y of p r e s e n s i t i z a t i o n t o HLA antigensofthe donor ( 4 ) . The r o l e of a n t i - I da n t i b o d i e st o HLA was a l s oe v a l u a t e d i n r e n a l and h e a r tt r a n s p l a n t a t i o n (5,6,7). In thesestudies,patients were monitoredfollowingtransplantation e found t h a t 5 y e a ra c t u a r i a l f o rt h ep r o d u c t i o n of A b 2 . W s u r v i v a l of h e a r t and kidney a l l o g r a f t s was s i g n i f i c a n t l y h i g h e r i n p a t i e n t s d e v e l o p i n g Ab2, compared t o p a t i e n t s without Ab2. Taken t o g e t h e r , o u r d a t a s u g g e s t t h a t a n t i - I d a n t i b o d i e s t o HLA p l a y an important role i n s u p p r e s s i n g t h e immune response t o HLA and t h a t such a n t i - I d may be of t h e r a p e u t i c i n t e r e s t i n transplantation.
273
REED
274
REFERENCES 1.
N. Suciu-Foca, E . Reed, C . Rohowsky-Kochan, P . Kung, D . W . K i n g . P r o c N a t l Acad S c i USA, 830-834 ( 1 9 8 3 ) .
2.
E . Reed, V . Bonagura, P . Kung,
m,
J . I m m u n o l , ~ ,2890-2894 (1983)
D.W.
.
King and N.Suciu-Foca.
M . Hardy, C . L a t t e s , J. B r e n s i l v e r , R . McCabe, K . Reemtsma a n dN . S u c i u - F o c a . T r a n s p l a n tP r o c , U , 735-738 (1985) .
3.
E . Reed,
4.
E . Reed, M . Hardy, A. B e n v e n i s t y , C . L a t t e s , J . B r e n s i l v e r , R . McCabe, K . Reemstma, D.W. Kingand N . Suciu-Foca. New
E n g l a n dJ o u r n a lM e d i c i n e ,3 1 6 , 1 4 5 0 - 1 4 5 5( 1 9 8 7 ) . Suciu-Foca, E . Reed, V . D . D ' A g a t i , D . J . B e n v e n i s t y , R . McCabe, J . B r e n s i l v e r , D . W . H a r d yT . ransplantation, 51, 5 9 3 - 6 0 1( 1 9 9 1 ) .
Cohen, A . I . K i n ga n d M.A.
5.
N.
6.
N.
7.
E. Reed, E . Ho, D . J . Cohen, W . Ramey, C . Marboe, V. D ' A g a t i , E . Rose, M.Hardyand N . S u c i u - F o c a .I m m u n o l o g i cR e s e a r c h , 12, 1-11 ( 1 9 9 3 ) .
Suciu-Foca, E . Reed, C . Marboe, Y . P . X i , Y . K . Sun, E . Rose, K . Reemtsma a n d D . W . K i n gT. r a n s p l a n t a t i o n , U, 716-724 ( 1 9 9 1 ) .
PART IV: IMMUNOLOGICAL EFFECTS OF BLOOD TRANSFUSION
This Page Intentionally Left Blank
IMMUNOLOGIC EFFECTS OF BLOOD TRANSPUSION Paul 1. Tartter, M. D. From the Department of Surgery of the Mount Sinai Medical Center New York, NY 10029
ABSTRACT Blood transfusion is associated with numerous clinical phenomena attributableto immune suppression. Homologous blood transfusion isassociated with declines in lymphocyte numbers andinhibition of lymphocyte function. Indialysis patients this immune suppression is accompanied by prolongation ofsurvival of subsequently transplanted allografts. For patients undergoing surgicalprocedures, the receipt of homologous blood increases the risk of postoperative infectious complications. Patients with malignancieshave significantly increased recurrence and mortality rates when removal of their tumor is accompanied by the administration of blood. "he clinical course of Crohn's diseasemay be beneficially influenced by transfusion at the time of resection of diseased bowel. Women suffering recurrent abortion may carry to term following transfusion of spouse leukocytes. Experimental studies, in addition to replicating the clinical studies, have documented that transfusion inhibits wound healing. Blood transfusion, the oldest form of transplantation, causes profound and prolonged alterations in immune function which result in clinical phenomena which can be either beneficial or detrimental to the recipient. INTRODUCTION Blood transfusion is one of the most common therapeutic modalities used in medicine and surgery. Recently, transfusion of blood has become associated with clinical
phenomena which
can be attributedto immune suppression. The observation that dialysis patients who receive blood transfusions prior to renal transplantation enjoy longer allograft survival resultedin prospective studies proving that transhsion of homologous bloodhas profound effects on the recipient's immune functions. Retrospective studies in manyareas support the hypothesis that immune suppression induced by blood transfusion causes clinical
phenomena attributable to the
receipt of blood. Blood transfusion is apparently one of the most significant risk factors for postoperative infections. In addition, patients with malignancies who undergo potentially curative surgery accompanied by blood transfusion have higher recurrence rates and mortality than patients who are not transfused. Finally, transfusion is related to a number of other clinical and experimental phenomena which may result in beneficial or detrimental outcomes. We will review here the immunologic effectsof transfusing blood in man and criticallyanalyze the literature linking these changes in immune function to clinical observations.
277
TARTTER
278
THE EFFECT OF BLOOD TRANSFUSION ON IMMUNE FUNCTION Sincehomologousblood
is never given to normalvolunteers,the
effect ofblood
transfusion on immune function in normal man is unknown. In patients who receive homologous blood, changes in immune response are evaluated in the context blood is given and extrapolated to the effect ofblood
of the disease forwhich
in the absenceof
disease.
the
Changes in
immunity consistently following transfusion for a variety of diseases can be assumed to be due to the transfusion and not to the
diseases. Changes in immune function following transfusion with
autologous blood or washedlfilteredhomologousbloodcan receiving routinelyprepared
homologousblood.
be compared to patients who are
The blood is given withinthecontextof
surgical procedure as a consequence of operative blood
a
loss which is due to trauma and trauma
itself is associated with changes in immune function.
In Vitro Lvmuhocpte Resuonsiveness Generally, inhibition of lymphocyte response to a given antigen or mitogen measured
by
incorporation of tritiated thymidine is accompanied by inhibition of response to all antigens and mitogens. Surgery, anesthesia,
blood loss and blood transfusion cause lymphocyte suppression in
clinical studies. Isolating the effect of homologous blood transfusion from the surgery, anesthesia and blood loss is not easy. In vitro lymphocyte responses decline in proportion to the magnitude of theprocedureandinproportion
notablyetherand
to theamount
ofblood
lost.Certainanestheticagents,
cyclopropane, are associated withmoreprofound
suppression ofimmune
function than halothane and nitrous oxide, for example (1). Patients with malignancies have low lymphocyte responses and declines with surgery are malignancies.
Operatedpatients
responsivenesscompared
more precipitous than for patients without
whoreceivehomologusblood
to untransfused patientsundergoingthe
have declines in lymphocyte same procedure.Thorough
well-controlled studies have also observed the opposite, causing Munster et al. to comment that continuedinvestigation
" intothe
effectof
PHA and ConA on post-traumaticlymphocyte
transformation in many laboratories has produced no conclusive and repeatable pattern.' ( 2 ) Prolongeddepressionininvitro
lymphocyte responsiveness is notedwithinhoursof
surgery and recovers over thenext several days. Theinhibition is duetobothintrinsicand extrinsicfactorssincelymphocyte
responsiveness can be partially restored by testing in plasma
from normal blood donors. Homologousblood transfusion adds to the depressed state of the lymphocytes, but may causestimulation in unoperated patients. The in v i v ~counterpart of h testing of lymphocytes is delayed cutaneous hypersensitivity to antigens. Delayed Cutaneous Hpersensitivity There existsa
correlationbetweenin
vivo andinvitrolymphocytetestingand
preoperative evaluation of h & lymphocyte function is predictive of postoperative infection and subsequentcourseafter
surgery. Anergy is associated withlow
serumalbuminandreduced
polymophonuclear neutrophil chemotaxis.Patientswithgastrointestinalbleeding, homologous blood, are often anergic
recipientsof
(3). Sepsis following surgery for gastrointestinal bleeding is
more common, hospital stay longer, and mortality higher in anergic patients. Patients who are initially anergic and remain anergic usually die.
IMMUNOLOGIC EFFECTS OF BLOOD TRANSFUSION
279
The effect of homologous blood transfusion on delayed hypersensitivity skin test response has been studied using tetanus and diphtheria toxoids, streptococcus, tuberculin, Proteus, candida and trichophyton antigens (4). Postoperative skin test response area decreased 57% in transfused patients compared to a 38% decrease in untransfused patients. Since transfused and untransfused patients differed significantly in duration of surgery, preoperative blood hemoglobin albumin, the authors reanalyzed their data with 64 pairs of patients matched
and serum
for these variables
with the same results. The predictive value of delayed hypersensitivity skin testing for sepsis and mortality has notbeenaccepted
by allinvestigators.
(5) agreethatanergicpatients
Brown etal.
have
significantly higher rates of sepsis and mortality than normal responders, however 'careful study of thetemporalrelationshipbetween
skin reactionsand
clinical events in individual patients
suggested that these differences were not of value in clinical practice. Abnormal reactions usually followed obviouscomplications
such as sepsis or secondary hemorrhageratherthanpredicted
them.' Anergy to skin testing may be related to a circulating serum factor which appears after trauma and causes lymphocytesuppression.
There is no proven association ofblood transfusion
withserum suppressiveactivity or with anergy.Infectiouscomplications
and hospitalstay
are
both significantly related to immunosuppressive serum and anergy. Lvmphocpte Subsets Lymphocytes, B cells, T cells, helper cells and suppresser cells drop significantly five days aftersurgeryandthedecline
is twice as greatinthe
transfused patientscompared
tothe
untransfused (6). Helper cell number declines in transfused patients cause the helperlsuppresser ratio to decrease significantly despite a significant decline in suppresser cell number. Changes in cellnumbersrecoversomewhat
by ten days so the differences betweentransfusedand
untransfused patients are no longer
statistically significant although cell numbers in transfused
patients are stilt lower than those in untransfused patients.
Lymphocyte responses to C o d and
PHA decline significantly in transfused groups, remaining below preoperative levels even one year Response to ConA and PHA and MLR's inuntransfusedpatientsare
followingsurgery.
significantly higherthanin Significantdeclines
transfused patientsat
inimmunoglobulinG,
90 days and 45
A and Mcells
arenoted
-
60 days respectively.
postoperatively inboth
transfused and untransfused patients. Other authors have not observed consistent changes in lymphocyte subsets in relation to transfusion. Changes with and without
in the numbers of lymphocytes in the various subsets in relation to surgery
bloodtransfusions studied in patients testedbefore
and aftersurgery and in
patients tested one week following transfusion alone, surgery alone or both reveal no evidence of suppression of immunity by surgery or blood transfusion (7). Generally surgeryis
followed by significantdecreases
numbers affectingalllymphocytesubsets
inperipheral
to somedegree.Declines
associated with a significant decrease in the helperlsuppresser ratio. patientsexhibitgreater
declines in lymphocytes duetothe
blood lymphocyte
in helper cell numbers are It is not clear if transfused
transfusion, due to the operative
trauma, or due to pre-existing anemia which caused physicians to transfuse blood.
TARTTER Natural Killer Cvtotoxicitp In aprospectivestudyofcolorectalcancer
patients, the number of natural
killercells
increased significantly in both transfused and untransfused patients who had potentially curative surgery (8).
Natural killer cytotoxicitydeclinedsignificantly
in untransfused patientswhile
increasing slightly in the transfused. Three months following surgery no differences in peripheral cell numbers or T cell subsets between the transfused and untransfused patients were noted. Removingleukocytes
from the blood to be transfused abrogates the changes innatural
killer
cytotoxicity (9) These studies conflictwith
to receive whole blood or filtered whole blood, removing 99.98% of
Patients were randomized
the leukocytes and platelets. Natural unfilteredwholeblood
(9).
the findings ofaprospectivestudyofcolorectalcancer
killer cytotoxicity declined significantly in patients receiving
and remained significantly depressed 30 days following surgery.Natural
killer cytotoxicity inuntransfusedpatientsandinpatients
receiving filteredblooddeclined
fully recovered by 30 days.
significantly withsurgerybut
Sincedeclines
innatural
killer
cytotoxicity can be prevented in cancer patients by simply filtering blood and since natural killer cytotoxicity is ofprovenprognostic
significance, filteredblood
may improve the outcome for
patients with malignancies. ImmuneFunctionFollowinnTransfusion
of DialysisPatients
The effect of transfusion on dialysis patientsisbothimmuneenhancingandimmune suppressing. Transfusion
is followed by the appearance of antibodies to antigens present on the
cells of the transfused blood and these antibodies are
capable of killing lymphocytes having these
antigens. Lymphocytotoxic antibodies are responsible for early graft failures and their appearance is called sensitization. Immune suppression accompanies sensitization.Suppressorlymphocytes begintoappearintheserum
oftransfusion
recipientsatthesametimelymphocytotoxic
antibodiesareappearingandtheirappearance
is probably also mediated by antibodies
-
antibodies which play a role in regulating immune function. Suppresser cells suppress lymphocyte responses to antigensonthe
cellsoftransfusedblood
andonthe
cells ofthetransplant.
Lymphocyte suppression following blood transfusion may be permanent or transient, but in most recipients the degree of suppression is enhanced by additional blood transfusions
and probably
maintained by the presence of the allograft following transplant. In dialysis patientswho
receive bloodtransfusionslymphocyteresponses
mitogens and homologous lymphocytes decline to
to antigens,
YO% one week following a single unit
incomparisontolymphocytereactivitymeasuredimmediatelybeforetransfusion
of blood
(10).
Lymphocyte reactivity declines progressively with additional units of blood given and returns to pretranshsions levels if blood is withheld for six weeks. Suppresseractivity
is enhanced following transfusion butnotatoneweekwhen
lymphocytereactivity is at its lowest point, indicating that suppressive activity and lymphocyte inhibition are separate events (11). Finally, natural killer cytotoxicity is significantly reduced following transfusion of dialysis patientsandremains
lowfollowing
transplant,although
cytotoxicity and graft survival has not been shown (12).
a correlationbetweennatural
killer
28 1
IMMUNOLOGIC EFFECTS OF BLOOD TRANSFUSION
These studies indicate that surgery depresses immune function because both anesthetic agentsand physical trauma causecirculating surgery with general anesthesia causing
levels of all lymphocyte subsets to declineafter a panlymphocytopenia. Lymphocyte function,
independent of cell number, is inhibited whether measured in vitro by lymphocyte responses to mitogens, antigens or homologous lymphocytes or measured in vivoby loss of response to skin testing. Lymphocyte functionalinhibition
may be related to disproportionatedeclines in T cell
subsets or relatedtotheappearanceofimmunosuppressiveserumfactorswhichinhibit lymphocytes.Transfusionpotentiateswhatever inhibition;surgery
mechanism isresponsible
for lymphocyte
accompanied by transfusion is followed by moreprofound
decreases in
lymphocyte numbers and in lymphocyte functional activity than surgery without transfusion. It is difficult to extrapolate these observations to retrospective clinical studies linkingtransfusion to increases in risk of infection or recurrence of malignancy. The study by Jensen et a1.(9) suggests that use of leukocyte-free blood will prevent transfusion-associated adverse clinical phenomena, but this study needs to be replicated. The data certainly favors avoiding the use of homologous blood.
BLOOD TRANSFUSION AND INFECTION Thehypothesisthat
transfusioncauses
immune suppression leading to infections is
confounded by the observation that the magnitude
of the injurydirectly
correlates with the
degree of immune suppression and the necessity for transfusion. Potential confounders must be considered in any study of infections
following surgery:
not significant or non-existent inanother.
confounders in one clinical situation are
Each field ofsurgery
has its own risk factors for
infection which are often associated with transfusion as well as with infection. The contribution of transfusion to the risk of infection independent of variables reflecting tissuedestructionandbacterialcontamination logistic regression (13).
can be calculatedstatistically
usingstepwise
This type of analysis is commonly used in medical studies, ignoring the
basic preceptthattheindependent
variables mustbetrulyindependent.Theindependent
variables are not genuinely independent: ;he magnitude of the procedure, the duration of surgery, the blood loss and the tissue damage are all related to one another and
at1 are related to the
number of units of blood given as well as to the risk of infection. The analysis is useful as long as one is aware that all conclusions drawn are subject to limitations. This analysis hasbeen
applied to 23 populations of patientsundergoingprocedures
ranging from bone marrowharvesting to coronary artery bypass graft. In 22 studiestransfusion was astatisticallysignificantriskfactor
for infection and in 17 of the 23 it was themost
significant determinant ofinfectiouscomplications
in stepwise logistic regression. In 14 studies
the p value for the relationship between transfusion and infection was 0.001 or less. Non-operative site infections are increased following bloodtransfusion,
indicatingthat
transfusion's association with infection is independent of the operative trauma (14-16). Several studies have demonstrated a dose-response relationship between transfusion and infection risk but the greatest increment in risk is noted between no transfusion and one unit of blood (14,16-19). Transfusion is a potent predictorofinfectionafter reflecting tissue destruction and contamination.
controlling for variables
TARTTER
282
Since blood transfusion is linked to the magnitude of the surgical procedure, comparing transfused patients to untransfused patients will always be confounded by infection risks due to factorsrelated
To control for these factors onemustcomparepatients
totheprocedure.
transfused with red cells &om different sources or prepared in a manner which minimize infection risk.
Patients transfused with homologousblood
recipients of equal
have infection rates several fold higherthan
values of autologous blood undergoing
Homologousbloodrecipients
the same operative procedure (20-23).
have significantly longerhospital a blood transfusion exceeds
infections.Thecostof
administration because of transfusion'sassociationwith
stays attributedtotreating
thecost
of collection,storageand
lengthof
stay.
In thiseraof
cost-
containment the association with prolonged stay may ultimately curtail the use of blood. Homologous blood can be filtered to remove donor leukocytes which may be contributing
A prospective randomizedtrialcomparingthe
toimmunesuppressionandinfection
risk.
infection rates among colorectal cancer
patients receiving filtered and unfiltered blood has been
conducted (9). There were 17 infectious complications among the 56 andoneinfectious
recipients of whole blood
4 8 recipients of filtered blood.
complication amongthe
Infections were
prevented by the seemingly simplistic addition of a $25/filter to every bag of blood transfused. These clinical studies are very convincing:homologousbloodtransfusion with increased risk ofinfection
in every clinical situation examined.
transfusion was a significant predictor of infection after consideration of and in the
is associated
In multivariate analyses other variables measured
majorityof those studies transfusion was the single most significant factor. Patients
receivinghomologousbloodexhibitedanincidence approximatelyfour
of infectiouscomplicationsthat
timeshigherthanpatients
receivingautologousblood.
transfusion with infection is found among patients undergoing surgery gastrointestinaldisordersand
was
The associationof
for cardiac, orthopedic and
for trauma as well as amongunoperatedpatients
transfusedfor
bums and gastrointestinal bleeding. The observation that nosocomial infections are increased in these studies argues strongly that the associationoftransfusion
with infection is not simply areflection
markerof tissue destructionandcontamination.
of transfusion as a
Infections that develop in transfused patients
away fiom the site of trauma or in the absence of trauma, cannot be attributed to the quantity of tissuedestroyed
or to thedegreeofbacterialcontamination.Filteredblood
leukocytes and prevent
can remove
postoperative infections. Since filtering blood can significantly reduce the
incidence of infection among transfused patients, all transfusedblood
will be passing through
filters in the very near future.
EXPERIMENTAL STUDIES RELATING BLOODTRANSFUSION TO INCREASED RISK OF INPECTION Patients are extremely heterogeneous and even in prospectiverandomizedtrials,factors whichinfluencepatients'participation randomization.
affect theoutcomedespitedouble-blindingand
In animalstudies using syngeneic strainswithidenticalhousing,lighting,
access
to food and water, control
over the extent ofinjury, use of antibiotics and exposure to other
variables theinfluenceof
a singlevariable
Waymack's laboratoryhas
intensively studiedparameters
such as bloodtransfusioncan
be measured. Dr.
which interactwith
transfusion in
OF BLOOD TRANSFUSION
IMMUNOLOGIC EFFECTS
283
affecting survival following septic challenge in animal models. Using contaminated burn modeltheyfound
a pseudomonas
that the effect of transfusion w a s not dose-related (24).
They also demonstratedwiththismodelthat
transfusionwithin
24 hoursofpseudomonas
challenge did not affect survival, suggesting that a time dependent interaction of the recipient and the transfused blood takes place resulting in increased susceptibility to bacterial challenge (24). Neither anesthesia(methoxyflurane) intravenousinjectionsin intravenousdose of
comparison to untransfusedunanesthesizedanimalsgiven
E. m (26).
increasedmortalitycompared cavity.
Thetiming
nor transfusion affected survival of animalsgiven the same
Both allogeneic transfusion and anesthesia caused significantly
to controlswhen
oftransfusionrelative
challenge interact in determining the
lo7 E.
were injectedintotheperitoneal
to septic challenge andthe
severity oftheseptic
significance of allogeneic blood for increasing susceptibility
to infectious agents (27).
E and F l a production by
Immunosuppressivethromboxaneandprostaglandins
macrophages is increased following allogeneic transfusion (28) and macrophage migration into the peritoneal cavity isreducedin
animalspreviouslytransfused
Macrophages from animalstransfused
withallogeneicblood(29).
with allogeneicblood
phagocytose and kill bacteria inculture.
also exhibitimpaired
ability to
Leukotrienes areimmunostimulatorymetabolites
of
arachidonic acid and their production is inhibited following allogeneic transfusion. Macrophages and macrophage supernatants from transfused rats suppresslymphocyteresponses
to PHA (30).
Significant elevations of serum corticosterone accompany declines in leukocyte counts in animals transfused with allogeneic blood in comparison to syngeneic recipients (31).
The experimentalstudies causes inhibitionof
reproducibly demonstratethat
cellular antibacterial mechanismswhich
bacterialpathogens.Themodelssupportthe
allogeneicbloodtransfusion
cause increasedsusceptibility
to
hypothesis that transfusion-induced immune
suppression leads to enhanced susceptibility to bacterial pathogens in the recipient.
CANCER RECURRENCE In1981
a letterinTheLancet
transfusionwhichare malignancies (32).
beneficialfor Therearenow
relationshipbetweenhomologousblood
suggested thatthe
immunosuppressive propertiesof
dialysis patients may bedetrimental
for patientswith
over onehundredpublishedstudiesinvestigatingthe transfusion and cancer recurrence. Meta-analysis of 20
colorectal studies representing Y,236 patients calculated cumulative odds ratios of 1.8 for disease recurrence, and 1.76 for death from cancer in transfused patients (33). Academicians will never be convinced by retrospective studies that transfusion is anything other than a marker of stage of disease andextentof
surgery. Since preoperative anemia oftenleads
to bloodtransfusion
and
anemia is often a sign of advanced disease in cancer patients, transfusion would be expected to be associated with early disease recurrence because it is associated withanemia. Advanced malignancies necessitate extensive surgery,requiremoretimeand
cause greater blood loss.
Procedure, duration of surgery and blood loss are associated with transfusion and may account for transfusion's association withrecurrence.Prognostic
factors cannot be adequatelycontrolled
in
retrospective studies. The significance of perioperative blood transfusion for patients with malignancies cannot be definitely proven without randomizing patients to receive blood or go untransfused. Given the
284
TARTTER
risks of homologous bloodtransfusion, randomizationofpatients
such astudy is unethical. Less controversialwould be
likely tobe transfused into an autologousblood
utilizing multiple institutions in the
program. A study
Netherlands with over 500 colorectalcancer patients (23)
found the relative risk of cancer recurrence for patients transfused with 1 - 2 units of autologous blood was 1.78 compared to untransfused patientsand homologous blood.
2.11 for recipients of 1
-
2 unitsof
Both autologous and homologous transfusions were buffjr coat poor, standard
for the Netherlands. Blood transfusion, whether autologous or homologous, was associated with significantly increased risk of cancer recurrence but the risk for both groups was comparable. of colorectal cancer patients by Weiss et al., (34) from
A randomized prospective study
Munich randomized 120 patients to receive either homologous or autologous blood if transfusion follow-up of 21 months (9
wereneeded.Withmedian
-
48). therecurrencerateamong
homologous recipients is 29% compared to 17% among autologous recipients and
was significant
in both B (p = 0.032) and C (p = 0.006) tumors. Multivariate regression identifiedhomologous blood as an independent prognostic factor (p = 0.008).
EXPERIMENTAL STUDIES OF TRANSFUSION AND TUMOR GROWTH Experimental studies control for tumor burden (disease stage) and extent of the procedure including blood loss. Allogeneic blood transfusion producesprofoundchanges systems ofexperimental animals which areanalogous studies haveobserved
in theimmune
to those observed inman. Experimental
promotion or inhibitionoftumorgrowth
transfusionsbecause the effect of transfusion on tumor growth
following allogeneic blood is route-,tumor-,
species-, and
strain-specific. In mice, tail vein inoculation of basal call carcinoma produces pulmonary nodules which are inhibited by prior allogeneic transfusion while no effect is seen if the tumor is given subcutaneously (35). In the same strain, growth of subcutaneous adenocarcinoma transfusionwhilepulmonarynodules
is inhibited by
to tumor
are unaffected. Timing oftransfusionrelative
inoculation also determines subsequent tumor growth. Studies of tumorgrowthin cancer patient. The tumor
experimental animals lack analogy to thesituationinthe
hasbeenpresent
for years inpatients with malignancies and some
immunologic interaction between the host and the tumor has preceded the effects of surgery and bloodtransfusion. In experimentalstudies,tumorinoculation generally followed allogeneic transfusion.
MISCELLANEOUSPHENOMENAASSOCIA”J3DWITH Recurrent Abortion
BLOODTRANSFUSION
One of the most exciting, intriguing and controversial areas in which transfusion affects theoutcomeand
hasa
therapeuticrole
is in thetreatmentofrecurrentabortion.During
pregnancy, lymphocyte function, as measured by responses to antigens, mitogens and homologous lymphocytes (MLR), is suppressed. Inhibitionof lymphocyte function is due to serum factors, blocking antibodies which develop in response to trophoblast antigens. When spouses share HLA antigens,trophoblastantigensarenot blocking antibodies are not
recognized by thepregnant
woman’s immune system,
produced, and the fetus is rejected. In 1981 Taylor and Faulk (36)
induced suppressivesera in womensuffering from recurrent spontaneous abortion and sharing
HLA antigens with their spouse by transfusing the women with leukocyte-enriched
plasma from
285
OF BLOOD TRANSFUSION
IMMUNOLOGIC EFFECTS multiple donors.Three
women hadnormal pregnancies and deliveries atterm.
Several groups
have replicated this work with spouse leukocytes and successful deliveries result in more than 10% of the women treated. Crohn's Disease
Crohn's disease is an inflammatory condition of the gastrointestinal tract which presents with diarrhea and crampy abdominal pain. nearlyhalf
of the patients
Recurrence of disease following surgery is common
will develop symptoms of recurrence within ten
-
years of surgical
resection of all diseased bowel. Immune function is abnormal and patients are often treated with immunosuppressive steroids. Transfused patients have significantly decreased total lymphocyte and t-cell counts following surgery despite being
clinically well. Increasing numbers of units of
blood received are associated with progressively lower numbers of lymphocytes at follow-up. Several groups have studied the effect of blood transfusion on the outcome Crohn's disease because the immunosuppressive effects of transfusion might benefit patients in the steroids affect the course to the disease.
same way
Most of the studies observed that untransfused patients
exhibited higher rates of recurrence than transfused patients (37-40). The studies suggest that transfusion may influencethe
courseof
have an immune or
diseases which arethoughtto
autoimmune basis and clinically respond to steroids.Crohn's
disease patients with more
severe
disease, those with tower hemoglobins and serum albumins, undergoing resection of more bowel, should have higher recurrence rates.Yet,these
patients when transfused have recurrence rates less bowel
comparable to untransfused patientswithhigherhemoglobinsandalbuminsand resected.
Wound Healing It has recently been recognized that lymphocytes contribute to wound healing which
is
primarily mediated by macrophages. Lymphocytes secrete lymphokines which enhance fibroblast replication,migrationand
collagen synthesis.
In vivo depletion oflymphocytesimpairs
skin
wound healing. Since transhsions inhibit lymphocyte function, transfusion-induced inhibition of lymphocyte function should lead to impaired wound healing (41). Rats undergoing ileocolic resection with primary anastomosis and transfusion with saline, syngeneic or allogeneic bloodwere sacrificed three and seven days following surgery and the burstingpressureofthe
anastomosis measured.Burstingpressure
was significantlylower
following transfusion with syngeneic or allogeneic blood in comparison to saline. Hydroxyproline contentoftheanastomoses
was reducedandanastomotic
abscesses werecommoninthe
transfused animals. This study clearly implicates blood transfinion in impaired wound healing.
Diabetes In man, insulin dependentdiabetes mellitus is associated with decreases inboththe number and functional activity ofsuppresser T lymphocytes. In the Bio-Breeding rat, diabetes develops whenthe
animalsdeveloppancreatic
pathogenesis.Diabetes
insulitis,suggesting
acell-mediated
is prevented in these animals by treating them with
immunosuppressive
agents such as anti-lymphocyte serum, steroids, cyclosporin, irradiation, neonatal blood transfusion (42).
immune
thymectomy, or
286
TAR'M'ER These studies indicate that homologous blood transfusion affects the outcome of clinical
diseases inboth
beneficial and adverse ways.
randomized clinical trials
- transfusions
Experimentalsituationsarenotsuitable
cannotbe given to prevent theonset
or autologous blood.
wound strength measured in man following receiptofhomologous experimental observations indicate
for
of diabetes or These
that the outcomes of numerous clinical diseases which have
not been studied may be manipulated by the use of homologous blood or that transfusion should be avoided. Several studiesindicate that changes in immune function following transfusion arc permanent.Thenumber
ofclinical
phenomena associatedwith
immunesuppressionand
attributable to blood transfusion is unknown.
SUMMARY Given the evidence presentedhereit homologous blood has
would be foolish to suggest that transfusion of
no immunologic consequences for the recipient. Blood transfusion is the
oldest form of transplant
- no one
would argue that transplantation between unrelated
individuals
has no influience on the immune system. In organ transplantation the immunologic sequelae are permanent and there
is evidence that the same is true following homologous blood transfusion.
Lymphocytopenia ispresentone
year following surgery for Crohn's disease if patients receive
perioperative blood transfusion (43). Colorectal cancer patients transfused more than seven years
prior to diagnosis have significantly reduced numbers of
lymphocytes and tower natural killer
cytotoxicity thancolorectalcancerpatients
who have never been transfused (44). Transfusion of
neonates causes suppression of lymphocyte
reactivity which is still demonstrable 2.5 to 30 years
later (45). There is evidence that transfusion at any timeprior
to electivesurgeryincreases
susceptibility to infectious complications (14) and otherwise healthy transfused individuals may be atincreased
risk of developingmalignancies
( 4 6 ) . All thelongtermconsequencesof
blood
transfusion are not negative: Survival of transplants is prolonged by pretransplant transfusion and some womensuffering
fiom recurrent spontaneous abortion
can deliver at term if
previously
transfused with their spouse's leukocytes. In the future we will be able to transfuse blood without causing immune perterbations and the consequent
clinical phenomena. Studiespresented
here suggest that removal of donor
leukocytes reduces the risk of infection and cancer recurrence.
The technology has not reached
the point of reducing the leukocyte number in transfused blood below 10li/unit. An alternative which is increasingly being utilized is autologous blood programs. Physicians are discovering that patients tolerate hemoglobin levels which were previously unacceptablylow and many patients preferbeinganemic
over the risks ofreceivinghomologous
blood.
Sincetransfusion
is an
identifier of high cost hospitalized patients, alternatives to routine blood use are being studied in hopes of safely reducing the costs of transfinion.
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ScandJ
Cfin Invest
TRANSFUSION REACTIONS: THE CHANGING PRIORITIES H. Perkins Irwin Memorial Blood Centers San Francisco, CA 94118 ABSTRACT Over the last dozen years the relative frequencies of specific transfusion reactions have markedly altered, in general for the better. Although AIDS remains thePublic's primary concern, the risk of AIDS from a transfusionis extremely low at this point. Hepatitis remains the most conunon infectious complication of blood transfusion, but only 1 in 6,000 units now carry a risk, whereas in the early 1980's the riskis believed to have been closeto 10% per patient. Transmission of HTLV-1/11 has also been markedly reduced by tests of donor sera. In contrast, cytomegalovirus has become of increased importance in view of the large number of patients immunosuppressed for transplantation and cancer therapy; bacterial growth in blood components appears to be increasingly common; and Chagas disease is likely to become a serious transfusionproblem in this country. More widespread use offilters which remove three logs or more of white blood cells from components should play a major rolein reducing transfusion reactions further. INTRODUCTION When I was assigned the topic of "Transfusion Reactions" I wondered what I could say about this topic that would be new or interesting, especially since many of the things shall I mention will be discussed in more detail by other speakers at this convocation. I decided to take advantage of this opportunity to emphasize the dramatic changes which have occurred in the frequencies and relative importance of various types of transfusion reactions, changes which deserve much more publicity than they have had. This will, in turn, lead to consideration of where we need to place our emphasis as we strive get to as closeto zero risk aspossible. And let me remind you that, the closer we get to perfection, the more the Public expects of us. Since I will limit my discussion to areas where there have been significant changes in the relative frequency of reactions, tables will provide a more complete list. AIDS AIDS remains the Public's number one concern related to blood transfusion despite the reduction in risk toextremely low levels (1). This concern has been abetted by the media which have attacked blood banks
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290 TABLE I VIRUSES TRANSMITTED BY TRANSFUSION HIV-112 Hepatitis A/B/C HTLV-1/11 Cytomegalovirus Epstein-Barr virus Parvovirus
repeatedly during the past year, repeating allegations about the slow response of blood banks and the government to the threat of transfusionassociated AIDS in the 1983-5 period. The reports are based on statements of expert witnesses hired by plaintiffs suing blood banks, claims whichhave been successfully rebutted in the courts. The reportsallege that blood banks are continuing to drag theirfeet. MONEY MAGAZINE in its May 1994 issue took the FDA and blood banks to task because they have not introduced the HIV p24 antigen test. Although more than onemillion tests onblood donors in this country and in Germany failed to identify a single donor whose serum was p24antigen positive but negative for anti-HIV-l, there have been several subsequently reported instances where an anti-HIV-negative donor transmitted HIV with blood later shown to be p24 antigen-positive. Blood banks now use an improved antibody test which shortens the antibody-negative window and might possibly have detected those donations, but the Public will not tolerate any risk for AIDS, and the question of testing for p24 antigen may need to be reconsidered. The decision to test will have to take into account not only the very small addition to blood safety but also the loss of donors from the inevitable false positives. Loss of safe donorsat a timeof increasing blood shortages may result in more patient deaths, not less. The psychological damage to a donor rejected because of a positive AIDS tests, even if falsely positive, is considerable. Blood banks lost the public's confidence when they grossly underestimated the riskof AIDS from transfusion in 1983. Both the American Association of Blood Banks (AABB) and the U . S . Public Health Service (which includes the Centers for Diseases Control [CDC], the Food and Drug Administration [FDA] and the National Institutes of Health [NIH]) reported an estimate that the risk wasless than one in a million, which was avery reasonable estimate based on the data available at the time(2). As moreand more cases of transfusion-associated AIDS were reported, however, it became obvious thatthe risk prior to 1983 had been somewhat higher than that, but there wasstill no reason to suspect that the self-exclusion policies had not reduced the risk below the one in a million level. A truer estimateof the risk became possible onlyafter a test for the human immunodeficiency virus (HIV) became available long enough for accumulation of meaningful data. In
TRANSFUSION REACTIONS: THE CHANGING PRIORITIES
29 1
1987 Peterman at the CDC estimated that 29,000 blood recipients had been infected prior to donor screening with anti-HIV (3), an estimate which is likely to be low. Busch in San Francisco was ableto show thatthe riskwith Irwin blood had peaked at 1.3% per unit at the end of 1982 and beginning of 1983 (4). The risk dropped with self-exclusion of gay donors; in San Francisco 86% of the riskhad been eliminated before the anti-HIV test became available. With anti-HIV testing routine, the remaining risk hasbeen from donors so recently infected that they do not yet have detectable anti-HIV. Calculations of that risk have progressively fallen. Several years ago the risk wasreported to be onein 225,000 (1); and it should be lower than that now based on the increasing sensitivity of tests in current use. The frequency of donors found anti-HIV-positive and permanently excluded has dropped to an average of five per 100,000 in the country, a figurewhich we have matched at Irwin despite the 4%frequency of infection in SanFrancisco. In short, the battle against transfusion-associated AIDS has been almost completely successful, in contrast with other epidemiologic causes of this dreadful disease. HEPATITIS Hepatitis was recognized to be a common complication of blood transfusion in the 19405, with some reports showing an overt frequency as high as 35% of recipients. The early 1970's brought two major changes. (1) The cause of Hepatitis B had been discovered, and the test for Hepatitis B Surface Antigen became available to eliminate almost all chronic carriers of the virus. (2) It is generally accepted that a more important change was elimination of paid blood donors from those blood banks which still relied on them. As a result of these two procedures, the incidence of clinically obvious post-transfusion hepatitis dropped to approximately 1% (10% when subclinical liver damage wasincluded), as evidenced in two majorprospective studies (5,6). Those same studies made clear not only that 90% of posttransfusion hepatitis (PTH) cases were subclinical, but also that 90% could not be explained by Hepatitis B. Identification of the Hepatitisvirus A and a test for it made clear that Hepatitis A was only very rarely responsible for PTH (7), since it lacked a chronic carrier state. The remaining cases were attributed to thenon-A, non-B virus(s). Discussions of the potential usefulness of surrogate tests such as alanine aminotransferase (ALT) and antibody to Hepatitis B Core antigen (anti-HBc) did not lead to general implementation of those tests. There was considerable debate about the value of atest with 70% false-negative and 70% false-positive results, and the clinical significance of an infection manifested only by elevated ALT levels was widely doubted (8). In fact, a very recent report on subjects enrolled in one of those early studies showed that recipients who developed hepatitis following transfusion did not have significantly higher mortality than those who did not, although they did have some increase in mortality due to liver disease (9).
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A few centers did introduce the ALT test in the early 1980s, but the NIH Clinical Center reported that this had no effect on thefrequency of PTH in that institution, where heart surgery cases were followed with repeated ALT tests for six months following transfusions (10). Calls for a national prospective study to determine whether the suggested surrogate tests would be useful were unsuccessful. The very expensive investigations were considered to be low in priority by those whoallocated NIH research funds. The recognition that AIDS was transmitted by blood, and the resulting increasingly restrictive exclusion of homosexually active malesundoubtedly had some effect on thefrequency of PTH. Although such men were very a small minority of blood donors, they were disproportionately infected with Hepatitis B (11). The effect could not have been great, however, since gays are not disproportionately infected with the Hepatitis virus C which was the cause of 90%of PTH cases (12). In 1986 the report of Koziol et al. (13) resulted in general acceptance that non-A, non-B hepatitis was not the relatively harmless disease it had been considered, emphasizing that even the subclinical cases had a 50% chance of chronic liver disease with 20% of them going on to develop cirrhosis. Both ALT and anti-HBc then became routine at blood banks. Review of the changing risk of PTH in the1980's shows a progressive fall, but there were no sharp drops coinciding with the changes taking place, and it is impossible to be sure what changes wereresponsible or most important. Finally, in 1990 the primary cause of non-A, non-B hepatitis, the Hepatitis Cvirus (HCV), was discovered, and progressively better tests have of become available to screen blood donors. Over thepast 50 years the risk PTH has steadily dropped to thecurrent value of approximately one in 6,000 donations. Given thecurrent sensitive test for HCV, it is likely that the ALTand anti-HBc surrogate tests no longer add any further protection. These tests result in the destruction of 2-4% of collected units, possibly for no useful purpose, and the psychological damage to donors with a positive test is considerable no matter how carefully they are counseled. Whether discontinuation of those surrogate tests should be recommended will be debated at an NIH conference early next year. THE HUMAN T
CELL LYMPHOTROPIC VIRUSES
(HTLV)
Blood banks began testing for antibody to HTLV-I as soon as it was recognized that this virus could cause acute T cell leukemia as well as myelopathy and a test became available (14). At the timeit seemed to many that we had jumped in without adequate justification to prevent an extremely rare disease whose transmissionby blood was not completely established. The precipitous response was attributed to criticism about blood banks' allegedly slow response to the threat of AIDS. Subsequent reports of myelopathy following transmission of HTLV-I by transfusion, though uncommon, have justified our attempts to prevent transmission of this virus. Although the association of acute T cell leukemia and HTLV-I is very clear, direct evidence that this complication has followed HTLV-I transmission by
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transfusion is lacking because it takes decadesfor the disease to manifest itself. Nonetheless, since transmission through transfusion clearly occurs (15), and the test eliminates carriers of the virus, it must be assumed it will reduce ultimate occurrence of leukemia. This will be difficult to measure, since transmission is very rare, and only a small per cent of infected recipients ultimately develop disease. HTLV-I1 is so closely related that current tests for anti-HTLV-I detect approximately 85% of those with anti-HTLV-11. There is still no unequivocal evidence that HTLV-I1 causes any disease, but there have been a few reports of myelopathy, and it is likely that our tests for anti-HTLV-I will be modified to make them more sensitive to anti-HTLV-11, reducing that small risk. CYTOMEGALOVIRUS (CMV) CMV has infected approximately 50% of blood donors, but without apparent clinical effects. This organism has its devastating effects in immunosuppressed individuals. Transmission of CMV by blood transfusion with resulting disease wasoriginally shown to occur in low birth weight premature infants at Stanford (16). Despite the unequivocal evidence in that study, there have been controversies about its implications generally. For one thing, there is an unexplained difference in the riskof CMV transmission in studies from different areas, resulting in the statement in the Standards of the AABB that CMV-antibody-negative blood components should be used for premature infants at risk "where transfusion-associated cytomegalovirus (CMV) disease is a problem" (17). The risks also appear generally lower in more recent accounts. Nonetheless, it is generally accepted that CMV antibodynegative components should be given to premature newborns weighing less than 1200 grams. Many neonatal intensive care nurseries have convinced their blood banks to give themonly CMV-negative components, with the result that all newborns tendto receive CMV-negative components. Where this wasdone, it undoubtedly lowered the riskof transmission of AIDS. The use of CMV-negative blood can also be justified for marrow transplant recipients(18). Systemic CMV infections with pneumonia have been a major cause of death in this group. These infections can be avoided in marrow recipients who are CMV-negative, if their marrow donor and all blood transfused are CMV-negative (19). More controversial is the question whether CMV-positive recipients will benefit from antibody-negative blood. CMVpositive recipients have a high probability of reactivating latent CMV even when transfused with CMV-negative blood, presumably due to allogeneic activation of the cells containing the latent virus. Nonetheless, there are multiple genetically different strains of CMV and superinfection is possible. There is some evidencethat superinfection lowers the prognosis. Finally, the recipients of solid organ grafts such as kidneys are at risk for CMV transmission and activation, with evidence that this canlead to some seriousmorbidity and graft loss (20). Transmission of CMV by transfusion occursfrom only a small proportion of CMV antibody-positive donors. There are as yet no practical tests to
discriminate the dangerous donors. It is not possible to meet the requests for CMV antibody-negative components which might be generated by all the conditions mentioned above, as well as others. An alternative approach which appears to be equally effective on a statistical basis is to filter blood components through filters with at least a threelog capacity for removal of white cells (19). CMV is a cell-bound virus. Filters nowin clinical trial, which remove fiveto six logs of leukocytes, should be even more reliable. VIRUSES RESISTANT TO STERILIZATION The success in lowering the riskof viral infections among recipients of blood bank components has been exceeded by the manufacturers of plasma derivatives who now have procedures to kill viruses while retaining the required activities of their products. They are also producing genetically engineered products which have never been exposed to human viruses. HIV transmission no longer occurs, and transmission of Hepatitis B and C viruses is unlikely if not impossible. There are viruses, however, which are resistant to the sterilization procedures now in use. Lacking lipid envelopes, they are not affected by procedures based on solvent-detergents. They are also resistant to heat. One of these resistant viruses is the Hepatitis A virus. I have already noted that this is amost unusual cause of hepatitis following transfusion of blood bank components because there is only a brief period of viremia during the acute phase. Plasma derivatives are made from pools of plasma from thousands of donors, and a rarely present virus is more likely to be included. The hemophilia world has been startled recently to learn of epidemics of hepatitis A attributed to factor VI11 preparations (21). Fortunately, the sources of these infected components have been quite restricted. Parvovirus B19 is another problem virus which lacks a lipid envelope and is resistant to heat. It is a common virus to which most of us have been exposed and are immune. It rarely causes clinical symptoms. However, it does interfere with red cell production in the marrow; and a recipient with a compensated hemolytic anemia may have avery abrupt and dangerous fall in hemoglobin when exposed to thisvirus. An immunologically impaired recipient of the virus may be unable to eliminate the virus, and a severe chronic anemia may result. SYPHILIS The first infectious disease which resulted in a test which blood banks could use to eliminate infected but apparently healthy donors wassyphilis. A serologic test for syphilis (STS) has been performed on all donated blood for many decades; for the most part a surrogate test has been used. And syphilis wasunquestionably transmitted repeatedly in the years beforethat became routine. Public health efforts to control syphilis, the discovery of effective therapy with penicillin and the frequent treatment of blood recipients with a variety of effective antibiotics have markedly affected the
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usefulness of the routine blood donor STS. The only report I can remember of syphilis transmission in the last 20 years involved a donor whose infection was so early the STS wasstill negative. A positive STS is found for approximately 50,000 donations a year in this country, most of which are false positives or occur in known cases of treated and non-infectious syphilis. More than a decade ago, the AABB Committee on Standardsdropped the requirement for a routine STS, and subsequently, the FDA Blood Products Advisory Committee recommended that the FDA drop its requirement for a routine STS. The test remains, however, now viewed as a surrogatetest for AIDS, and requirement for that test has been restored to theAABE Standards. PROTOZOA Malaria has not been a major problem for blood banks in this country in recent decades, but it continues to be a low level nuisance. One problem results from strains (Plasmodium malariae and Plasmodium ovale) which can persist for decades in asymptomatic carriers. Preventionof malaria transmission by blood transfusion relies on exclusion through history of donors or visitors from countries where malaria is endemic, and has been directed to preventing transmission of P. falciparum and P. vivax. Elimination of donors for three years after a malaria attack or use of malaria prophylaxis has been the basis for the prevention, along with a six month deferralafter being in a malaria area without prophylactic medication. These criteria have been recently challenged, and controversy remains. Chagas diseaseis a majorproblem for blood banks in South and Central America, and increasing numbers of immigrants from endemic areas make it likely that this will become a significant problem for blood banks in this country (22). Infection with the causative agent, Trypanosoma Cruzi, has serious late effects on vital organs. Thus far, the few blood transfusion cases reported in North America and dissatisfaction with tests availableto screen donors have not led to any suggestions for general screening. As the number of donors from endemic areas increases and effective testsbecome available, we can anticipate introduction of routine procedures intended to eliminate donors at risk for transmitting Chagas disease. BACTERIA In contrast to our increasing success in preventing transmission of viruses, there is increasing evidence that bacteria create problems far beyond our earlier recognition. Some of the increase may be more apparent is easier when than real. Recognition of bacterial infections in recipients other causes of chill-fever reactions have been controlled, and requires sufficient suspicion of the transfused blood as a causative agent that culture of blood remaining in the bag is carried out promptly. And some of the increase is undoubtedly real because of the increasing use of procedures where blood is held at room temperature for a number of hours (23). There is also evidence that current use of additive solutions, removing almost all
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OTHER
TABLE I1 INFECTIOUS DISEASES TRANSMISSIBLE BY BLOOD
Protozoa Malaria Chagas disease Babesiosis Spirochetes Syphilis Relapsing fever Lyme disease ( ? ) Rickettsiae Rocky Mountain Spotted Fever Q Fever Bacteria Contamination with cold-growing organisms Yersinia enterocolitica Klebsiella Brucellosis Other Jakob-Creutzfeldt disease
of the plasma from a red cell unit, deprives that unit of antibody and complement which had antibacterial properties (24). It was recognized decades ago that 3-5% of blood collected contain bacteria despite all efforts to create an aseptic area of the skin for the phlebotomy. Bacteria in the hair follicles and sweat glands cannot be removed. Early studies showed that almost all of these bacteria were destroyed by leukocytes and antibodies present in the blood, and that their growth was inhibited by the low temperatures at which blood was stored. Those bacteria which persisted in growing and causing trouble were organisms which grew preferably in the cold, and released large amounts of endotoxin. Some of these organisms metabolize citrate. They often resulted in gross changes in the color of the blood or clots which should have led to discarding the unit. The need to store platelet concentrates between 20 and 24 deg. C provides atemperaturemoresuitable for growth of most bacteria. Recognition of the frequency with which platelet concentrateswere contaminated despite all efforts, and the fact that bacterial growth enters a phase of rapid growth after 5 days led the FDA to reduce the permitted storage period of 7 days which was allowed at one time. Even 5 days at room temperature offersmany bacteria the opportunity to grow into numbers which can be lethal for the recipient. In recent years therehave been increasing reports of serious morbidity or death following transfusion of red cells contaminated with Yersinia
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enterocolitica. Since these organisms generally take as long as three to four weeks to reach dangerous concentrations, consideration was given to shortening the permitted storage period for red cell components. This idea was dropped when evidence was cited that stored components are less likely to transmit HIV, HTLV-I and other viruses. In Dodd's excellent review of the infectious risksof blood transfusion, he indicated that the risk of significant harm to patients by bacterial contamination of blood components was less than one in a million. In the last two years, our blood bank has had one fatal Klebsiella infection attributed to a platelet component and one Yersinia enterocolitica infection related to a red cell component (stored only 19 days). We collect Those numbers aretoo small to be approximately 100,000 unitslyear. meaningful, but we need to keep our eyes open to the possibility that bacteria are causing more frequent and serious problems than we recognize. It appears possible to reduce thelikelihood of bacterial transmission by leukodepletion of the component at the proper time, leaving the unit at room temperature long enough for the bacteria to be ingested by leukocytes before filtration (25). If the filtration is done too late, the leukocytes may have lysed and released viable bacteria. Since leukocytes play a role in removing contaminating bacteria, there must also be concern about the possibility that leukodepleted blood will have less protection against bacteria which remain (26). Further studies are needed to determine how and when to get the most benefit from leukodepletion. INCOMPATIBILITY REACTIONS Hemolytic reactions caused by recipient antibodies to donor red blood cells still occur, and rare deaths occasionally result. As ever, the primary either misidentification of the sample taken cause hasbeen clerical error from the patient for typing and crossmatching or transfusionof the red cells to the wrongpatient (27). Several blood banks in our area introduced the practice of a final check sample from the patient, which proved completely effective in preventing further mishaps of that type. The risk of such events was always so low, however, that the current strong administrative pressures to reduce expenses may lead to elimination of that additional safety step. On the other hand, concern about clerical mishaps has led to development of devices which claim to completely prevent giving the wrong blood to a patient. When I first became involved in blood banking in 1959, there was considerable emphasis on the variety of red cell antibodies which could be found, and on theneed to identify them all and provide blood for transfusion which lacked the corresponding antigens. Along with this, techniques to detect patient alloantibodies became increasingly sensitive, culminating in In 1976 the antiglobulin crossmatch and screening vs detection cells. Giblett, in her Landsteiner lecture, raised the questionof whether we were trying to detect antibodies which were of no clinical significance (28). And the resulting trend to simplify continues. Crossmatching is now often limited to a simple onestep phase which basically checksonAB0
-
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PERKINS TABLE I11 INCOMPATIBILITY REACTIONS Red blood cells Immediate hemolytic reactions Delayed hemolytic reactions White blood cells Chill-fever reactions Graft-versus-host disease Platelets Refractoriness Chills, fever, hypotension Post-transfusion thrombocytopenic purpura Plasma Urticaria Anaphylaxis Immunomodulation
-
as long as theantibody screen did not demonstrate theneed compatibility for more sensitive approaches. And others now are suggesting that we can drop that simple crossmatch, if we check the records on the patient in the computer (29). Again, fiscal constraints have played a major role in these decisions. Chill-fever reactions caused by antibodies to donor leukocytes were recognized more than 30 years ago. In general, removal of donor leukocytes below 5 x 10' per component prevents such reactions for all but the most highly sensitized patients. Currently available filters, which lower the leukocytes to less than 5 x lo6, should prevent all such reactions. A rare but serious problem caused by donor leukocytes is graft versus host disease (GVHD). Since we have recognized that recipientswhoare severely immunocompetent cannot reject donor leukocytes, and are thus susceptibleto this complication, we have prevented it by prior irradiation of the component. More recent recognition that the same phenomenon may occur in a recipient with normal immune competence if the donor, by chance, has HLAcompatible leukocytes, has led to irradiation of components from blood relatives. Under these conditions, GVHD should be an extremely rare event. It is likely that protection against GVHD can be accomplished by adequate removal of leukocytes. The current 3 log filters have permitted a few cases of GVHD to occur, but filters now in the investigative stage may well prove completely effective. Platelets cause chill fever reactions due to contaminating white blood cells reacting with recipient antibodies. These reactions can be prevented by filtering out most of the leukocytes. Many reactions to platelets which have been hard to explain (fever and vasomotor reactions) are likely due to cytokines released from leukocytes. Again, removal of the leukocytes prior
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TABLE IV MISCELLANEOUS COMPLICATIONS Circulatory overload Air embolism Iron overload Massive transfusion effects Hypothermia Citrate toxicity Acid-base imbalance Potassium imbalance Hemostatic abnormalities
to storage may prevent these reactions (30). The number one problem with platelet transfusion is the refractory state which develops when recipients are repeatedly exposed to donor antigens. The recognition phase of the immune response requiresleukocytes; so again, removal of the leukocytes may prevent alloimmunization. IMMUNOMODULATION The most interesting effect of transfusionsto be newly recognized is the immunomodulation that occurs. This wasfirst identified by the fact that patients who received solid organ transplants had better graft survival if they received previous blood transfusions. These data areunequivocal, and there can be noargument that transfusions impair immune responses. There has been much controversy, however, in some related areas. If immune responses aredepressed bytransfusions, it seemed reasonable that transfused patients would be less able to handle post-operative infections. And, if it is true that immune responses control the spread of cancer cells, transfused patients should have more cancer recurrence after surgery. There are many reports of animal experiments and human evaluations which indicate that these two hypotheses are true. While the data relating to postoperative infections are fairly consistent, the reports of transfusioneffectsoncancer recurrence are morecontradictory; and it is difficult to convince critics in either situation that one can adequately compensate for confounding effects. There remains the possibility that the transfusedpatient was the sickest and most at risk. Nonetheless, the preponderance of evidence favors the possibility that the immunomodulating effects of transfusion result in increased postSincethe operative infection and increased cancer recurrence (31). immunomodulating effect appears to be from the donor's white blood cells (31), which can be removed by filtration, this offers another possible benefit of filtered components. For the sake of completeness, Table IV includes additional complications oftransfusion.
300
PERKINS TABLE V INDICATIONS FOR LEUKODEPLETED COMPONENTS Proven effective Prevention of chill-fever transfusion reactionscaused by recipient antibodies to donor leukocytes Almost certain clinical benefit Reduction of alloimmunization which can make patients refractory to transfused platelets Elimination of cell-bound viruses E.g., cytomegalovirus and HTLV-1/11 Improved quality of red cells during storage (32) Less hemolysis Better in vivo recoveries Possible clinical benefit Removal of bacteria contaminating blood components Prevention of immune modulation by transfused blood products Possibly less risk of post-operative infection Possibly decreased cancer recurrence Prevention of transfusion reactionscaused by cytokines released from leukocytes during storage Prevention of activation of latent viruses resulting from allogeneic reactions (33) E.g., HIV-l/2, HBV and HCV Possible clinical benefit with filters not vet licensed Prevention of graft-vs-host disease
CONCLUSIONS There has been amarked change in the over-all rate of reactions to blood transfusions. We nowhave the safest blood supply by far the world has ever seen, and we are preventing many types of serious reactions. The of AIDS and hepatitis has been reduction in the risk of transmission particularly dramatic. On the other hand, other organisms such as CMV, Trypanosoma Cruzi, parvovirus and bacteria are causing increasing concern. Finally, given the increasing evidence that donor white blood cells are harmful in a variety of ways, and given increasingly better technics to remove them, we may be able to eliminate many of the remaining problems with leukodepletion (TABLE V). For optimal effect, this should be done prior to storage of the blood component.
301
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in
BLOOD TRANSFUSION, BLOOD STORAGE AND IMMDNOMODULATION M.S.
Mincheff and H.T.Meryman Transplantation Department Holland Laboratories, American Red Cross Rockville, MD 20855 ABSTRACT Allogeneic blood transfusion is the most frequent allotransplantation procedure performed on a routine basis with no prior HLA-typing. Roughly 50% of therecipients of unprocessed red cells and platelets become alloimmunized. Evidence also exists for some degree of transfusion-induced immunosuppression. Prior transfusion has been shown to enhance kidney transplant survival and evidence of an increase in tumor recurrence and of infectious complications has also been presented. The presence of donor antigen-presenting cells appears to be a prerequisite for alloimmunization and they must be both viable and capable of presenting a costimulatory signal in order to induce IL-2 secretion and proliferation of responding CD4 T cells. APCs presenting antigen but no costimulatory signal can induce non-responsiveness in CD4 T cells, a possible mechanism of transfusion-induced immunosuppression. APCs in refrigerated blood continue to present antigen but progressively lose their ability to provide costimulation. By day 14 costimulatory capacity is absent and transfusion of such blood should not alloimmunize but could induce some degree of immunosuppression. Further refrigerated storage in excess of 2 to 3 weeks leads to induction of apoptosis in contaminating leukocytes. We have found that alloantigens expressed on such cells do not appear to be recognized by responder T cells and transfusion of blood stored in excess of 3 weeks should neither alloimmunize nor immunosuppress. INTRODUCTION Evidence abounds that allogeneic blood transfusions can induce immunization against HLA antigens. Alloimmunization is seen in roughly 50% of transfusions ofuntreated red cells or platelets (l). It leads t o nonhemolytic febrile reactions during subsequent transfusions and represents a serious problem in patients who require frequent platelet support. Whereas immunization by transfusions has been recognized for decades, only in recent years has it been appreciated that transfusions may also induce a degree of immunosuppression (2-8). The beneficial effects of pre-transplant blood transfusions onkidney allograft survival have been well documented both in humans and animals (3,s-9). Although donor-specific transfusions have been shown to beparticularly effective (10-13), significant protective effect has been seen even following a single transfusion from an unrelated blood donor (14). The development of both alloimmunization and immunosuppression seems to be independent of thenumber of transfusions or theantigenic load administered. The observation that leukodepletion can substantially reduce the incidence of seroconversion supports the assumption that it is contaminating leukocytes that are responsible for alloimmunization (15). Enhancement of renal allograft survival has been documented following transfusions of whole blood, of buffy-coat-rich red cells and even following leukocyte-poor 303
304
MINCHEFF AND MERYMAN
transfusions although the number of residual leukocytes does seem to be a determining factor (16). NORMAL FUNCTION OF THE IMMUNE SYSTEM Prior to a discussion of the mechanisms by which transfusion might induce alloimmunization and immunosuppression, a review of current thinking regarding the normal function of the cellular and humoral immune systems may be helpful. The immune system has evolved to seek out and destroy agents, primarily pathogenic microorganisms, which threaten the homeostasis of the macroorganism. The first line of defense is the phagocytizing granulocytes. These cellspossess a powerful killing machinery and are easily activated by microbial products. However, since they lack antigen-specific receptors, they are not selective in their effector function. Further spread of an antigenic challenge engages specialized cells such as B cells, T cells and antigen-presenting cells (APCs), some of whichhave evolved a highly sophisticated system for antigen recognition. In the humoral immune system, B cells produce antibodies that bind to native antigen either as soluble antibodies or acting as B cell receptors (17). These antigens are usually protein molecules with orwithout carbohydrate or lipid residues. In the cellular immune system, T cell receptors (TCRs) recognize antigenic fragments only when expressed in association with Class I or Class I1 major histocompatibility complex (MHC) molecules at the cell surface (18,19). Class I1 MHCmolecules are expressed primarily by APCs whereas Class I molecules are expressed by all nucleated cells and by blood platelets. A pathogen (X) that enters through the skin or mucous the barrier will be phagocytized by APCs such as tissuemacrophages. The associated microbial or viral protein antigens are digested in intracellular acidic vacuoles (endosomes and lysosomes). The resulting peptides (X,, &, X 3 ) , ranging from 5 to 20 aminoacids, are displayed at the cell membrane bound to Class I1 MHC molecules which are synthesized within the APC (MHC" + X,, MHCA + XI, MHC" + X,). Peptides which are not derived from exogenous proteins but are synthesized within the cytosol of virally infected or tumorcells are expressed in association with Class I MHC molecules (20). Binding of peptides to MHC molecules induces mutual conformational changes which can berecognized by specific clones of T cells whose receptors are capable of relatively high affinity binding to that particular MHC-peptide complex (21). CD4 (cluster determinant) T cells recognize peptides complexed to Class I1 molecules, while CD8T cells recognize peptides complexed to Class I molecules. The recognition of Class I1 MHC-peptide by CD4 T cells may lead to different sequellae depending on the affinity of cell receptor engagement and on the presence of accessory signals. Ligands which bind with a relatively high affinity and trigger a biologic response are classified as agonists. By contrast, low affinity binding ligands that cause no TCR clustering and no T cell responses are classified as antagonists (22). Antagosists compete, sometimes extremely effectively with agonists, leading to functional receptor inactivation and selective inhibition of T cell function. High affinity binding triggers a series of early activation steps in the CD4 T cell such as Ca2+ influx and phosphatidylinositol turnover which result in IL-2 receptor expression but not IL-2 secretion (24). There is now good
BLOOD TRANSFUSION AND STORAGE
305
evidence that additional, costimulatory signals are required for IL-2 secretion and CD4 T cell proliferation. Such costimulatory signals canonly be provided by "professional" APCs and most probably consist of other surface molecules such as CD80 (BBllBt), CD40, CD54 and CD58 (25,26,27). CD4 T cells which have recognized the antigenic MHC-peptide complex but do not receive the costimulatory siganal do not proliferate but instead become anergized and can not be restimulated. Both TCR antagonism and T cell anergy can result in the development of antigen-specific tolerance, a form of immunosuppression. In thenormal course of events the first encounter of naive, "virgin" CD4 T cells with antigen probably occurs in the paracortical regions of lymphoid tissue. The bone-marrow derived dendritic cells which reside there are particularly effective at presenting MHC-peptide and the costimulatory signals (28). Presentation of antigenic HHC-peptide by these cells results in IL-2 secretion and CD4 T cell proliferation and maturation. Resting B cells circulate in the blood and migrate across high endothelial venules (HEV) to sites of trapped antigen in secondary lymphoid organs such as the lymph nodes, the spleen, the tonsilsand the Peyer's patches (29). Those B cells which bear immunoglobulin receptors specific for a given trapped antigen, enter the T-cell rich paracortical regions, capture and process the antigen and express MHC-peptide complexes. B celle, however, are incapable of providing the costimulatory signals. Since both the B cells and the dendritic cells come from the same individual and therefore express identical MHC-peptide complexes, if the antigen specific CD4 T cells have already been activated by dendritic cells and are secreting IL-2, these T cells can then interact with antigen-bearing B cells in a cognate fashion and induce them to migrate into B-cell follicles to become either plasma cells and secrete antibodies, or tobecome memory cells. However, if the CD4 T cells encounter the antigen for the first time on resting B cells, since the B cells CannnOt supply the costimulatory signals, the T cells will become tolerized while theB cells undergo apoptosis (30). ROLE OF ANTIGEN-PRESENTING CELLS IN BLOOD TRANSFUSION Blood transfusion is the most frequent allotransplantation procedure performed on a routine bases with no prior HLA-typing. Following a transfusion containing donor leukocytes, the immune system of therecipient will encounter APCs and B cells expressing foreign Class I and Class I1MHC, as well as platelets expressing foreign Class I MHC. Recipient B cells can bind to the foreign MHC-peptide complex but antibody secretion will not result unless those recipient B cells which recognize the antigen receive additional signalling (help) from recipient CD4 T cells. The activation of recipient CD4 T cells may result from either of two proposed mechanisms: a. The indirect recotanition model proposes that alloantigens from the donor are processed by recipient APCs (31,32,33). According to this model, Class I antigens (the classical transplantation antigens) are presumed to function as conventional antigens which can be processed and presented by APCs. The model therefore proposes that donor MHC molecules shed or released after cell death are internalized and degraded by recipient APCs. Recipient T cells then recognize these alloMHC peptides expressed in conjunction with recipient Class 11MHC. The cells actively participating in allorecognition are thus APCsand T cells, both of recipient origin (34). This routeof
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MINCHEFF AND MERYMAN
allosensitization may occur in some cases of long-term alloantigen exposure and may be the cause for chronic graft rejection. It may also explain some of the seroconversions in patients receiving repeated transfusions of leukocytedepleted platelets. However, this route ofallorecognition cannot be themajor mechanism following blood transfusion since the majority of patients receiving enormous loads of Class I on purified platelets, or Class I and Class I1 on W-B-irradiated platelet concentrates, do not alloimmunize (35,36,37). b. The direct recoanition model proposes that recipient T cells recognize donor HHC molecule + peptides directly on donor APCs, i.e. the cognitive phase does not involve participation of recipient APCs. The ultimate outcome of thisencounter depends on the functional status and the immunologic experience both of thedonor antigen-presenting cells which contaminate the donated blood and the recipient lymphocytes which recognize the antigen. Fresh blood contains a heterogeneous population of antigen presenting cells such as dendritic cells, monocytes and B cells. Current evidence suggests that there may be twopopulations of blood dendritic cell8 (38). A minor subpopulation expresses costimulatory activity, probably as a result of exposure to lymphokines following passage through tissues (38). We suspect that thesecells are the main cause of alloimmunization since their elimination results in prevention of alloimmunization (reviewed in reference 1). The majority of blood dendritic cells, however, are immature and have recently migrated from the bone marrow (38). Such cells are poor stimulators in mixed leukocyte cultures (MLCs) but become very potent upon maturation in vitro (4.38). Blood monocytes are also good stimulators, but this may be a reflection of their activation during in vitro separation and purification. B cella do not elicit primary MLR responses, do not express costimulatory activity and have been shown to induce tolerance (39). ROLE OF DURATION OF BLOOD STORAGE Blood preservation solutions currently in use donot adequately support leukocyte metabolism and white cells degenerate during blood storage. We have found that mature dendritic cells deteriorate rapidly and mononuclear cella, isolated from 6-10 day-old blood will not stimulate in an MLR (4). However, monocytes and probably immature dendritic cells remain viable following 6-10 days of refrigerated storage and will express costimulatory activity if preactivated (matured) in vitro (4). In contrast mononuclear cells that have been isolated from blood stored longer than 13 days cannot provide the costimulatory signal even following preactivation in vitro (4). For that reason we have proposed that blood stored for 13 days and longer should not alloimmunize and a clinical study is in process to validate this hypothesis. Supporting evidence has been reported by Light et a1 (40). In their study 3 out of 6 candidates for kidney transplants who received fresh blood became alloimmunized, while seroconversion was seen in none of the13 patients who received stored blood. We have also shown that blood stored in excess of 13 days contains antigenic, class I1 positive MNCs whose antigens are readily recognizable by responder T cells in culture. Such recognition does not lead, however, to T Cell proliferation due to lack of costimulation and we have speculated that it may be associated with the development of immunosuppression.
307
BLOOD TRANSFUSION AND STORAGE
Immunosuppression by this mechanism would be specific for the MHCpeptide complexes expressed by the donor APCs. Since recipient T cells K, or + Y,, Y2, recognize various binary complexes of "alloMHC + X,, q, Y,, where X8 and Yo are peptides which originate from different antigens, both alloimmunization and immunosuppression should be polyclonal. On theother hand similarity between MHC? + X, and MHC' + Y2 binary complexes i s possible. On this basis immunosuppression by a single blood transfusion could cover multiple specificities and induce sufficient tolerance so that no alloimmunization will develop following multiple subsequent transfusions of fresh blood. Our clinical trial is designed t o explore this hypothesis as well. Recent evidence from our laboratory suggests that leukocytes in preserved blood ultimately undergo apoptosis during refrigerated storage. There are variations in individual donors, but almost all cells are apoptotic by day 25 in blood collected and stored in CPDA-1. Induction of apoptosis seems to beenhanced by serum deprivation and leukocytes isolated from ADSOL supplemented packed red cells undergo apoptosis from day 14 to day 21. Studies in vitro show that such cells continue to express Class I and Class I1 MHC molecules but the recognition of such MHC molecules in culture doesnot lead to early activation steps and IL-2 receptor expression by responder T calls. We speculate that apoptotic cells present in transfused blood will neither alloimmunize nor induce immunosuppression through induction of anergy. However, MHC-peptide complexes present on apoptotic cells may behave as CD4 T cell receptor antagonists and still induce inhibition of T cell function (22,23). Experiments in an animal model are currently ongoing to determine the irnmunogenicity andthe toleragenicity of apoptotic APCs.
....
....
SUMMARY AND CONCLUSION In summary we propose that theoutcome of a blood transfusion will depend on theduration of refrigerated blood storage. Transfusion of fresh blood containing viable, professional APCs results in alloimmunization. With progression of blood storage, the immunogenicity of blood APCs gradually falls while toleragenicity prevails. Finally, following apoptosis induction and progression, both immunogenicity and toleragenicity of blood leukocytes may be lost and the transfusion of blood containing such cells may have no immunologic consequences.
ACKNOWLEDGEMENTS We wish to express our deep appreciation to both Veena Kapoor and Michael Hammett for their excellent technical assistance. This research was supported in part by a grant from The G.Harold and Leila Y. Mather's Charitable Foundation and by NIH grant BSRG 2 507 PR05737.
1.
H.T.
2.J.H.
Meryman. Oh and H.M.
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THE TUMOR GROWTH-PROMOTING EFFECT OF ALLOGENEIC BLOOD TRANSFUSIONS
M. A. Blajchman and J. 0. Bordin The Departments of PathologyandMedicine,McMasterUniversity;and The CanadianRedCrossSociety,Hamilton,Ontario,Canada; U N 325 ABSTRACT
Over the past decade, many studies have suggested that allogeneic blood transfusions(ABT) may adverselyaffectarecipient. The ABT-associated deleterious effectsinclude the development of transfusionreactions,graft-versus-hostdisease, alloimmunization,andimmunomodulation.While the ABT-associatedimmunosuppressive effects might be beneficial for recipients of kidney allografts, in reducing the relapse rate in patients with Crohn's disease, and in ameliorating the rate of abortion inwomenwith recurrent spontaneousabortions;evidence is accumulating that the immunosuppressionassociated to perioperativeABTmightadverselyaffectoverall prognosisinpatientswithamalignancyundergoingcurativecancersurgery.In addition, the ABT-associated immunomodulation has been reported to be associated with an increasedriskforpostoperativebacterialinfections. Data from both inbred and outbred experimental animal models indicate that ABTpromotetumorgrowth.Evidence is available that thisABT-promotingtumor growth effect can be adoptively transferred to naive animals, using splenic immunocytes. Furthermore, data from the experimental animal models indicate that the ABTeffecton the growthof tumors is due to the presence of the donor leukocytes in the transfused allogeneic blood, and that this deleterious effect can be ameliorated by the pre-storage leukodepletion of the allogeneic blood. Importantly, recent evidence suggests that post-storage leukodepletion inefficacious is in preventing the ABT-associatedtumorgrowthpromotioneffect.Whileresultsfrom studies in experimentalanimalscannotnecessarilybeextrapolated to the clinical situation, these studies suggest that ABT promote tumor growth and that pre-storage leukodepletion ameliorates this effect. Properly-designed prospective clinical studies are nonetheless required to ascertain whether patients with a maligancy undergoing curativesurgeryshouldreceiveleukodepletedallogeneicbloodproductsand the appropriate timing for such leukodepletion.
311
AND
312
BLNCHMAN
BORDIN
INTRODUCTION
Many reports have appeared over
the past decade suggesting that allogeneic
blood transfusions (ABT) are associated with immunosuppression in recipients Suchimmunosuppressiveeffects
(14).
may be clinicallybeneficialinincreasingallograft
survival in renal transplant recipients, decreasing the recurrence rate of spontaneous abortion in affected women, and reducing
the relapse rate in patients with
inflammatory bowel disease; however, concern has been raised that this ABT-induced immunomodulationcouldadverselyaffect
the prognosis of patients undergoing
curative surgery for a malignant tumor
(4-7). To date most of the reported clinical
data are fromnon-randomizedretrospectivestudies,andnodefinitive
data proving
thisABT-associatedtumorgrowth-promotingeffectinmanhavebeenprovided.In contrast, the relationshipbetweenABTandtumorgrowthhasbeenexamined extensively in experimental animal models and indicate,
in both inbred and outbred
animalmodels,thatABTenhancesolidtumorgrowthandmetastaticnodule formation (8-12). EFFECT OF ABT ON ANIMAL TUMOR GROWTH
The effect of ABTonsolidtumorgrowthhasbeeninvestigatedinallogeneicallytransfusedmice
that wereinoculatedintramuscularlywith
either syngeneic
malignant melanoma (B16) or mastocytoma (P815) cells. In both situations animals that receivedABTdevelopedlargertumorsthancontrolsyngeneicallytransfused mice (9). Experimentsperformed
to evaluate the effect of the tumorcelldose
showedthat the ABT-associatedtumorgrowthpromotioneffect whensmallnumbers
was mostevident
(1.25 to 2.5 x 10’) of either B16 or P815 tumorcellswere B16
inoculated into hostanimals.Similareffectswereobservedwhensyngeneic
tumor cells were inoculated intravenously and the numbers of pulmonary metastatic nodules were enumerated (8,9). The importance of the timingof the ABT on tumor growth
in experimental
animalshasalsobeenexamined.Initialstudies,inbothinbred(mice)andoutbred (rabbits)animals,providedevidencethatunmodifiedABThaveatumorgrowth promoting effect when given prior
to the infusion of syngeneic
tumor cells (10). To
try to simulate the clinical situation we recently explored the role of ABT in animals that receivedsuchtransfusionssubsequent
to the inoculation of the tumorcells.
These data show that ABT also enhance tumor growth
in animals with established
313
EFFECT OF ABT
GROWTH-PROMOTING TUMOR
tumors (12). Using a varietyof transfusion protocols in two animal models, the tumor growth-promoting activity of ABT has thus been observed to occur in experiments when the ABT precededtumorcellinoculationandalso administeredsubsequent
to tumor cell inoculation.
when the ABT were Our observations in these
experimental animal models are summarized in TABLE I. Other investigators, using inbred animals (mice) only, also provided evidence that
ABT given after tumor cell
engrafment enhanced tumor growth. In this report, however, some groupsof animals, transfused with allogeneic blood
1 and 10 days after the tumor cell inoculation, did
not developlargertumorsthancontrolmicegivensyngeneicblood
or saline.This
latter data must be viewed with caution, however, because some groups conmprised lessthan
10 animals (11). Overall, the available data providesolidevidencethat
ABT enhance both the size of solid tumors and the number of metastatic pulmonary nodules observed compared
to that seen in control animals.
CLUES AS TO THE MECHANISM OF ABT-INDUCED ENHANCED TUMOR GROWTH EFFECT
Although ABT have been shown experimental animal models, yetbeenelucidated.
to enhance tumor growth in several
the mechanism of this biological phenomenon has not
ABT havebeenshown
to beassociatedwithimmunologic
abnormalities in humanrecipients (1,3,4). The observedabnormalitiesinclude lowering of the ratio of helper to suppressor T lymphocytes, a decrease in killer (NK)cellactivity,adecreasein
natural
the delayedtypehypersensitivity,and
suppression oflymphocyteblastogenesis
the the
(13-15). Inanimals,decreasedinterleukin
2 (IG2) generation by lymphocytes has been reported following ABT (16). In contrast to that seen in humans reduced NK cell activity against tumor cells mice (5). Furthermore, ithasbeenproposedthat
was not seen in
ABT mightcauseimmunologic
unresponsiveness due to the inactivation of alloreactivelymphocytes;induction suppressor T cells;and/orinduction
of
of anti-idiotypicantibodies (1,4,17).
It has been argued that the presence of passenger leukocytes bearing Class I1 majorhistocompabilitycomplex ("IC)
antigens in the transfusedallogeneicblood
is important inmodifying the immune response of ABT recipients (1,4). In support
of this hypothesis, we have recently demonstratedthat the enhancement of metastatic tumornodulesformation inexperimentalanimalscan be prevented by the prestorage leukodepletion (99.7% removal) of the ABT (10,12). Furthermore, we have shown in the rabbit model, that leukodepletion (99.6% removal) of ABT after storage
BLAJCHMAN AND BORDIN
314
TABLE I Summaryofexperimentalanimalstudiesperformedin the authors’laboratory to examine the effect of the timingofsyngeneic(SBT)andallogeneic(ABT)blood transfusions on tumor growth in experimental animals. Day 0 represents the day each animalwasinfusedintravenouslywithtumorcells. Animal Model
Dayof Transfusion
Median Number of Metastatic Pulmonary Nodules SBT (Number of animals)
(Number of animals)
P ABT value 0.0006
Rabbit
-10 and -7
Rabbit
+4 and +9
Mouse
-10 and -7
1.0
(22)
5.0
Mouse
0 and +4
3.0
(7)
48.0
Mouse
+4 and +9
6.5
(14)
90.0 (15)
0.0003
Mouse
+9 and + l 1
9.0
(14)
36.0 (12)
0.002
27.5
3.0(17)
(30)
0.003
68.0 (27)
20.0 (17)
but before transfusion (post-storage leukodepletion) does
(34)
0.01
(8)
0.009
not prevent ABT-induced
pulmonary metastatic nodule formation (12) (TABLE 11). It is also possible that prestorage leukodepletion may prevent the accumulation of soluble biologic mediators that are activelysynthetizedandreleased
by the leukocytes present in donor
allogeneicbloodduringstorageandthatsuchsubstances the immunomodulationobservedfollowingABT hypothesisthat
are somehowinvolvedin (18). In further support of the
the ABT-tumorgrowthpromotioneffect
is due toallogeneic
leukocytes, we recently observed that animals receiving allogeneic leukocytesdevelopedsignificantlyhighernumbers
buffycoat
of pulmonary metastatic nodules
than animals that received either plasma or leukodepleted whole blood (12). Further clues as to the mechanism of the ABT-inducedtumorgrowthwere provided by observations that thiseffectcanbeadoptivelytransferred
to naive
animals, using spleen cells harvested from allogeneically transfused animals. In latter studies, the number of pulmonary metastatic nodules observed
these
in bothmice
and rabbits that had received spleen cells from allogeneically transfused animals
was
significantly greater than that observed in animals that had received spleen cells from animals that weretransfusedwithsyngeneicblood
(10). Importantly,thistumor
TUMOR GROWTH-PROMOTING EFFECT OF ABT
315
TABLE I1 Summaryof studiesperformed inexperimentalanimals(rabbits) to examine the effect ofunmodifiedsyngeneic(SBT), non-leukodepletedallogeneic(ABT), prestorageleukodepletedallogeneic(PRE-LD-ABT), or post-storageleukodepleted allogeneic(POST-LD-ABT)bloodtransfusionsonpulmonarymetastaticnodule formation. On day 0 each animal was inoculated with tumor cells. Transfusions were given either on days -10 and -7 (animalswithnon-establishedtumors) or days +4 and +9 (animalswithestablishedtumors).
Animals with EstablishedTumorsNon-EstablishedTumor
Animalswith
Type ofBlood Transfusion
Number of rabbits Per group
Number of pulmonary metastatic nodules median (range)
Number of per group
SBT
11
11.5 (3-72)
12
17.5(5-28)
ABT
20
42.8(1-150)
20
50.0 (5-86)
PRE-LD-ABT
20
15.3(1-98)
20
20.0(2-50)
POST-LD-ABT
20
30.5 (1-100)
18
39.0(22-86)
growth-promotingeffectcould
not beadoptivelytransferred
Number of pulmonaryrabbits metastatic nodules median (range)
to naiveanimals
by
spleencellsderivedfromanimalsthathadbeentransfusedwithpre-storage leukodepleted ABT (10). In addition, we have observed recently that both B and spleniclymphocytesmustbetransferred tumorgrowthpromotingeffect
T
to the naiveanimals to produce the ABT
innaiveanimals(unpublishedobservations).
While the precisemechanism of ABT-associatedimmunomodulationhasnot been completely elucidated, our
data from both inbred and
outbred animal models
suggest that the ABT-associated tumor growth promoting activity is immunologically mediated and that this effect is related to the presence of allogeneic donor leukocytes in the transfused allogeneic blood products.
It is'important to caution, however, that
results from animal data should not be extrapolated to the clinical situation without proper study. Our results suggest nonetheless that pre-storage leukodepletion might be effective in ameliorating
the tumor-growth promoting effect of ABT.Finally,
wouldemphasizethatproperlydesignedprospectiveclinicalstudies
we
are required to
316
BLAJCHMAN AND BORDIN
ascertainwhether
patients withamalignancyundergoingcurativesurgeryshould
receive leukodepleted blood products and
the appropriate time for such
leukodepletion. ACKNOWLEDGMENTS
Dr. J. 0. Bordin is the recipient of a Post-Doctoral Scholarship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPq, Brazil.Thiswork was supported, in part, by a grant from the Miles/CRCS R&D Fund.
REFERENCES 180-193 (1989).
1.
H.T. Meryman.Transfus.Med.Rev.
2.
DJ. Triulzi,J.M. Heal and N. Blumberg,inTransfusionMedicinein S.T. Nancy, ed, American Association of Blood Banks, Arlington, VA, 1-23.
the 1990s, (1990) pp.
3.
J.W.Alexander.Transfusion,
4.
J.O.Bordin,N.M.
5.
D.M.A. Francis. Br. J. Surg. 78-
6.
M. Chung, O K Steinmetz and P.H. Gordon. Br. J. Surg. @J 427-432 , (1993).
7.
E. VamvakasandS.B.Moore.Transfusion
8.
5 195-196(1991).
Heddle and M.A. Blajchman. Blood, in press
(1994).
1420-1428(1991).
-33 754-765(1993).
S. Shirwadkar, M.A. Blajchman, B. Frame, F.W. Orr and D.P. Singal. Transfusion
a 188-190(1990).
9.
S. Shirwadkar, M.A.Blajchman,B. Oncol. 118,176-180(1992).
10.
M.A. Blajchman, L. Bardossy,R.Carmen, 1880-1882(1993).
11.
D.M.A. Francisand G.J.A. Clunie.J.
12.
J.O. Bordin, L. BardossyandM.A.Blajchman.Blood,in
13.
J. Kaplan, S. Sarnaik, J. Gitlin and J. Lusher. Blood -4 6
14.
P.I. Tartter, T.M. Heimann and AH. Aufses Jr. Am.J. Surg. 151,358-361 (1986).
15.
J.P.Waymack, K Balakrishnan, N.McNeal, S. Gonce, P.Miskell,G.D. 204, 681-685(1986). and J.W. Alexander. Ann. Surg.
Frame and D.P.Singal.J.Cancer.Res.Clin. A. SastryandD.P.Singal.Blood
Exp. Surg.Res.
237-241(1993).
press (1994). 308-310(1984).
Warden
TUMOR GROWTH-PROMOTING EFFECT OF ABT
16.
R.N. Stephan,J.M. Kisala, R.E. Dean, 123,235-240 (1988).
17. M.E. Brunson and 18. R.N.I. Pietersz, (1993).
317
AS.Geha and I.H. Chaudry. Arch. Surg.
J.W. Alexander. Transfusion 651-658
(1990).
I. Steneker and H.W. Reesink. Transfus. Med. Rev. 17-24
This Page Intentionally Left Blank
THE ROLE OF CYTOKINES
IN
HEMOLYTIC
TRANSFUSION
R. D. Davenport Department of Pathology, University of Michigan Medical Ann Arbor, MI 48109.
REACTIONS
School,
ABSTRACT Experimental evidence is accumulating to support a central roleforcytokinesinthepathophysiologyofhemolytic transfusionreactions.Theproductionoftumornecrosis factor, interleukin-8, and monocyte chemoattractant protein AB0 incompatible red occurs in whole blood in response to cells, a modelofacutehemolytictransfusionreactions. Peripheral blood mononuclear cells may produce interleukin-lp, tumor necrosis factor, interleukin-8, monocyte chemoattractant protein, and interleukin-l receptor antagonist in response to IgG-coated red cells, a model of delayed hemolytic transfusion reactions. Cultured umbilical vein endothelial cells respond to conditioned plasma from ABO-incompatibility reactions by expressing the procoagulant tissue factor and the leukocyte in v i t r o adhesion molecules ELA"1 and ICA"1. These endothelial cell responses can be inhibited by neutralizing antibodies to tumor necrosis factor, suggesting that TNF may have a central role in intravascular coagulation and end-organ injury that may occur in acute hemolytic transfusion reactlons.
The term cytokine embracesa large variety of proteins that are involved in cellular communication. Particularly, a number ofcytokineshaveprofoundeffectsontheimmuneand inflammatory responses. There are at least 35 wellcharacterizedcytokines,includingtheinterferons,hematopoietic growth factors, interleukins and tumor necrosis factors, with many more candidates being described. Out of this molecular menage, we know very little regarding the possible role of most cytokines in hemolytic transfusion 319
320
DAVENPORT
reactions.However,thosecytokinesforwhichthereis clinical or experimental evidence of involvement in HTR can be grouped into three general categories, as summarized in Table I. The full extent to which these molecules may be involved in transfusion reactions is only beginning to be elucidated. Pro-inflammatory cytokines (IL-l, TNF, I L - 6 ) : First, there are pro-inflammatory cytokines, particularly interleukin-l (IL-1), tumor necrosis factor (TNF) , and interleukin-6 (IL-6). These are produced by mononuclear phagocytes but, also, are increasingly being recognized as products of a variety of non-immune cells. They also have a broad spectrum of target cells, including phagocytes, lymphocytes, endothelial cells, and many stromal and epithelial cell types.
The biology of IL-1 has been well reviewed.l One of the first biological activities of IL-1 to be characterized was PGE2by its ability to induce fever. This response is mediated production in the hypothalamus, and may occur through the intermediate production of IL-6. In addition to fever, IL-1 has important systemic effects. IL-1 will stimulate hematopoiesis and is involved in the recruitment and activation of neutrophils and platelets from the bone marrow, which may also bemediatedthroughtheinductionofotherintermediate factors, such as IL-8. At sufficient concentrations in blood IL-1 will cause circulatory collapse, shock and death. also is involved in cellular and humoral immune respons several levels. Lymphocyte activation is a complex process that involves antigen presentation and accessory signals, which, in part, is dependent on IL-1. Splenic B-cells that are incubated with IL-1 before a mitogenic stimulus have an augmentedproliferativeresponse.IL-1willenhancethe generationofimmunoglobulin-secretingcellsinmitogenstimulated B-cells cultured with T-cells, or B-cells stimulated with CowenI Staphylococcus aureus. IL-1 also will increasethenumberofactivatedcells,cellcycleprogression, and IL-2 production by mitogen-stimulated T-cells. IL-1 has been shown to induce the expression of numerous genes from many cell types. Some inducible gene products that may be significant in the setting of hemolytic transfusion reactions include the cytokines IL-1, TNF, IL-6, IL-8, MCP, and IL-lra;
2 x
CYTOKINES AND HEMOLYTIC TRANSFUSION REACTIONS
Table p
Pro-inflammatory cytokines : Interleukin-l (IL-1) Tumor Necrosis Factor (TNF1
321
I.
Fever Hypotension, shock, death (synergy) Mobilization of leukocytes from bone marrow Activation of T- and B-cells Induction of cytokines (IL-l, IL6, IL-8, TNF, MCP) Induction of adhesion molecules Induction of procoagulants
Interleukin-6 (IL-6) Fever Acute phase protein response B-cell antibody production T-cell activation Chemokines : Interleukin-8 (IL-8) Chemotaxis of neutrophils Chemotaxis of lymphocytes Neutrophil activation Basophil histamine release Monocyte Chemoattractant Protein (MCP) Chemotaxis monocytes of Induction ofrespiratory burst Induction ofadhesion molecules Induction of IL-1 Anti-inf lannnatory Competitive inhibition IL-1 of Cytokines : Interleukin-l Receptor type I and 11 receptors Antagonist (IL-lra, I M P )
complement proteins; cellular adhesion molecules; and the potent procoagulant tissue factor. Inducible adhesion molecules on vascular endothelium function to localize various classes of leukocytes to sites of inflammation. However, when the endothelium is inappropriately activated, this may contribute to organ damage. Endothelial leukocyte adhesion molecule-l (ELAM-1) serves to bind neutrophils through the carbohydrate ligand sialyl Lewis-x. Intercellular adhesion molecule-l (ICAM-1) will bind monocytes and neutrophils through the ligands LFA-1 (CDlla/CD18) and Mac-l (CDllb/CD18). Both of these molecules are not present on unstimulated endothelial cells, but may be induced in response to several stimuli, including IL-1 and TNF.4 In many ways, tumor necrosis factor has similar activities to IL-l. TNF is also a potent pyrogen, as demonstrated by
322
DAVENPORT
clinical human trials.6 TNF is similar to IL-1 in the ability toenhanceB-cellproliferation,generationofantibody secreting cells, and T-cell proliferation in concert with mitogen stimulation. IL-1 and TNF display synergy in the shock response, in that the lethal dose in experimental animals is an order of magnitude less when they are administered together as compared to either one given alone. The effect of TNF on vascular endothelial cells is also similar to IL-1, in that it will induce the expression of adhesion molecules and chemotactic cytokines. 8 TNF appears to be particularly potent in altering the balance of coagulation and fibrinolysis to favor clotting. Normal human subjects given an intravenous injection of TNF demonstrate activation of the extrinsic system of coagulation. 9 This may be due largely to the induction of tissue factor expression, which is the ligand for factor VII, on monocytes and endothelial cells by TNF. In addition, TNF will cause the internalization of thrombomodulin by endothelial cells. Thrombomodulin, when it binds thrombin, C which, in turn, inactivates is a potent activator of protein factors VIIIa and Va. Interleukin-6 is similarly produced by phagocytes in responsetobothimmuneandnon-immunechallenges.l1In adddition to beinga pyrogen, similar to IL-1 and TNF, IL-6 is involved in several stages of B-cell development, in that it stimulates both proliferation and differentiation. Antibody production by differentiated B-cells is enhanced by addition of IL-6 and markedly reduced by neutralizing antibodies to IL6. IL-6 playsa role in responses to red cell antigens, since both primary and secondary antibody responses to sheep red cells by mice are significantly enhanced by IL-6, both in vivo and in vitro.12 IL-6 is necessary for the growth of many hybridoma cell lines, a fact that has been used for bioassays. The actions of IL-6 on T-cells are not as well characterized as those on B-cells. IL-6 will stimulate the proliferation of mature T-cells in concert with T-cell receptor ligation and, in large measure, can replace the function of mononuclear phagocytes as an accessory signal for T-cell activation. IL-1 potentiates this response, not only by inducing IL-6 production but, also, by increasing the responsiveness of T-
CYTOKINES AND HEMOLYTIC TRANSFUSION REACTIONS
323
cells to IL-6. The acute phase protein response of the liver, in which the synthesis of certain proteins is increased, including fibrinogen, complement factors, and C-reactive protein, while others such as albumin are reduced, appears to be due principally to IL-6. Chemokinee (IL-8, MCP) : A second class of cytokines that may be involved in hemo-
lytic transfusion reactions has been termed chemokines. These are produced by mononuclear phagocytes similar to the proinflammatory cytokines, but have a much more restricted range of target cells, usually one or two cell types, for which they are potent chemotactic factors. Interleukin-8 (IL-8) is representative of this class, in that it is primarily a chemotactic and activating factor for neutrophils in the PM-nM range. 13 IL-8 also is a chemotactic factor for T-lymphocytes and, at greater concentrations, for endothelial cells. At higher doses than are required for chemotaxis, IL-8 will stimulate neutrophils to degranulate and produce reactive oxygen metabolites. IL-8 also is chemotactic for basophils and will stimulate the release of histamine. Monocyte chemoattractant protein (MCP) is similar to IL-8, in that it is produced by mononuclear phagocytes in response to similar ~timu1i.l~ However, MCP appears to be restricted to monocytes, for which it is a chemotactic factor and will induce the respiratory burst. Recently, it has been shown that MCP is sufficient for the induction of adhesion molecule as IL-1 and IL-6 production by monocytes. l5 expression as well Because IL-1, subsequently, will stimulate MCP gene expression by the same cells, this may represent a potential positive feedback loop by which an initial pathologic stimulus may be amplified many-fold. MCP will cause the cell surface expression of CDllb and CDllc, the alpha subunits of the integrin molecules Mac-l and p150/95 on monocytes. These moleculesformdimerswithCD18andmediatebindingto stimulated endothelial cells. Anti-inflanuuatory cytokines (IL-lra):
The third class of cytokines for which there is current evidence of involvement in hemolytic transfusion reactions may
324
DAVENPORT
beconsideredasanti-inflammatory.Interleukin-lreceptor antagonist(IL-lraor I M P ) is a representativeofthis category. Like the other cytokines we have considered, IL-lra is produced by mononuclear phagocytes.16 However, IL-lra is quite different, in that it appears to have no biological activity in and of itself. Rather, IL-lra isa competitive antagonist of IL-1 binding to type I and type I1 receptors. ILi n v i v o and i n v i t r o effects of IL-1. Thus, Ira will block the IL-lra may prevent or down-regulate cellular activation events mediated by IL-1 in human disease states, including transfusion reactions. Our knowledge of the production of cytokines in immune hemolysis comes largely from i n v i t r o models. The production of pro-inflammatory cytokines and chemokines has been demonstrated using a model of AB0 incompatibility in which packed RBC are incubated with fresh heparinized whole blood 1). (Fig. TNF appears rapidly in plasma, with peak TNF bioactivity, as WEHI-l64 assay, occurring at 2-4 hours determined by the following addition of red cells. l7 24 By hours of incubation, plasma TNF levels have returned to baseline. There ais dosedependent relationship of TNF with the concentration of red cells, in that TNF levels parallel the degree of hemolysis. AB0 TNF gene expression in buffy coat cells is induced by incompatible red cells, and appears to be superinducible by the protein synthesis inhibitor cycloheximide, suggesting that thegeneisunderthecontrolofshort-livedrepressor proteins, as has been demonstrated for other stimuli. The chemokines IL-8 and MCP also are generated in whole blood in response to incompatible red cells. 18' l9 These a somewhat delayed time course relative to TNF, in that significant levels of IL-8 are not detected for 4 hours and MCP is not detectable in the first 6 hours following addition 24 hours, both chemokines are of red cells. However, by present in the plasma in substantial amounts. Cytokine production in whole blood in response to ABO-incompatible red cells in this model requires a heat labile plasma factor, most likely active complement. when blood is reconstituted from washed cells and heat-inactivated plasma,
follo
325
CYTOKINES AND HEMOLYTIC TRANSFUSION REACTIONS
3000
..........
"_
MF IL-8 MCP
2000
lo00
a 4
8
12
Incubation Time
16
20
24
(hours)
FIGURE 1.
Temporalcourseofcytokineproductioninwholeblood followingadditionof-0-incompatibleredcells.Plasma levels of TNF,IL-8, and MCP are shown.
both the hemolysis of incompatible red cells and cytokine production are abolished, although such blood retains the ability to agglutinate -0-incompatible red cells. Cytokine production in reconstituted whole blood still occurs in response to endotoxin. Thus, heat treatment of plasma does not non-immune stimulus. prevent leukocytes from responding a to While there is little clinical evidence to date for the a involvement of cytokines in hemolytic transfusion reactions, recent case report has shown TNF production in a group 0 patient who inadvertently received 100 m1 of group A red
326
DAVENPORT
cells.20 This patient was ona protocol in which TNF levels and neutrophil elastase were measured as part of a study of physiologic responses to cardiopulmonary bypass. Both of these became elevated following the incompatible blood transfusion. The time course of TNF appearance in plasma was similar to that observed in v i t r o . The release of neutrophil elastase in this patient increased progressively over 24 hours, as might expected be IL-8, if which can induce neutrophil degranulation, were produced in circulation, as suggested by the whole blood model. The authors also stated that no other patient they had studied showeda significant rise in plasma TNF levels in the course of surgery. Thus, it appears that TNF production in the setting AB0 of incompatibility is not simply a laboratory phenomenon. As with the case of AI30 incompatibility, the evidence for cytokine production in IgG-mediated red cell incompatibility . There appear to be come mainly fromin v i t r o models (Fig. 2) two categories of cytokine responses in this case. High level responses include IL-8, MCP and IL-lra which reach concentrations in the medium 1 of ng/ml or more by 24 hours of incubation. Low level responses include IL-lp, IL-6 and TNF that are in the pg/ml range. We have found that IL-lp is detectable in the culture medium in a progressively increasing fashion in response to anti-D coated red cells.21 TNF is not significantly elevated in the supernatant, except at 6 hours. However, cell-associated TNF can be demonstrated by immunocytochemical staining in monocytes engaged in erythrophagocytosis. These findings suggest that one possible reason fortheclinicaldifferencesbetweenacuteanddelayed hemolytic transfusion reactions may be that, in the former case, TNF is released into systemic circulation where it can have diverse effects on many cell types, whereas in the latter case TNF effects may be confined to local effects at the site of erythrophagocytosis, primarily the spleen. IL-6 production by monocytes also occurs in response to IgG-coated red cells. Since IL-1 and IL-6 are B-cell growth and differentiation factors, the production of these two cytokines may promote the production of red cell allo- and auto-antibodies that are associatedwithdelayedhemolytictransfusionreactions.
327
CYTOKINES AND HEMOLYTIC TRANSFUSION REACTIONS
..........
00
Low level responses Hlgh level responses
I
4
8
l
I
I
1
12
16
20
24
Incubation Time (Hours)
Similar to the case of AB0 incompatibility, IL-8 and MCP are in the produced in a progressive fashion reaching high levels culture medium. There is some controversy, however, surrounding whether TNF, IL-1, or both are produced by monocytes in response to IgGcoated red cells. Others have found that TNF, but I L - 1 ,not is produced by monocytes in response to either Rh(D) or coatedredcells.22Onepossibleexplanationforthis discrepancy is that monocytes used in this study were adherent toglassslides,whereaswehadusedperipheralblood
ma-
328
DAVENPORT
mononuclear cells incubated in Teflon chambers to which they do not adhere. The adherence of monocytes to foreign surfaces may be an activating event that can stimulate mRNA expression forTNFandIL-8 .23*24 However,sincethebiological activities of IL-1 and TNF are so closely related, and each may stimulate gene expression of the other in mononuclear phagocytes, it may not matter which of these pro-inflammatory cytokinesareproducedfirst in vivo duringhemolytic transfusion reactions. Production of cytokines by monocytes in this model appears to be Primarily the result FcpI of interactions with red cell bound IgG, in that it can be inhibited by soluble human IgG but not by Fab fragments of monoclonal antibodyIV.3 that is specific for the FcWII receptor. This correlates with evidence that erythrophagocytosis by monocytes is mediated by F C ~ R I .This ~ ~ observation leads to something of a paradox, however, since at normal levels of immunoglobulins in circulation, the monocyte FcpI is most likely occupied by nonspecific monomeric IgG.26 Perhaps this explains why it is so unusual to see erythrophagocytosis on peripheral blood smears or from patients with delayed hemolytic transfusion reactions autoimmune hemolytic anemia due to IgG antibodies that do not fix complement despite, large numbers of IgG coated red cells in circulation. An
interesting feature of IgG-mediated hemolysis is the production of the IL-1 inhibitor IL-lra.27 Significant levels of IL-lra appear in the culture supernatant in a parallel fashion to that on IL-1. Immunocytochemical stains demonstrate strong reactivity for IL-lra in monocytes engaged in erythrophagocytosis. Northern blot analysis of mononuclear cell RNA shows that IL-1 gene expression precedes that of ILIra in response to IgG coated red cells. However, neutralizing antibodies to IL-1 do not suppress either IL-lra or IL-lP gene expression in this setting. Therefore, it appears that IL-lra production is a primary response to the IgG-coated red cell stimulus, rather than an autocrine phenomenon induced by initial IL-1 production. Treatment of mononuclear cells with thesteroiddexamethasoneinhibitsIL-lraproductionin response to IgG-coated red cells. These data suggest the
CYTOKINES AND HEMOLYTIC TRANSFUSION REACTIONS
329
possibility that the marked clinical variability of delayed hemolytic transfusion reactions, as well as autoimmune hemolytic anemia, may be accounted for, in part, by the relative balance of IL-1 and IL-lra production. Mediators that are produced during hemolytic reactions may have effects on many cell types. One cell likely to be closely involved in these reactions is the endothelial cell. Because the endothelium lines the entire vasculature, CytOkineS in systemic circulation will have immediate access to these cells. Under normal circumstances, the endothelium provides a semipermeable barrier and an anticoagulant surface that are essential to maintaining the microenvironment of circulation. However, under the influences of IL-1 and TNF, endothelial cells will express leukocyte adhesion molecules and chemotactic factors, and alter their surface characteristics to favor thrombosis. Since substantial amounts of TNF are produced in whole blood in response to AB0 incompatible red cells, it seems likely that this pro-inflammatory cytokine may have a central role in vascular pathology of acute hemolytic 3. When cultured transfusion reactions, as represented in Fig. human umbilical vein endothelial cells are stimulated with conditioned plasma derived from whole blood incubatedAB0with incompatibleredcells,theywillexpressimmunologically (R. D.Davenport, reactiveICA"1andELAM-lmolecules unpublished data). In addition, these cells will elaborate the leukocyte chemotactic factors IL-8 and MCP into the culture medium.Northernblotanalysis of endothelialcell mRA indicates that gene expression for ICAM-1, ELAM-1, IL-8 and AB0 MCP are strongly induced by the conditioned plasma for incompatibility reactions. Control plasma from whole blood incubated with compatible red cell does not induce gene expression or protein production for any of these molecules. Thus, two of the necessary conditions for leukocyte infiltration of tissues are established in these reactions. The addition of neutralizing anti-TNFto the conditioned plasma at thetimeofendothelialcellstimulationwillcompletely abrogate the production of both adhesion molecules and chemotactic factors by the endothelial cells.
330
DAVENPORT
0
4
16
12
20
24
Incubation Time (hours)
FIGURE 3. Temporalcourseofendothelialcellresponsesto ABOincompatibility conditioned plasma. Procoagulant activity is determined by a one-stage recalcified plasma clotting time, and expressed as thromboplastin units relative to a rabbit brain thromboplastin standard. Expression of the leukocyte adhesion molecule ICAM-1 is determined by modified ELISA technique. Control plasma from whole blood incubated ABOwith compatible RBC fails to induce either procoagulant activity or ICA"1 expression.
Inaddition,culturedendothelialcellsincubatedwith AB0 incompatibility reactions will conditioned plasma from produce a procoagulant that initiates clotting through a factor VII-dependent mechanism (R. D. Davenport, unpublished data). This procoagulant activity can be blocked with specific mRNA antibodies to tissue factor. Analysis of tissue factor expression by quantitative polymerase chain reaction technique shows that gene expression increases approximately 100-fold in response to conditioned plasma. Similar to the case with adhesion molecule and cytokine induction, neutralizing antiTNF added to the stimulating medium will prevent induction of procoagulant activity. The expression of tissue factor by endothelial cells in the course of a hemolytic transfusion reactions could result in intravascular coagulation. In these
33 1
CYTOKINES AND HEMOLYTIC TRANSFUSION REACTIONS
various ways, the endothelial cell appears to be an active participant in the constellation of pathophysiologic events of hemolytic transfusion reactions. REFERENCES C. A. Dinarello. Blood 77, 1627-1652 (1991) L. D. Butler, N. K. Layman, R. L. Cain, et al. Clin. Immunol. Immunolpathol.5 3 , 400-421 (1989) 3. J. S. Pober. Am. J. Pathol.m, 426-433 (1988) 4. A. Mantovani, F. Bussolino,E. Dejana. FASEB J. 6, 25911. 2.
2599 (1992)
5. K. J. Tracey, A. Cerami. Crit. Care. Med. a, S415-S422 (1993)
6.
P. B. Chapman, T. J. Lester, SE. . Casper, et al. J. Clin. Oncol. 5, 1942-1951 (1987) 7. S . Okusawa, J. A. Gelfand, T. Ikejime, et al.J . Clin. Invest. U, 1162-1172 (1988) 8. Pober JS. 9. T. van der Poll, H. R. Buller, H. ten Cate, al. et N. Engl. J. Med. 3 2 2 , 1622-1627 (1990) 10. C. T. Esmon. ScienceU , 1348-1352 (1987) 11. S. Akira, T. Taga, T. Kishimoto. Adv. 1mmunol 5 4 , 1-78 (1993)
12
..F.
Takatsuki, A. Okano, C. Suzuki, et al. J. Immunol. 141,
3072- (1988) 13. C. A. Hebert, J. B. Baker. Cancer Invest. 11, 743-750 (1993) 14. K. Matsushima, C.G . Larsen, G. C. DuBois, et al.J. Exp. Med. 169, 1485-1490 (1989) 15. Y. Jiang, D. I. Beller, G . Frendl, et al.J. Immunol. 2423-2428 (1992) 16. W. P. Arend. Adv. Immunol.5 4 , 167-227 (1993) S. L. Kunkel. Br.J. 17. R. D. Davenport, R. M. Strieter, Haematol. 540-544 (1991) 18. R . D. Davenport, R. M. Strieter, T. J. Standiford, et Blood 2 4 , 2439-2442 (1990) 19. R. D. Davenport,M. D. Burdick, R.M. Strieter, et al. Transfusion. 3 4 , 16-19 (1994) 20. J. Butler, D. Parker, R. Pillai, et al. J. Br. Haematol. 2 2 , 525-526 (1991) 21. R. D. Davenport, M. Burdick, S. A. Moore, et al. Transfusion U, 19-24 (1993) 22. M. Hoffman. Vox Sang 184-187 (1991) 23. S. Haskill, C. Johnson, D. Eierman, et al. Imunol. J. 1690-1694 (1988) 24. K. Kasahara, R. M. Strieter, S. W. Chensue, et al.J. Leukoc. Biol. 287-295 (1991) 25. S. J. Ruegg, T.W. Jungi. Immunology 513-520 (1988) 26. R. G. Q. Leslie. Biochem. Soc. Trans.12, 743-746 (1984) 27. R. D. Davenport, M. D. Burdick,R. M. Stricter, et al. Transfusion 3 4 , 297-303 (1994)
m,
x,
m,
m,
a,
a,
al.
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THE ROLE
OF CYTOKINES AND ADHESIVE MOLECULES IN NON-HEMOLYTIC TRANSFUSION REACTIONS
FEBRILE
Edward L. Snyder, M.D. Professor of Laboratory Medicine Yale University School of Medicine Director of Blood Transfusion Service Yale-New Haven Hospital 06504 New Haven, CT ABSTRACT
Febrile non-hemolytic transfusion reactions occur not infrequently following transfusion. Our understandingof the molecular biology of these reactions has increased dramatically over the past few years. A variety of biological response modifiers have been shown to play a role in these reactions. These chemical messengers include cytokines, complement fragments, antibodies and adhesion molecules. Many of the clinical symptoms associated with these reactions are attributable to activation and generation of these substance This review article will cover the role of cytokines in generation of nonhemolytic febrile transfusion reactions and the role of activation of adhesion molecules in the generation of TRALI (non-cardiogenic pulmonary of these chemical messengers edema). Our ability to modulate the generation could help us control clinical symptoms associated with these transfusion reactions. ROLE
OF
CYTOKINES
The incidence of febrile reactions is believed to be one percent in the overall patient population and ten percent in chronically transfused individuals, with an estimated frequency of 0.5% /unit transfused(1). Those individuals who are at greatest risk for febrile reactions are multiply transfused patients and multiparous women. When a non-hemolytic transfusion reaction begins, there is a lag time between the onset of chills and other symptoms and the peak body temperature attained. Furthermore, the patient may remain febrile long after he/she has begun to better, feel clinically. This clinical symptom complexis believed to be due to the time needed to synthesize the chemical messengers required to stimulate the hypothalamic thermogenic response and then to the time needed to remove these and other biological response modifiers (BRM), including cytokines and complement factors, from the circulation (2-5).
These BRM produce many of the symptoms
333
334
SNYDER
of fever, chills, pain, dyspnea, and vasomotor instability seen during (F'NHTR). febrile, non-hemolytic transfusion reactions
A study by Mangano, et al., ( 6 ) concluded that a third generation leukocyte depletionfilterwasnotcompletelyeffectiveinpreventingfebrile transfusion reactions. This result is most explicable if one considers the role
of
cytokines
in
the
pathogenesis
of
non-hemolytic
transfusion
reactio
Cytokinesarechemicalmessengersinvolvedincellularactivation, proliferation,differentiation,chemotaxisandphagocytosis.Thefirst cytokine which was found to be involved in febrile reactions was called endogenous pyrogen, now known as interleukin-l (2,5). (IL-1) Sources ofIL-1 include activated macrophages, T, B and K cells and activated vascular endothelium. In addition, cytokines can up-regulate or down-regulate other cytokines so that
there
is
a
feedback
an effect on another. Cytokines such as
mechanism
whereby
one
cytokine
can
have
IL-1 produce various changes in
different organs, such as generation of a thermogenic response in brain tissue, activation of lymphocytes, resorption of bone and activation of the endothelium. Since macrophages do not normally store or express
IL-1, it
must be synthesized following cellular activation which occurs following exposure
to
microorganisms,
antigen-antibody
complexes,
phagocytic
challenge,
toxins or complement activation. Monocytes
or
phagocytes
release IL-1,which
binds
activated to
by
receptors
ingestion in
the
of
bacteria,
hypothalamic
are
stimulated
regulatory
center.
This, in turn, results in production of prostaglandinE,. PGE, raises the patients
thermostatic
set
point
and
produces
a
fever.
This
mechanism
of
PGE,
production utilizes a pathway which includes arachidonic acid metabolism involving cyclooxygenase. Aspirin (acetylsalicylic acid) inhibits cyclooxygenase.Thisisthemechanismwherebyaspirinproduces
an
antipyretic effect. The aspirin interferes with arachidonic acid metabolism andinterruptstheproductionofPGE,,thethermogenicmolecule.The differential diagnosis of febrile non-hemolytic reactions include hemolytic reactions, bacterial contamination of the blood component, leukocyte-related febrile non-hemolytic reactions and drug-induced fevers. All of these processes involve inflammatory reactions and differentiation among them can be quite difficult. Other cytokines that are involved in non-hemolytic reactions includeIL-8 which is produced from monocytes, endothelial cells, and several other cell types ( 7 ) .
Cytokines
can
be
generated
by
recipient
leukocytes vivo, in post-
335
FEBRILE NON-HEMOLYTIC TRANSFUSION REACTIONS transfusion vitro
from
during
donor
leukocytes
component
in
vivo,
or
by
donor
leukocytes
present
i
storage.
A recent paper by Stack and Snyder (8) showed that IL-8 levels increase in stored platelet concentrates over time and that this rise is proportionate both to the number of white cells present in the concentrates and the duration
of
storage.
The
highest
levels of IL-8 produced
were
found
to
be
in
units of platelets that were stored for five days and had white counts of greater than 4,0OO/pL. Stack and Snyder( 8 ) also showed that if platelets were filtered at Day 0 to remove leukocytes, by Day5 , while the control group showed elevated levels IL-8, of the filtered group failed to generate detectablelevelsofcytokines,becauseofremovalofthecytokine( 9 ) the generation of generating white cells. This same group also showed
IL-8 in
red
blood
cell
units,
although
the
levels
were
much
lower,
likely
to the storage of red cells at refrigerated temperatures. While
studies
have
clearly
shown
that
cytokine
levels
are
increased
in
units of blood, whether there is a causal relationship between the presence of cytokines and the occurrence of febrile transfusion reactions is not obvious and has been the subject of several studies. Heddle et al. (10) studied
the
frequency
of
FNHTR
by
component
type
and
found
that
platelet
associated with a higher percentage of reactions, (30.8%) with random donor platelets, compared to 6.8% with red cell concentrates (P<0.0005).
Their
group also showed that older units of platelets and red cells, and those units
with
higher
white
counts,
are
more
likely
to
produce
febrile
reac
Muylle (11) studied the effect of storage time on the incidence of reactions to platelet transfusion. They found that platelets stored less than three
days, showed a reaction rate of 8.7%, while platelets stored for over three days, had a transfusion reaction rate of 17.6%. Another of Muylle’s studies revealed (12) that units of platelets given to those individuals who had febrile reactions, contained higher levels of TNF alphaIL-6, and than did platelet units given to patients whono had febrile reactions. Muylle et al. also showed (12,13) that the higher the white count, the greater the levels of TNF alpha, IL-1 p and IL-6. Also, the longer the storage time, the higher the levels. All
of these studies support the concept that cytokines can
indeed cause FNHTR due to the formationof cytokines, in vitro, prior to transfusion. Cole et a1.(14) showed that the association of cytokine generation in stored blood components was true for a variety of other cytokines including
SNYDER
336
macrophage inflammatory protein (MIP-l). Stack and Snyder (15) showed that units of platelets contaminated with
E-coli generated high levels of
cytokines, providing that the platelets were not leukoreduced prestorage. Removal of white cells produced a decrease in the production of cytokines, even
if
the
platelets
were
contaminated
with
bacteria.
While it is known that pre-storage leukodepletion will prevent cytokine generation
by
virtue
of
removal
of
the
white
cells canbefore synthesize they
these biological response modifiers, preliminary data from our laboratory showed on
levels of IL-8can
that
preliminary
data
filtered through a Pall
also be reduced
from
our
post-bedside
laboratory, IL-8 levels
in
filtration.
platelet
Based
concentrates
PXL-8 bedside leukodepletion filter, initially 450 pg/mL post-
decreased from about 5000 pg/mL prefiltration to about
filtration. The cytokines likely adhere to the filter media, either by electrostatic or some other, yet to be elucidated, binding mechanism. Similar findings have also been shown by Shimiuzu to be true for complement to
factors.(l6) The actual mechanism of how cytokines function
produce
their effects is known for someof the BRMs. Kagan (17) showed that TNFalpha actually forms a sodium channel that intercalates into the cell membrane;
the
channel
in
the
cell
membrane
leads
to
ion
fluxes
which
produc
cell destruction. ROLE OF ADHESION There
are
five
Ukiyama (18):
of adhesion sets
molecules
MOLECULES as
discussed
in
a
recent
review
by
cadherins, the immunoglobulin gene super family, integrins,
selectins and cell surface proteoglycans. Immunoglobulin gene super family membrane glycoproteins are involved in antigen recognition, inflammation, cell adherence, cell migration and cell-cell signaling. Major of this members group include ICA"1, ICA"2, LFA-2 and 3, MHC Class I and11, the T cell receptor,
andCD4, CD8.
Thus,
these
molecules
are
involved
in
many of aspects
biology. The ICAM molecules act as ligands to CDlla/CD18 which are involved in
cell
adhesion (18).
The integrin family, present on all human cells, links the intracellular cytoskeleton with the extra cellular matrix. Integrins are involved in leukocyte transmigration, platelet aggregation, and tissue repair. Integrin of molecules are composed of alpha and beta sub units. The most prominent
the integrins is CDlla/CD18 (also knows as LFA-1) which binds ICA"1 to and ICA"2.
CDllb/CD18 (also known as Mac-l and CR3) binds toICA"1 and G3bi.
NON-HEMOLYTIC FEBRILE TRANSFUSION REACTIONS
337
CDllc/CD18 (also known as p150,95) binds to fibrinogen and perhaps C3bi. These proteins are found on all leukocytes, monocytes and granulocytes. The well-knownmemberoftheintegrinfamily,GPIIb/IIIa,alsoknownas CD41/CD61, which is found on platelets binds fibrinogen, fibronectin, Von Willebrand factor, vitronectin and thrombospondin. There are a large number of other adhesive molecules in this family (18). The selectin family is found on white cells and endothelial cells and platelets (19-21). They regulate leukocyte migration, leucocyte binding to endothelium, binding inflamed to tissues and binding the to reticuloendothelial system. Members of this family include molecules known as
L-Selectins
which
are
found
on
lymphocytes
and
bind
to
saccharides
r
to sLea (20,21). P-Selectin,also known asCD-62 is found on platelets and endothelium and E-Selectin is found on endothelium. These selectins are involved
in
a
variety
of
cellular
biological
activities.
The
general
concep
is that a white cell will roll along the endothelium without sticking until it encounters an activated endothelial cell. At this point Selectins are generated. Once this occurs, the white cells slow down and begin rolling along
the
result
in
activated leukocyte
vascular sticking
endothelium. and
Integrins
transmigration
are
then
generated
which
through endothelialwall. the
This involves activation of receptors for CD11/CD18 and generation of ESelectin, P-Selectin, L-Selectins. These
molecules allow the activated
leukocytes to bind to the endothelium (19-21). This concept of activated white cells generating various adhesive molecules resulting in leukocyte transmigration is believed to play a major pathogenesis of TRALI (transfusion-related acute lung injury (22). In this condition,
there
is
migration of activated
granulocytes
into
the
role
in
interstitial
space between the alveolar epithelium and the capillary endothelium. This results in capillary leakage syndrome and non-cardiogenic pulmonary edema, also
known
as
TRALI. In addition, this same mechanism is believed to play a
role in the etiology
of
reperfusion injury
(19).
During organ
transplantation prior to revascularization or aortic cross-clamping tissue ischemia occurs. This ischemia activates the endothelial cells. When fresh blood
is
reperfused
through
the
of the vasculature, or after removal resultingentry
ischemic
organ as following such
reattachment
of the surgical cross clamp the
of granulocytesintotheactivatedareasresultsin
neutrophil activation and generation of the adhesive molecules described above.
Along
with
leukocyte
activation
release
of
neutrophilic
enzymes
o
338
SNYDER
This in turn, results in tissue destruction or injury, hence the term reperfusion injury (19).
It has been postulated that removing white cells during such surgery would serve a useful purpose in this regard, by preventing reperfusion injury. Removal of white cells does, indeed, prevent activation-induced damage. How these
studies
completely
would
clear
relate
since
it
to is
what
happens
difficult
to
clinically man, however, in render
a
human
is
not
patient
neutropen
for long periods of time. Studies performed using antibodies to CD11/CD18 have shown that animals treated with such antibodies are susceptible to overwhelming sepsis (23).
This occurs because the same adhesive molecules
are needed to inactivate bacteria in infected animal models. Adhesive
interactions
relate
to
the
concept
of
cell-cell
communication
among
platelets, leukocytes and endothelial cells. It is clear that cytokines play major roles in stimulation of febrile reactions and activation of white cells. This, in turn, plays a major role in non-hemolytic transfusion reactions as well as generation of a variety of adhesive molecules which allows the cells to localize on the tissue to produce of their many effects (24). All
of these cellular alterations normally occur during infection.
When they occur in response to transfusion responses, however, the response isoftenclinicallyinappropriateandundesirable(25).Methodsare currently being sought to ameliorate or modulate these adverse reactions to prevent further complications in transfusion recipients. References 1.
R.H. Walker, Am J Clin Pathology,8 8 , 374-378 (1987).
2.
C. Dinarello, Hospital Practice, 2 4 , 111-128 (1989).
3.
R. D. Davenport, R.M. Strieter, T. J . Standiford, and S . L. Kunkel, Blood, 2,2439-2442 (1990).
4.
R. D. Davenport, R. M. Strieter and Haematology, 18, 540-544 (1991).
5.
C. A. Dinarello, Blood,7 7 , 1627-1652 (1991).
6.
M. M. Mangano,L. A. Chambers, M.S . Kruskall, Am. J. Clin. Path, 95, 733-738 (1991).
7.
K. S . van Zee, L. E. DeForge, E. Fischer, etal, J. Immunol, 146, 34781482 (1991).
8.
G.
S.
Stack and E. L. Snyder, Transfusion,3
L. Kunkel, British J. of
4 ,
20-25 (1994).
NON-HEMOLYTIC FEBRILE TRANSFUSION REACTIONS
339
9.
G. Stack, L. Baril, P. Napychank, E. L. Snyder, Blood, 8 2
,
10.
N.M Heddle,L.N. Klama, L. Griffith, et al., Transfusion, 3 3 , 794-797 (1993).
11.
L. Muylle, E. Wouters, R. De Bock and M. E. Peetermans, Transfusion Medicine, 2, 289-293 (1992).
394a (1993).
12.
L. Muylle, M. Joos, E. Wouters, al, et Transfusion,2 , 195-199 (1993).
13.
L. Muylle, M. E. Peetermans, Vox Sang 66, 14-17 (1994).
14.
S . Cole, G. Stack, Blood, 8 2
15.
G. Stack,S . Cole, S . Campbell, E. Snyder, S . Edberg, Transfusion, 2, 50s, (1993)
,
S.
398a (1993).
Mizuno, et al, Vox Sang, 66, 161-165
16.
T. Shimizu, C. Uchigiri, (1994).
17.
B. L. Kagan, R. L. Baldwin, D. Munoz, B. J. Wisnieski, Science,2 5 5 , 1427-1430 (1992).
18.
Uchiyama andK. C. Anderson, Transfusion Medicine Reviews, U, 84-95 (1994) .
19.
K. Beinvenu and D. N. Granger, Blood Cells, 19, 279-289 (1993).
20.
U. H. von Andrian, J. D. Chambers, E. L. Berg, al, et Blood, 8 2 , 182191 (1993).
21.
D. V. Erbe, S . R. Watson,L. G. Presta, B. A. Wolitzky, Journal of Cell 1227-1235 (1993). Biology,
22.
6 4 , 1118-1132 (1989). D. W. Swank,S . B. Moore, Mayo Clin Proc,
23.
S.
24.
R. Busund,R-0. Lindsetmo, L-T. Rasmussen, etal, Arch Surg,126, 591597 (1991)
m,
R. Sharar, Surgery,110, 213-220, (1991).
m, 1037-1040 (1994).
25. P. A. Mackowiak, Annals of Internal Medicine,
This Page Intentionally Left Blank
NEONATAL ANEMIA: PATHOPHYSIOLOGY AND TREATMENT Ronald G.Strauss Departments of Pathology and Pediatrics University of Iowa College of Medicine DeGowin Blood Center, University of Iowa Hospitals& Clinics Iowa City, Iowa 52242
ABSTRACT All neonates experience a decline in circulatingblood red cell (RBC) massdue to diminished erythropoietin (EPO) levels. This effect is more pronounced in small, premature infants and can lead to severe anemia and need for RBC transfusions-particularly, if repeated phlebotomy is required to monitor acutely-ill neonates. Although optimal RBC transfusion therapy hasbeen a long-term challengefor neonatologists, the emergence of recombinant EPOas promising therapy for neonatal anemia is the majorissue for 1994. Accordingly, this reportfor the 12th International Convocation on Immunology (Transfusion Immunology and Medicine) will focus on this aspectof neonatal transfusion medicine. Although several controlled trials to evaluate EPO as therapy have been completed, definitive answers to all questions regarding efficacy and possible toxicity have not been provided. However, therapywith EPO plus iron and adequate nutrition is likely tobe proven effective for the relatively late anemiaof stable prematures. To date, EPO has not been shown, of acutely-ill, premature infants. convincingly, to alleviate the anemia present early in the life JNTRODUCTION Newborn infants, particularly prematures with birth weight < 1.3 kg, are given multiple red bloodcell (RBC) transfusions during thefust weeks of life. The mechanisms responsible varyat different periodsof time during early infancy. During the f i t 2-3 weeks of life, severe respiratory disease may predispose to repeated blood sampling for laboratory studies and, consequently, lead to replacement transfusions. During later weeks, the inability to adequately mountan effective erythropoietin(EPO) response to fallingRBC values (hematocrit or hemoglobin concentration) may lead to additional transfusions to treat the “anemia of prematurity.” Remarkable advances in diminishing the severity of acute respiratorydisease (e.g., maternal corticosteroid administration, surfactant therapy for premature infants and use of 341
STRAUSS
342
less damaging mechanical ventilators) have reduced the need, in some neonates,for early RBC transfusions. To illustrate,the pattern of RBC transfusion given to small premature neonates at the University of Iowais shown in TableI. Although not shown in Table I, most transfusions given currently are to very small infants. When the 31 infants studied during 1993 were subdivided, 94%of those with birth weight< 1.0 kg received RBC transfusions vs. only 27% of those weighing 1.0 to 1.3Itkg. is important to note that this reduction in RBC transfusions occurredwithout use of recombinant EP-a drug that actually shows greatest promise as a means to alleviate the later anemia of prematurity. The degree to which EPO will influence anemia early inlife the of severely ill prematures, who experience substantial phlebotomy blood losses, remains to be shown. PATHOPHYSIOLOGY OF NEONATAL ANEMIA in circulating RBC During the f i t weeks of life, all infants experience a decline volume (mass), generally expressed as hematocrit or blood hemoglobin concentration. This decline resultsboth from physiological factors (e.g., the rapid rate of growth and needfor a commensurate increasein RBC mass to accompany the increase in blood volume) and, in sick premature infants, from phlebotomy blood losses. In healthy term infants, the nadir hemoglobin value rarely falls belowg/dL 9 at an age of approximately 10-12 weeks (1). This decline occurs earlier and is more pronounced in premature infants, even in those without complicating illnesses,in whom the mean hemoglobin concentration falls to approximately8 g/dL in infants of 1.0 - 1.5 kg birth weight and to7 g/dL in infants < 1.0 kg (2,3). Because this postnatal drop in hemoglobin level is universal and is well tolerated in term infants, it is commonly referredto as the "physiologic" anemiaof infancy. However, in premature infants, this decline in hemoglobin maybe associated with abnormal clinical signs severe enough to prompt transfusions. Accordingly, the acceptance of this anemiaas a normal, benign event has been questioned (4.5). A key reason that the nadir hemoglobin values of premature infants are lower than those of term infants the is former group's relatively diminished EPO output in response to anemia (2,3,6-9). Although anemia provokes EPO production in premature infants, the levels achievedare lower than those observed in older persons with comparable degrees of anemia (8). When related quantitatively, rising EPO levels and falling blood hemoglobin concentrations correlateweakly in premature infants (9).Low EPO output, although expected as part of physiologic anemia, may limit compensation for anemia in the newborn due to "nonphysiological" mechanisms (i.e., RBC loss due to bleeding, hemolysis, etc.). Erythroid progenitor cellsin the blood (10) and bone marrow (1 1)of premature infants are quite responsive to EPO in vitro. Although erythroid colonies derived from fetal and adult subjects display different growth patterns in culture (12), the bulk of information available
343
NEONATAL ANEMIA
TABLE I RBC transfusions given to premature infants at Iowa
Infants49 < 1.5 kg % given RBCs Mean per 10.0 infant*
Range*
-> 4 Tx* -> 15Tx*
1-40
1985 53
ND 12.9
2-45
ND ND
deBe
m
1993
78
67
61
52
8.2
82%
26%
2-46 51% 14%
31** 3.8
1-12
37%
0%
* Only transfused infants considered in the calculations. ** Infants with birth weight < 1.3 kg studied in1993.
supports the hypothesis that it is the inadequate production of EPO, rather than an abnormal response of erythroid cellsto this growth factor, thatis the major causeof physiologic anemia. The mechanisms responsible for the diminished EPO output by premature neonates are undefined. One mechanismof apparent importanceis the belief that premature infants must relyon the liveras the primary siteof EPO production, during the first several weeks of life, ratherthan on kidney (13). A s established by animal studies, the sequence of EPO production in normal fetuses is liver followed by kidney. In lambs, EPO production by fetal liver begins to decrease after 120-130days of gestation (term= 145-150days), at which time renal EPO production increases (14). However, at term,70-75% of EPO producedin response to anemia continues be to produced by the liver.This dependencyon hepatic production of EPO is important because the liver is less sensitive to anemia and tissue hypoxia than is the kidney (14)--hence, the relatively poor EPO response in fetus theand neonate to the falling RBC mass. The switch to renal EPO production in islambs complete by about 40 days afterbirth. Viewed from a teleological perspective, decreased EPO production under conditionsof tissue hypoxemiain utero may offer an advantage to the fetus. If this were not the case, normal levels of fetal hypoxemia could stimulate high EPO output, with resulting polycythemia and hyperviscosity in utero. Following birth, however, diminished EPO responsiveness to tissue hypoxemia could result in ineffective erythropoietic compensation--not unlike that routinely observed as the physiologic anemia of infancy. As another mechanism possibly contributing to low EPO levels, plasma levels of EPO, undoubtedly,are influenced by metabolism (clearance) as well as production. Thus, pharmacokinetic studiesof EPO duringthe perinatal periodare likely to be important both for understanding the physiology involved and for designing successful therapeutic trials
344
STRAUSS
(i.e., optimal dose, schedule and route of administration) for useof recombinant EPO. Data in human infants (15,16) suggest that low plasma EPO levels may result from increased clearance and/or volume of distribution of this hormone in neonates relative to adults.In addition, pharmacokineticsof subcutaneous doses are variable to suggest erratic absorptiona fact that must be considered when evaluating EPO therapy studies. Physiological factors influencing EPO biology arecritical in the pathogenesisof neonatal anemia. In addition, a contributing practical factor is the needfor repeated blood sampling to monitor critically neonates. ill Small prematures, whoare the most critically ill, require the most frequent blood sampling and suffer the greatest proportional loss of RBCs because their circulating RBC volumes are small. Although attempts have been made to minimize sampling,to employ percutaneous monitoring devices and to miniaturize testing assays, themean volume of blood removedfor sampling has been reported to range from 0.8 to 3.1 mwkg per day during thefiist few weeksof life. When the volumes removedare expressed in termsof total RBCs lost, the quantities during an entire hospitalization range from 30% to more than300% of the total circulating RBC volume of neonatesat birth. Assuming a daily RBC loss of 0.8 m u g by phlebotomy, about2% of the total circulating RBC volumeof a neonate islost per day via blood sampling. Because the of diminished erythropoietic response to anemia, repeated losses of this magnitude could lead to severe anemia, unlessthe RBCs are replacedby transfusions. RECOMBINANT EPOTO TREAT NEONATAL ANEMIA Because infants mount an ineffectiveEPO response to anemia, the administration of recombinant EPO hasbeen touted as promising therapy (17-19). However, initial studies revealed mixed results, and several reasonable criticisms were obvious. Most early studies enrolled fairly small numbers of subjects, and only a few were of a randomized, placebocontrolled design. In addition, EPO administration (dose, schedule and route) were quite variable. Althoughthe explanations forEPO failure or success in the treatmentof neonatal anemia willbe established by further studies, key factors leading to failure in these early studies are likely related to less than optimal EPO administration (dose and schedule), to pharmacokinetic differencesbetween newborns and adults, and/orto inadequate availability of iron during erythropoiesis stimulated high to rates by EPO therapy. With respect to the last, some anemic adults with kidney disease respond to EPOifonly treated with parenteral iron (20). Except for one relatively recent study (21) in which intravenous iron was given, all other studiesof infants have employed oral iron. The belief that highdoses of EPO, administered aloneor combined with parenteraliron, will consistentlybe effective in managing neonatal anemia remains be to established. However, in supportof using larger therapeutic doses,high "pharmacologic" dosesof EPO have been effective when administered to patients with disease processes associated with anemia is that generally unresponsive to EPO, when it is givenin the lower more "physiologic"doses as used to treat
NEONATAL ANEMIA
345
anemic renal patients(22). In addition, high dosesof EPO have been found effective in increasing hemoglobin levelsin newborn rats (23). Although of limited valuein establishing the efficacy of EPO as treatment for neonatal anemia, the early reports of EPO therapy in neonates provided information of value for the designof later trials,and they will be briefly reviewed.The three reportsof Halperin et al. (24-26) employed historical controls and noted only marginal effects when infants were for one month at varying doses. In the most given EPO subcutaneously three times per week as 75 (N=3), 150 (N=6), 300 (N=5) recent report(26), 18 stable prematures were given EPO or 600 ( N 4 ) units/kg/dose starting at21-35 days of age. All infants were given oral iron (28 mg/kg/day) and vitamin E(5-20 mdday). Serum EPO increased with therapy, butthe effects of EPO were quite variable.A dose-dependent reticulocytosis occurred, compared to historical controls. Hematocrit values remained stable in EPO infants, whereas values fell in historical controls. Only threeof the 18 infants, those with low baseline hematocrits of 2123%.were given transfusions-presumably, fewer than expected per historical controls. Although all infantswere given oral iron, values of serum iron, transferrin saturation and femtin fell in mostinfants-exceptions were those giventhe larger doseof 7-8 mg/kg/day. Thus, ironmay not have been sufficient to sustain effective erythropoiesis. Blood platelet counts increased transiently 13 in infants, and neutrophil counts decreased modestly. < 0.5 x lo%, no infectious Although threeof 18 infants exhibited absolute neutrophil counts complications were noted. Peripheral blood was also studied for clonogenic hematopoietic precursors during EPO therapy, and granulocyte-macrophage colonies decreased by about 60%(from 845/mL to 355/mL). A s noted by the authors, interpretationof this study mustbe extremely cautious,as no concurrent control groupof infants was evaluated(26). In another early study, Beck et al. (27) conducted an uncontrolled trial in which stable, older premature infants were given weeklydoses of EPO intravenouslyat 10 (N=2), 25 (N=3), 50 (N=3), 100 ( N 4 ) and 200 ( N 4 ) unitskg per week. Hemoglobin levels rose, but without an EPO dose-dependent effect. Reticulocyte counts increaseddose at levels of 25,50 and 100 unitskg, but not at10 or 200 unitsfig. Neutropenia was common, as all but one infanthad transiently lowered neutrophil counts, with several patients exhibiting values below 1.0 x lO91L. No infections occurred.A mild increase in platelet count occurred,but it femtin fell, but not to was not relatedto EPO dose. Serum iron, transferrin saturation and iron deficient values--suggesting that EPO doses were too low to exert effect much on iron be drawn utilization via stimulationof erythropoiesis. Overall, definitive conclusions cannot fromthis study becauseof the lack of controls, the small number of subjects, use of varying EPO doses and the inconsistent responses observed. Studies in which control infants were evaluated concurrently to assess the role of recombinant EPO in the management of neonatal anemia are summarized in II. Table The first randomized, placebo-controlled trial of Shannon et al. (28) found no overall effects on hematocrit, transfusion requirements, overall reticulocyte count, calculated erythrocyte mass or rate of growth. However, infants with low phlebotomy losses (< 20 mL during the
STRAUSS
346
TABLE I1 CONTROLLED STUDIES OF EPO IN NEONATAL ANEMIA
l3xmQsG
Desicm
EPO Effects early reticulocytes none on transfusions
Infants Studied age 21 days, stable
Randomized 10s. 1oc*
2 times/wk (IV)
30
age 20 days, stable
Randomized 4s. 4 c
100-200 Ukg 5 times/wk (SC)
higher reticulocytes higher hematocrits fewer (?)transfusions
31
age 24 days, stable
Randomized 77s. 80C
100-200 u/kg 5 times/wk (SC)
higher reticulocytes higher hematocrits fewer transfusions
32
age 41 days, stable
Randomized 10s 9c
200 Ukg 3 times/wk (SC)
higher reticulocytes higher erythropoiesis fewer transfusions
21
age 2 days, not severelyill
Randomized 1lS, 11c
400 Ukg 3 times/wk (IV)
higher reticulocytes higher hematocrits fewer transfusions
33
age I7 days, not severely ill
Randomized 25s. 19C
150 Ukg 2 times/wk (SC)
higher reticulocytes fewer transfusions**
34
age 21 days, stable
Randomized 14s. 15C
100 u/kg 3 times/wk (SC)
higher reticulocytes higher hematocrits fewer (?)transfusions
35
age 10 days, mixed severity
Nonrandomized 100-300 Ukg 31S, 20C 3 times/wk (SC)
higher reticulocytes higher hematocrits fewer (?) transfusions
! M A
28
~~
*
~
~~
~~
100 u/kg
~~~~~~~
~~
~
S = study, C = control (e.g., 10 study, 10 control infants).
** Fewer
transfusionsin uncomplicated infants, but not in complicated ones.
NEONATAL
ANEMIA 347
study) may have benefited,as none of four treated with EPO needed transfusions compared to three of five controls. Another possibleEPO effect wasthe more rapid riseof reticulocytes in EPOvs. control infants. Serum EPO levels and blood platelet, leukocyte and neutrophil counts did not differ in EPO vs. placebo infants. The lack of clinically important benefits was likelydue to the low dose of EPO given and the probable Occurrence of iron deficiency. Although all20 infants were prescribedoral iron (3 mg/kg/day), six didnot receive complete therapy. Additional information was provided about these20 infants in another report(29). The findings in this initial study led to a small randomized pilot study in which eight infants were given either EPO subcutaneously as 100-200 unitskglday or placebo, five days per week for six weeks(30). All infants were given oral iron as6 mg/kg/day. Although the number of subjects was quite small, EPO infants exhibited higher reticulocyte counts and hematocrits than did controls. A benefit on transfusion requirements possibly occurred, but the number of subjects was too small for meaningful statistical analysis. This pilot study (30) led to a multicenter, randomized, placebo-controlledfortrial which only preliminary data are available at this time(31). Although the data support the efficacy of EPO in treating the anemia present in older, stable infants, the findings are surprisingly (31). modest At least one RBC transfusion was given to57% of EPO infantsvs. 69%of controls. The mean number of RBC transfusions given was 1.1 for EPO vs. 1.6 for control infants, and the mean volume of RBCs given was 16.4 mL for EPO vs. 23.9 mL for controls. Clearly, EPO did not eliminate the need for RBC transfusions-even in older, stable infants in whom phlebotomy blood losses were minimal. Ohls and Christensen(32) randomized stable premature infants with anemia to receive either EPO or RBC transfusions given to raise the hematocrit initially to 2 40%. The transfused infants,in a sense, served as controls, and they were transfused again if the hematocrit fell toI30%and signs of anemia were present. Recipients of EPO received oral iron (2 mg/kg/day) and 25 units of vitamin E. It was not stated whether transfused infants also received these nutrients. Infants receiving EPO showed increases in serum erythropoietin, hematocrit, reticulocyte count and bone marrow erythroid activity compared to transfused infants--findings not unexpected, since the RBC transfusions as given were likely to suppress erythropoiesis (1,3,8). EPO infants did not require transfusions during the study. Mean blood neutrophil counts were significantly lower in EPO infants than controls (1.8 vs. 3.9 x lO9/L), but only one infant in each group fell below 1.0 x 109/L. EPO increased CFU-E in the marrow, but BFU-E, CFU-GM and CFU-MIX were different no in EPO vs. transfused infants. Although EPO seemed to exert effects on marrow erythropoiesis, the findingsare difficult to apply broadly to infants encountered in practice because control subjects were transfused to fairly high hematocrit levels--a situation that may have rendered them somewhat transfusion-dependent due to marrow suppression. It would have been interesting to document the transfusion requirements and erythropoietic responseof a more comparable groupof control infants(e.g., those given placeboplus iron and vitaminE and
348
STRAUSS
permitted to experience modest degrees of symptomatic anemia without being given RBc transfusions). me randomized trial of &nielli et a1 provides promising resultsin neonates studied shortly after birth (21). Study infants were given EPO and20 mgkg of iron intravenously from day two oflife until discharge. Use of intravenous ironis a unique feature of this study. Control prematures were given neither EPO nor, unfortunately, iron. Thus, two variables (EPO and iron) that might affect response were present rather than only one @Po). Infants were comparable, including phlebotomy losses. However, they were fairly large in size. with a mean birth weight slightly over 1.3 kg, and the criteria for transfusion were quite liberal, when compared to those used in the United States. Thus, theapplicabilityof the findings to neonatal transfusion practice might be debated. Nonetheless, the need for RBC transfusions was significantly decreasedin EPO versus control infants-the number of RBC transfusions was 0.8 vs. 3.1, and the volume ofRBCs transfused was 14 mLkg vs. 48 mL/kg. Blood neutrophil and platelet counts were unaffected. In the studyof Soubasi et a1 (33). attempts were made to identify factors in neonates that might predict a successful responseEPO. to Infants were randomized within the fxst week of life to either an EPO or a control group. There was no mention that control infants received a placebo, and the possibility of bias due to lack of blinding mustbe considered when reviewing results. Althougha llinfants were prescribed oral iron (3 mgflrglday beginning day15 of life), it could be discontinued at the discretion of the attending physician. All infants received vitaminE (5 units/day) and multivitamins. For analysis, infants were divided intotwo groups-"uncomplicated" or "complicated" with the latter definedas requiring mechanical ventilation for at least three days. Reticulocyte counts were increased by EPO, but hematocrits did not differ between EPO and control infants because they were less in maintained by RBC transfusions. The need for transfusions was significantly "uncomplicated" EPO infants versus controls (three of nine EPO infants received transfusions versus sixof seven controls), In contrast, therewas a high incidence of transfusions in all "complicated" infants, withno difference between EPO and control infants. No differences in platelet or leukocyte units were found between groups, and severe neutropenia (< 1 x 10%) was not observed. Thus,at the dose evaluated,EPO offered a benefit only to "uncomplicated" neonates, and not to those "complicated" by more severe illness-suggesting that EPOmay not solve the problem of early anemiain severely-ill neonates. Bechensteen et al (34) evaluated EPO instable premature infants,in whom unusual efforts were made to ensure optimal protein and iron balance.A randomized, but not placebo-controlled, studywas conductedat four centers. Breast milk feedings were supplemented with 9 of human breast milk protein, and ferrous fumarate (18 mg of elemental iron)was given daily. The dose oforal iron was doubled if the serum iron fell -c 90 WglaL (16 pmoVL)-a situation which occurred in26 of the 29 infants. After one week of therapy, reticulocyteand blood hemoglobin concentrations were significantly higher in the
NEONATAL
ANEMIA 349
EPO group. None of the 14 EPO infants were transfused with RBCs versusfour of the 15 controls. However,in the absenceof blinding to diminish bias, differences in transfusion practice are difficult to interpret. Neutropenia was not observed. Although modestdoses of EPO seemed to benefit stable premature infants, who were well nourished with protein and iron, it remains to be demonstrated whether acutely ill infants, studied earlier in life, will respond similarly. Messer et al. studied increasing dosesof EPO given to premature infants from age 10 days until approximately seven weeks (35). Control infants were those whose parents refused EPO therapy, but permitted thembetoobserved. The needfor RBC transfusions was judged by attending physicians, who were not blinded to therapy. Although the decline in hematocrit valueswith advancing age was slower in the EPO infants, the importance of the finding is difficult to judge because hematocrits were influenced by frequent RI3C transfusion+19% of EPO infantsvs. 45% of controls were transfused. Transient neutropenia (c 1.5 x 109/L) occurred in 61% of EPO and50% of controls (not significantly of EPO. The authors suggested different). Iron utilization increased with increasing doses who were relatively stable and that EPO wouldbe most effective for older premature infants, able to take large doses of oral iron (15 m&g/day). CONCLUSIONS In summary, EPO deficiency is a major mechanism responsible for neonatal anemia, and therapy for this disorder with recombinant EPOis promising-paxticularly for stable, older premature neonates. EPO may also have a role in the anemia that accompanies bronchopulmonary dysplasiain older infants(36). Although enticing, a great deal mustbe learned about the efficacy and potential toxicityof EPO beforeit is widely prescribedas standard therapy for the anemia of prematurity. Additional studiesare warranted with careful consideration of EPO dose, schedule, route of administration and use in combination with other hematopoietic growth factors. In addition, the full spectrum of potential toxicity must be defined before EPO is widely prescribed-in this regard, neutropenia and thrombocytopenia due to stem cell competition have been observed in conditions of EPOdriven hematopoiesis (37.38). It is anticipated that manyof these issues will be addressed by the final published reportof the multicenter study of Phibbs, Shannon et al. that shouldbe completed during 1994.
ACKNOWLEDGMENTS Supported in part by Research Career Development Award K04 HD00255, Transfusion Medicine Academic Award K07 HL01426 and ProgramProject Grant POlHL46925-01Al from the National Institutes of Health.
350
STRAUSS
.REFERENCES 1. 2.
3.
4.
5. 6.
7. 8.
9.
R. G. Straws, Vox Sang.,x, 1-9 (1986). P. R. Dallman, Ann. Rev. Med.,2,143-160 (1981). J. A. Stockman, Pediatr. Clin.North Am., 3,111-128 (1986). C. A. J. Wardrop,B. M. Holland, K. E. A. Beale, J. G. Jones and0. P. Gray, Arch. Dis. Child., 2,855-860 (1978). B. M. Holland, J.G. Jones andC. A. J. Wardrop, Hematol. Oncol. Clin. North Am., 1, 355-366 (1987).
m,
J. A. Stockman, III,J. F. Garcia andF. A. Oski., N.Eng. J. Med. 647-650 (1977). M. S. Brown, R. H. Phibbs, J. F. Gardia and P. R. Dallman, J. Pediatr.,1111,612-617 (1983).
J. A. Stockman, III, J. E. Graeber. D. A. Clark, K. McCellan, J.F. Garcia andR. E. Kavey, J. Pediatr., 786-792 (1984). M. S. Brown, J. F. Garcia, R. H. Phibbs and P. R. Dallman, J. Pediatr., 793-798
m,
m,
(1984).
10. K. M. Shannon, G. S. Naylor, J. C. Tordildson, G. K. Clemons, V. Schaffner, S. L.
Goldman, K. Lewis, P. Bryant andR. Phibbs, N.Engl. J. Med.,U,728-733 (1987). 11. S. M. Rhondeau, R. D. Christensen, M. P. Ross, G. Rothstein andM. A. Simmons, J. Pediatr., U ,935-940 (1988).
12. S. T. Holbrook, R. D. Christensen and G. Rothstein, Pediatr. Res.,24,605-608 (1988).
13. M. S. Brown, J. F. Garcia, R. H. Phibbs andP. R. Dallman, J. Pediatr.,M,793-798 (1984). 14. E. D. Zanjani. J. L. Ascensao, P. B. McGlave, M. Banisadre andR. C. Ash, J. Clin. Invest.,a.. 1183-1188 (1981).
15.
V.Ruth, J. A. Widness and K. 0. Raivio, J. Pediatr., U,950-954 (1990).
16. M. S. Brown, M. A. Jones, R. K. Ohls andR. D. Christensen, J. Pediatr., 622,655-657 (1993). 17. R. D. Christensen, Pediatr.,U,793-796 (1989).
18. J. A. Stockman, III, J. Pediatr., 112,906-908 (1988). 19. R. H. Phibbs, K. Shannon and W. C. Mentzer, in Erythropoietin: FromMolecula Structure to ClinicalADplication, Vol. 76.. C. A. Baldamus, L. Scigalla. L. Wieczzorek
and K. M. Koch, eds. Karger, Basel, (1989) pp. 324-329. 20. J. W. Eschbach, J.C. Egrie, M. R. Downing, J.K. Browne and J.W. Adamson, N. Engl. J. Med., U,73-78 (1987). 21. V. Carnielli, G. Montini, R. DaRiol, R. Call’Amico and F. Cantarutti, J. Pediatr., 121, 98-102 (1992).
22. H. Ludwig, E. Fritz, H. Kotzmann, P. Hocker, Gisslinger H. and U. Barnas, N. Engl. J.
Med., 322.1693-1699 (1990). 23. J. M. Koenig and R.D. Christensen, PediatrRes., 2,583-587 (1990).
NEONATAL ANEMIA
24
351
29.
D. S. Halpern, P. Wacher, G. Lacourt,M. Felix, J. F. Babel, M. Aapro andM. Wyss, J. Pediatr., 116.779-786 (1990). D. S. Halperin. Amer. J. Pediatr. Hematol. Oncol.,U,351-363 (1991). D. S. Halperin, M. Felix, P. Wacker, G. Lacourt, J. F. Babel and M. Wyss, Eur. J. Pediatr., 651,661-667 (1992). D.Beck, E. Masserey, M. Meyer andA. Calame, Eur.J. Pediatr., 661-667 (1992). K.E. Shannon, W. C. Mentzer, R. I. Abels, P. Freeman,N. Newton, D. Thompson, S. Sniderman, R. Ballard and R. H. Phibbs,J. Pediatr., .U!, 949-955 (1991). R. H. Phibbs, K. M. Shannon and W. C. Mentzer. ACTA Haematol., 87 &pp1 11.28-
30.
K.M. Shannon, W. C. Mentzer, R. I. Abels, M. Wertz, J. Thayer-Moriyama, W. Y.Li
25.
26. 27.
28.
m,
33 (1992).
and et al., J. Pediatr.,120,586-592 (1992). (1994). 31. R. H. Phibbs and J. F. Keith, Pediatr. Res., in press (abstract) 32. R. K. Ohls andR. D. Christensen, J. Pediatr., 119,781-788 (1991).
33. V. Soubasi, G. Kremenopoulos,E. Diamandi, C. Tsantali and D. Tsakiris, Pediatr. Res., 3,675-679 (1993). 34. A. G. Bechensteen, P. Haga, S. Halvorsen, A. Whitelaw, K. Liestol, J. Lindemann, M.
Hellebostad, 0. D. Saugstad, M. Grown, L. Daae, H. Refsum andE. Sundal., Arch. Dis. Child., 6 9 , 19-23 (1993). 519-523 (1993. 35. J. Messer, J. Haddad, L. Donato, D. Astruc, J. Matis., Pediatrics, 36. R. K. Ohls, D. D. Hunter andR. D. Christensen, J. Pediatr.,D,996-1000 (1993). 37. R. D. Christensen, J.M. Koenig, D. H. Viakochil and G. Rothstein, Blood,24,817-822
a,
(1989).
38. G. R. Saade, K. J. Moise, Jr., J. A. Copel, M. A. Belfort andR. J. Carpenter, Jr.,Obstet. Gynecol., B, 987-991 (1993).
This Page Intentionally Left Blank
QUALITY OF PLATELET CONCENTRATES Joseph D. Sweeney, M.D.*, Stein Holme, Ph.D.
+, and Andrew Heaton, M.D. + +
ABSTRACT Despite the current emphasis in transfusion medicine on regulatory compliance and cost containment, there is continuing activityin quality improvementof blood products. Quality can be assessed by measuring both benefit and risk. High quality products are those in which the benefit is maximized and the risk minimized. Risk, in the context of platelet transfusions, is minimized by reducing infectious agents, sources of allergic reactions, and other factors likely to cause adverse reactions in recipients. Benefit can be better described as potency. Potency is the ability to produce a desired effect. For platelet concentrates, potency has both quantitative [platelet yield] and qualitative [platelet viability, survival, and function] components. There are many activities which may influence the potencyof the final transfused platelet product and these are summarized in Figure 1.It is helpful to review each step in order to assess the potential impacton the potency of the final transfused product. COLLECTION, TRANSPORTATION AND PREPARATION Scant attentionhas been paid to this phase of platelet manufacture. Clearly, the potency of the end product is limited by the platelet count of the donor (range 150-450 x 109/L) and the volume of blood drawn. This results in a potential yield of between 5 - 20 x 1OO ' platelets for a blood donation of450 f50 mLs whole blood, buton average this is approximately 1.2 10". x Subsequent processing steps will resultin a reduction in the platelet content of the intermediate products (to be discussed). Duration of the donor draw time could impact on product quality. Lengthy draws [arbitrarily greater thantwentyminutes]
are sometimesassociatedwith
inappropriate thrombin generation. Thrombin generation will cause platelet aggregation and clumping and greatly impair the quality of any subsequent intermediate products.(l) Post draw short term storage and may also be important. Previous work by Pietersz et al suggests that rapid cooling from the immediate post draw temperatureof approximately 35" C to 20' C is an appropriate step and should be performed as soon as possible after collection
+
* Blood Bank, The Miriam Hospital, Providence, RI 02906; American Red Cross, Norfolk, VA 23510; Irwin Memorial Blood Centers, San Francisco, CA 94118
++
353
354
SWEENEY, HOLME, AND HEATON
Activities Which Influence Potency Pre-storaae Leukodepletion Gamma Irradiation Pooling Viral Attenuation Additive Solutions
Apheresis Platelets I
Whole Blood
Post-storaqe Leukodepletion Gamma Irradiation Pooling Washing
I
I
I
I
I
Manufactured Product
Stored Product
Final Transfused Product
FIGURE 1 Activities which influence the potency product.
final transfused
of the intermediate products and the
of the whole blood unit.(2) The next step is whatis described as the "hold time", i.e., the time from sample collection until subsequent primary processing in product manufacture. Work by Holme et al indicated thathold times of between two and eight hours resultin essentially similar subsequent platelet yields in the manufactured product.(3) More recently, studies have been performed comparing eight andtwenty-fourhourholdperiodsinwhichessentiallyidentical platelet yields were obtained.(4) Although earlier literature suggested that platelet production should optimally occur early after platelet collection, current thinking would indicate that this may be detrimental.(5) More research may need to be done, however, in this area in order to optimize conditionsto improve the platelet yield of the manufactured product. Platelet Preparation The manufactureof platelet concentrates from whole blood donations can proceed through either of two routes. These are outlined in Figure 2. For all of current North American productionandmuchofEuropeanproduction,plateletconcentrates
are manufacturedfrom
platelet rich plasma (PRP) as an intermediate product. This product is subsequently subjected to a hard centrifugational step (hard spin) resulting in the production of a platelet concentrate and aplatelet
poor plasmaproduct.In
the pastfewyears,therehas
been interestin
QUALITY CONCENTRATES OF PLATELET
355
Collection -
Shipping End Processing
Transportation
r
8 hours
-
Whole Blood Donations (472 558 mLS) Red Cell Concentrate
Soft Spin
24 hours
3
Hard Spin
Hard Spin
4
United Slates
1
Soft Spin
Ipcl-b + lpppl
+
Pool
(PRP- PC)
4
l
Soft Spin
[ecl Europe (BC- PC)
Manufacture, or primary processing, protocols for platelet concentrate production.
manufacturing platelet concentrates using the buffy coat as an intermediate product. In this schema, the whole blood collection is initially subjected to a hard spin with three intermediate products. The buffy coat is then subjected to a
the generation of
soft spin, either as a single
buffy coat or as a pooled buffy coat, which results in the generation either of a single platelet concentrate or of a multiple pooled concentrate. The advantages of the buffy coat method are the generation of finalproducts with lowerwhitecellcontent
(6), potentiallyharvesting
subpopulations which are intrinsically more effective hemostatically (7),less activation (8) and amenability to automation. Several studies have addressed the influence of these different centrifugational steps on theyieldandquality
of the manufacturedproduct(Figure3).Differentcentrifugational
protocols may result in products withwidelydifferentyields.(9,10)Inaddition,damage
to
platelets as indicated by a r e l e a s e of either LDH implying cell membrane damage, or of pthromboglobulinreleaseindicatingreleaseof
granule contents, may occur with excessive
centrifugational force. (1 1,12,13) Optimization of the centrifugational conditionsafter the hold period, therefore, is an essential prerequisite in order to improve quality by maximizing yield and minimizing platelet injury. Another issue
is what is known as the "rest period", i.e., the
period from completion of the hard spin to re-suspension. The current suggestion is that this should be approximately one to two hours in order to achieve the best quality product.
s
SWEENEY, HOLME,AND HEATON
356
Collection
Shipping End Processing
jq-j$iK $ iJF U
Transportation
Integral
Brake Time
Soft Spin (PRP)
Hard Spin (Platelet Pellet)
0
9
" 1
" l
" 1
0
0 10 210321432543554765876987 98 9 Mmuter
Factors
l.Soft spin suboptimal - reduce PRP yield 2. Soft spin membrane damage - LDH release 3. Hard spin suboptimal - (capture 95% PRP platelets) 4. Hard spin granule release - PTG release 5. Process-induced platelet activation 6. Rest period from hard spin to resuspension
FIGURE 3 Schematicrepresentationofcentrifugationalprotocolsusingplateletrichplasma as an intermediate productfor platelet concentrate manufacture. Important variables impacting quality are listed.
With regard to the production of a manufactured product from an apheresis device, this differs from whole blood donation in that the collection, transportation, and manufacturing steps are essentially combined. Thus, the end product from the apheresis collection is essentially a manufactured product intended for in vitro storage (see Figure 4). The factors which influence the quality of the product are numerous, but are largely determined by the pre-donation platelet count of the donor, the volume of blood processed, the efficiency of the device, and devicespecific factors. In general, where parallel comparisons have been made of similar devices, products of similar potency havebeen reported.(l4) Recent data, however, would indicate that
certain devices may have advantagesin terms of improved platelet yield and lowered whitecell contamination.(l5) Verification of this would require multi-site studies in order to confirm an advantage for a specific type of device.
IN VITRO STORAGE The in vitro storage of platelets has been the major focal area of investigational effort aimed at improving the quality of platelet productsover the past two decades.(l6,17) In order
357
QUALITY OF PLATELET CONCENTRATES Apheresis Platelets Collection
Shipping ~
End Processing
Transportation
0
Factors
0
0 0
Pre-donation platelet count Volume of blood processed Efficiency of device Device-specific factors FIGURE 4
Theprocessofcollectionandmanufacture are concurrentinplatelets,pheresis. variables influencing the harvest quality are listed.
to appreciate the concepts related to the storage of platelets,
important
it is essential to understand the
metabolic aspectsof platelets during in vitro storage.(l8) These metabolic aspectsare outlined in Figure 5. Platelets differ fromred cells inhaving mitochondria,andtherefore,generate carbon dioxide from the TCA cycle and consume oxygen during
active oxidative
phosphorylation.Althoughplatelets
there is no clear
are storedin aglucoserichmedium,
consensus as to whether the presence of glucose is essential for the stored platelet. There is considerable evidence that glucose is not metabolized through respiration, but that platelets preferentially metabolize other metabolic fuels such as fatty acids or amino acids.(l9) The glucose rich environment may, in fact, be detrimental in thatit may promote an active glycolytic pathway resulting in the production of excess lactic acid. This may lower extracellular pH if insufficient buffer is present. The absence of oxygen in the mediumwhich can occur when certaintypes of containers[to be discussed] are usedwill also enhancetheglycolytic rate through the Pasteur effect. Generation of carbon dioxide from the TCA cycle will also add to the hydrogen ionload, unless the carbon dioxidecan diffuse from the container. There has been recent interestin the addition of sodium acetateas a metabolic fue1,(19,20,21) and somein vitro data indicating that it diverts ATP generation from the glycolytic pathway toward oxidative phosphorylation, thus slowingdown lactate formation. This may allow for longer term in vitro platelet storage with acceptable preservation of quality.(22) Investigation effort has emphasized three aspects of in vitro storage of platelets: 1) The physical conditions, 2) Containers, and 3) The liquid environment.
358
SWEENEY, HOLME, AND HEATON ~
Collection Transportation
Shipping End Processing
Manufacture
Metabolism
FIGURE 5
Majorsuggestedaspectsofenergymetabolism during the in vitro storage of plateletsin autologous CPD or CP2D plasma or in acetate containing additive solutions.
With regard to the physical conditions, the major interest has beenin the temperature of storage and the question of platelet agitation. Although platelet concentrates
were originally
stored at the same temperature as red cells, i.e., between 1and 60, this fell out of favor when it was recognized that a higher temperature storage (in the region of 20 - 249, appeared more appropriate for the long term in vitro storage.(23) The advantages of the 4' storage were those of lesser bacterial growth, better preservation of pH and better in vitro aggregation responses. However, in vivo viability was greatly Curtailed.(24,25)
The optimal temperature for storage
seems to be within a fairly narrow corridor between 20 and 24'.(26)
Temperatures of lessthan
20' for any duration may be detrimental depending on the actual temperature and duration of
exposure. Temperatures of 18O - 2OoC for up to 24 hours
may be reasonably well tolerated,
whereas temperatures in the range of 12' for a relatively short period of time, i.e., less than 8 hours, may result in considerable compromise of platelet function.(27) Although this data has been generated from immobilein vitro conditions, it has largely been extrapolatedas simulating conditions associated with the shipping to, and storage at, the site of transfusion. The mode of agitation of platelet has beenof
interest for manyyears.
agitationhas been to facilitategasexchange.
The major rationale for platelet
It is unclear,however,whethermetabolite
359
QUALITY CONCENTRATES OF PLATELET
diffusion andlor maintaining platelets in a mobile liquid milieu is advantageous overall for the platelet. There is, however, much less data tosupportthe latter possibilities.Interestingly, incompatibilitybetweendifferentcontainersanddifferentagitatorshasbeendemonstrated indicatingthat the area of plateletagitation may be morecomplicatedthanwasoriginally thought.(28) Short term discontinuation (<24 hrs) may not be deleterious (29). The container in which the platelet is stored has significant implications for the final quality of the product depending on this duration of storage.(3O) were essentially polyvinyl chloride (PVC) containers
First generation containers
which were plasticized with
diethylhexylphthalate (DEHP). This containerhad previously been found useful for the in vitro storage of red cells at lower temperatures. It was soon realized, however, that this container was unsuitablefor gas exchange, and allowed onlyfor three day plateletstorage only at 20 - 24' C.With
the advent ofsecondgenerationcontainers,extended
storage of plateletsbecame
feasible with good preservation of quality. (31-33) A number of containers became available, such as thin-walledPVCDEHPcontainers,(example,XT612), trimellitate(TOTM,examplePL124O,CLX),andbutryl-trihexyl-citrate PL2209), non-PVC containers polyolefin [PL732],
other plasticizerssuch
as
@THC, example
or ethylene vinyl chloride (EVC).All
of
these second generation containers have shown good preservation of platelet function duringthe storage of platelets (Figure 6). The critical factors with regard to container suitability hasbeen the ability to allow gas diffusion through the container wall. Other pertinent issues are those of
platelet concentration ,leukocyte contamination, and volume. Certain containers allowfor higher plateletconcentrationsduring
the fullstorage
period.(l6)
The question of leukocyte
concentration as a detrimental factor hasbeen raised. Many in vitro studiessuggestedthat leukocytes stored with platelets were harmful to the platelet duringthe storage period.(34-36). Most of these studies, however, were performed in first generation containersand in situations where the white cell count was particularly high [> 1 x 109].(35) Under these circumstances, with a limited oxygen environment, the leukocytes would compete with platelets for available oxygen, thus driving the platelet to generate ATP by glycolysis through the Pasteur effect. This resulted in a lower pH. Modem manufacturing protocols tend to produce platelet concentrates with lower white cell concentrationsand thus an effect of white cells is less likely.In addition, the higher white cell concentrations previously reported a higher percentage of granulocytes, and mayhavebeen
responsible for damageby release of proteases.(37)Currentmanufacturing
protocols tend to produce platelet concentrates which are mostly contaminated with mononuclear cells. A certain minimum plasma volume still needs to be maintained [arbitrarily greater than 34 mLs] in order to insure adequate buffering capacity during the storage of platelets.(38) An interesting feature associated with the second generation containers has been the paradoxical high pH injury which occurs early in storage due to release of carbon dioxide from the permeable
360
SWEENEY, HOLME, AND HEATON Shipping
II End Processing I
J
L
Container IS' Generation 2"d Generation
Plasticizer DEHP Thin-walled DEHP (XT612) TOTM (PL1240) TOTM(CLX) BTHC (PL2209) (PL732)
Polymer PVC PVC PVC PVC PVC polyolefin
Critical Factors Gas diffusionthough container wall 0 Platelet concentration 0 Leukocyte concentration 0 Plasma volume 0
FIGURE 6 Types of containers,usedhistorically,andcurrentlyinuse,to concentrates. Critical factors affecting quality are listed.
store manufacturedplatelet
containers. This results in an elevation of pH before the platelet produces a significant amount of lactic acid in order to counteract this tendency.
The last issue with regard to the
in vitro storage has been the question of
the liquid
medium in which platelets are stored. Conventionally, most platelets are stored in autologous CPD or CP2D plasma which supplies plasma solutes, glucose, phosphate, and considerable bufferingcapacity.
This would appearto
be asuitable
medium for in vitro storageof
platelets.(l6,17) There has been considerable interestin the area of storing plateletsin additive solutionsanalogous to the situation withregard
to red cells. These additivesolutions are or other
crystalloid solutions containing glucose, phosphate, bicarbonate, acetate, components.(39-45) Considerable controversy exists regarding the need
for glucose in these
media, and is beyond the scope of this particular review.(l9) In essence, it would appear that some glucosemay be helpful to the platelet during storage, but that this needs to co-exist in the presence of a buffering system suchas bicarbonate or phosphate.(21) In addition, as mentioned previously, sodium acetate would appear to be a valuable metabolic fuel for the platelet and may substitute for glucose and allowfor more extended in vitro storage.(22,45) It will be important, however, to addresstheissue
of bacterialcontaminationbydevisingaprocessing
step for
bacterial attenuationof platelet products before more extendedstorage of platelets from five to ten days is likely to become accepted practice.(46)
361
QUALITY OF PLATELET CONCENTRATES SHIPPING, STORAGE, AND END PROCESSING
After a period of in vitro storage, the platelets are generally shipped from the site of manufacture, such as a blood center, to a transfusing facility.
At the transfusion facility, the
platelets may be stored for a variable period of time from hours to days, and then
may be
subjected to some end processing steps. Little practical work has beendone with regard to the question of platelet shipping. This process has beensimulatedby
the in vitro storage of platelets under conditions where,
for
example, temperature is less well controlled,or the plateletsare immobile.(27,29) Under these circumstances, it would appear that platelets can be left in a non-agitated state for up to twenty four hours, providing there is reasonable temperature control, i.e., between 18 and 24O. What
of course may happen inactual practice is variation in exposure to low temperatures for different periods of time, and the impact of this on quality is likely to be complicated and has not been extensively studied. The storage of platelets at the site of the transfusion facilitymay occur in a different environment from that of the manufacturing facility. Storage in transfusion facilities may occur in a non-agitated state,or by agitation on a benchat ambient temperature,or agitation in an incubator. Although the ideal environment is agitation in an incubator, device cost and volume of platelet transfusionsin the facility make the acquisition of such a device impractical. A last issue is the question of end processing (or secondary processing), which occurs in the
transfusion facility prior to transfusion. It is common to pool platelet products into a pooling container (usually600 mL capacity) prior to issue. These platelets may also be irradiated andlor washed or subject to leukodepletion commonly at the bedside. Although gamma irradiation appears without effect (47,48), the impact of other secondary processing mayalter the potency of the final product. MEASUREMENTS OF PLATELET POTENCY There have been many approaches towards the measurements of platelet potency, and these are best divided into the quantitative and qualitative methods (Figure 7 ) . The enumeration of plateletsshould be arelativelysimpleprocess,usingelectronic particle counters. It is less well generally appreciated that particle counters based on aperture impedance methods and those based
on laser diffraction and hydrodynamic focusing methods
may produce significant differencesin the platelet concentrationby as much as 10 - 15%. Thus, the method chosen in order to quantitate platelets can have significant impact on the reported yields of the intermediate or final product. Comparing
data, therefore, between centers with
regard to yield optimization, requires knowledge of the method of platelet measurement
(see
Table I). There is greater opinion divergence with regard to the qualitative assessment of platelet potency. These tests
are largely divided into in vitro tests, and the in vivo and ex vivo tests.
362
SWEENEY, HOLME, AND HEATON
Measurement Of Platelet Potency Collection
Shipping End Processing
Transportation
0
Quantitative:
Platelet yield orcontent Generally 5 - 10 x 10" Measure of potential
0
Qualitative:
/nvitro
- Multiple tests
In vivo or ex vivo
- Viability (Recovery) Survival Functionality
FIGURE 7 Platelet potency has both quantitative and qualitative components. A multiplicity of tests are available for qualitative assessment of potency, but in vivo viability is generally regarded as the benchmark test.
Table I Total Platelet Counts in Platelet Concentrates as Reported in the Literature Reference
Variation
Number of
Mean Count
S.D.
Coefficient
Studies
%
34
290
7.5
2.1
28.2
49
138
8.0
2.4
29.4
50
194
9.2
2.8
30.4
51
98
8.0
2.5
31.5
52
95
7.2
2.1
29.6
The in vitro tests are multiple and the aspects of platelets affected bystorage are schematically represented in Figure 8. a list of tests which are commonly performed in vitro are outlined in Table 11. It is important to appreciate that noneof the in vitro tests has received exclusive recognitionas being the most usefultest, and thus, in practice, investigators examining different manufacturing or storage conditionshavetendedtouse
a battery of tests.(16,17,21) However, it may be
363
QUALITY OF PLATELET CONCENTRATES
Measurement Of Potency Structure
Function
Dense Body
Resting
kb
LDG ;oy
Membrane
.. *Glycoproteins
o'o
\"
O
Shape Change
GPllblllla
0 . ) Lysosome
O
Activated Platelets
Fibrinogen a-Granules (PTG, vWF)
p-Selectin
FIGURE 8 by Schematic representationof aspects of platelet 'structure and function which have been used investigators to assess changes associated with platelet processing, especiallyin vitro storage.
Table 11 Measurement of Potency In Vitta Tests
*
Tests of metabolicprocesses:pH,
p02, pCOz,glucoseconsumed,
lactate generated,
platelet ATP.
*
Physiological"stress"tests:aggregation,change
in shape in response to ADP,osmotic
challenge (hypotonic shock response).
*
Structural:morphology score (microscopic),swirlingplatelets,presence clumps, gelanalysis
of membraneproteins
of macroscopic
or wholeplateletlysates
(1D/2D),
microvesicles, electron microscopy.
*
Leakage tests:
LDH (cytoplasm), bTG and
vWF (a-granules), lysosomal enzymes,
intracellular Caz+ shifts.
*
Surface alteration: loss (GPlb, GPllbllla) or gain@-Selection)ofglycoproteins.
364
HOLME,
SWEENEY,
AND HEATON
generally stated that measurements such as the response to osmotic challenge, agonist induced alterationsinshapechange,
and surfaceexpression of glycoproteinshasshownreasonable
correlation with in vivo tests. The question of the predictability of in vitro measurements has been the subject of a recent review.(53) Measurements of platelet potency can also be performed using
in vivo tests; these are
outlined in Table 111. The use of radio-isotope labeled platelets in healthy volunteers is generally considered optimal,(54) as it removes confounding variables that are known to occur in thrombocytopenic patients.(55) Both [recovery and survival] can be measured. There is much less data, and much more concern, with regard to the value of the assessment of platelet functionality after in vivo infusion.Approachesinthis
area have been attemptstomeasure
correction of the bleeding time either in thrombocytopenic subjects
ex vivo aggregation or or in aspirinized healthy
volunteers.(56,57) An important aspect which has not been adequately appreciated is both the intra-subject
andinter-subjectvariation.
This hasbeen
aconfoundingfactor
in attemptingtoidentify
relationships between in vitro tests and the in vivo viability studies,and this has resulted in the failure of many of the in vitro tests to adequately predict in vivo recovery. Although some of this variation is no doubtrelated to a true intra-subject variability (approximately 30%), a contributory factor in theestimateofrecovery
is the likelyinaccuracyinpredicting
volume of the donor based on published nonograms.
blood
Errors in blood volume calculation will
give rise to errors in recovery, and thus the in vitro tests performed may not show
the same
association as might be the case if more accurate measurements of blood volume were attainable. In addition, the percentageof infused platelets which distribute in the splenic pool will vary from donor to donor. Until these problems are adequately addressed and resolved, the likelihood is that none of the in vitro tests currently performed will allow reliable predictability of in vivo viability. The question of platelet survival as a better indicator ofin vivo events is confounded to the same extentby intra-subject variability. Thus, it is likely that the search will continue for some time for the elusive in vitro testwhich can replace manyof the in vivo measuresof viability.(53) Approaches to Studying Processing or Storage Effects on Platelet Potency Approaches to the study of platelet potency need to begin with an appropriate analysis of the process to be analyzed and the end product which will actually be examined. A review of the literature indicates that there has been many approaches to the analysis
of processing changes and the subsequent effect on platelet potency. Some studies have been reported without the presence aofcontrol populationor where historicalcontrols have beenused for comparison.Differentstudydesigns
can beemployed,
for example,atwo-grouped,
365
QUALITY OF PLATELET CONCENTRATES
Table III Measurement of Potency In Vivo Tests Healthv Volunteerg Viability (% recovery)
Thrombocvtopenic Patients
-
Initial 3 hours sample post-
10 60 minute increment
infusion of labeled platelets
in platelet count
("'In, Q)
survival
Serial samples for 10 days
Increments at 16 - 24 hours
Functionality
Correction of ASA prolonged
Bleeding time correction
Bleeding time; ex vivo aggregation or adhesion
Studying Processing/Storage Effects on Platelet Potency Name 2 Group Historical Data
Control
Control
2 Group
Concurrent Unpaired
R Control
One Group Paired Sequential
Variation *
# Studies'
Time lntersubject lntrasubject
?
lntersubject lntrasubject
64
lntrasubject
24
One Group Paired Concurrent
7
Studies which detecteda 10% difference with 0.95 probability at peO.05
FIGURE 9 Impact of study design on the numberof tests necessaryto detect a difference in potency (in vivo recovery) using two different processing methods.Based on data on file, American Red Cross, Norfolk, VA.
366
SWEENEY, HOLME, AND HEATON
Studies Of Processing Effects on Potency 2. Leukodeplete by filtration
3. Irradiate
Manufacture Soft Spin
Collection Transportation
uuSpin
1
2
3
4
Storage
5
Shipping End Processing
Study Stored Products
l.Leukodeplete PRP by filtration
FIGURE 10 Approachtothestudyofprocessingeffectsonthepotency concurrent paired study designs. See References 59, 6 0 , 61.
of plateletconcentratesusing
concurrent, unpairedstudyandaone-grouppairedsequential
study, or aone-grouppaired
concurrent study. The latter study has largely been performed in vitro, and in vivo when double isotopelabelingtechniques
are available, suchandIndiumand
Chromium.(58,61)Paired
sequential studies or two-group concurrent studies can be performed in virro, or in vivo using single isotopic tests. Assessing the number of studies whichneeds to be performed is a function of the degree of difference to be detected (power) at any defined statistical probability level
(Figure 9). Adopting this approach towards an analysis of processing of the various activities associated with the generation of a final end product will ultimately result in data which could meaningfully influence the quality of products ultimately transfused (Figure 10). REGULATORY ASPECTS OF QUALITY AND FUTURE DIRECTIONS The current regulatory requirements related to platelet manufactureare indicated in Table IV. It should be emphasized that theseare the minimum requirements for platelets. Products which hopefully exceed these requirements are currently in widespread use. The greater challenge is to continue to improve platelet quality and it is likely that this trust will continue, driven by market competitive
forces and physician expectations. Suggested
future directions for
improvement in platelet quality over the next few years are indicated in Table V.
367
QUALITY OF PLATELET CONCENTRATES
Table IV Regulatory Aspects of Platelet Manufacture
*
Potency:
(1)
plateletcontent
>5.5 x 10" (or >3.0 x 10" for apheresis
products) in 75% products (quantitative) pH
> 6.0 (qualitative)
Color Negative culture for bacteria Contaminating RBC (<5 mLs) Contaminating leukocyte < 1 x
lo9 (apheresis products)
Table V Future Directions for Platelet Quality
-
1994 2000
*
Improve Potency
*
Yield enhancing processes
*
Additive solutions
*
Betterunderstandingoftherelationshipbetweenvolumes,containers,and platelet content
*
Reduce Risk
* *
Leukoreduction
*
Bacterial growth retardation
*
Viral attenuation
Irradiation (UVB and gamma)
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E. Nelson,R.carmen.
THE QUALITY OF RED BLOOD CELLS
*
W. A. L. Heaton Irwin Memorial Blood Centers 270 Masonic Avenue San Francisco, California 94118 and Department of Laboratory Medicine University of California, San Francisco
ABSTRACT The evolving practice of medicine has required a number of changes in red cell product manufacture to ensure that the final product is more specifically tailored to the needs of the individual patient. As a result of the increasing concern over the risks of transfusion pharmaceutical standards of manufacture are now applied to blood component preparation. Studies have been undertaken to define the optimum method of blood processing, and newer technologies are emerging to allow acquisition of a more consistent dose of red cells in a fashion which may minimize thelesion of collection. Use of high efficiency 3+ generation filter technologies reduces leukokine build up during storage and improves the quality and purity of the stored blood product. The combination of new plasticizers for packaging and improved red cell additive solutions should allow the blood center to supply a more functional red cell with longer storage shelf life. Overall these developments should result in the provision of a more consistent dose of fully functional red cells to the recipient who will be less exposed to theundesirable sequelae of transfusion thanpreviously.. INTRODUCTION The last decade has seen a major shift in the way that red blood cell transfusion products are prepared. Early blood storage studies had focused on phlebotomy of whole blood into combined anticoagulant nutrient solutions (1). Subsequently, plastic packs weredeveloped which facilitated separation of packed red cells concentrates (2). Although early plastic formulations were occasionally associated with variable red cell survival, development of polyvinyl chloride containers plasticized with di-ethyl-2-hexyl phthalate resulted in more consistent red cell storage
* A modified version of this paper was presented at the Royal College of Physicians of Edinburgh, Consensus Conference on Red Cell Transfusion, May 9 6, 10, 1994, with publication in the Proceedings on the Conference. 371
372
HEATON
characteristics (3). Developmental studies focused on improving the anticoagulant/preservative to minimize the collection lesion; improving 2,3 Diphosphoglycerate preservation, and prolonging the storage duration of red cells ( 4 , 5 , 6 ) , with the result that CPDA-1 became the standard US red cell preservative in 1977. Since separation of the red cells from the plasma also reduces the amount of nutrient available to the red cells it was necessary to restrict the red cell hematocrit to ~ 8 0 %to avoid compromising red cell post transfusion recoveries (7). Later developments of combined anticoagulantfpreservatives (CPDA-2 and CPDA-3) exhibited sufficiently variable recoveries that it was not until the development of red cell additive solutions that further extensions to the red cell storage period were licensed (8,9). The publication in 1977 of two studies on red cell additive solutionsresulted in a majorshift in red cell preparation methods as a result the majority of red cells in the USA are now prepared using derivatives of these developments (10,ll). Red cell additive solutions represented a new concept in that the separation of the anticoagulant from the nutrient properties allowed for specific optimization for the component of interest. In addition the volume of the solution could be optimized to improve the flow properties of the final product, and the processing steps necessary to add the nutrient solution allowed for additional manipulation of the unit for example leukoreduction by in line filtration (11,lZ). Subsequently, there havebeen developments in red cell collection processing, component separation, additive solutions, plasticizer technology and post processing handling that all affect the quality of thestored red cell. These developments will form the subject of this review.
-
RED
CELLCOLLECTION AND COMPONENT SEPARATION
The first red cell anticoagulantfpreservative Acid Citrate Dextrose (ACD) represented an innovation in that the low pH ( ~ 5 . 5 ) Of the citric acidfsodium citrate was adequateto prevent caramelization of the glucose during heat sterilization by autoclaving (1). The ACD citrate concentration had been optimized for glass containers and is greater than thatrequired to chelate the calcium anticipated to be in the whole blood (13). Acceptable red cellpreservation can be achieved with half the citrateload and studies have shown improved Factor VI11 preservation in the plasma (14). More recently a number of studies have confirmed both improved Factor VI11 recovery, acceptable platelet preservation and preservation of normal levels red cell DPG for an additional week whenthe subsequent red cell additive was an additive similar to AS-3 (15,16,17). These studies also emphasized the importance of careful quality control in packed cell manufacture since post transfusion recoveries werelow, averaging 52%, in the control group which had low glucose concentrations at the end of storage (16). This problem is avoided with the useof additive solutions since the additiveis mixed only with the red cells. Half strength citrate anticoagulation has become a higher priority where the red cells are t o be subsequently stored in an additive solution which contains citrate as a substitute for chloride (18).
QUALITY OF RED BLOOD CELLS
373
In orderto keep therecipient citrate load to a level equivalent to that in currently licensed additives the primary anticoagulant was modified to decrease the citrate content and post storage red cell recoveries were shown to be excellent (19). Early red cell viability studies had shown that the high osmolality of ACD was associated with a significant lesion of collection (4). Post Storage in vivo recovery studies had shown that 28 day ACD-RBC recovery of the first 100 mL of red cells collected was on only one-third of that of the whole unit and that this lesion could be avoided through the use of less hypertonic storage solutions. It was the appreciation of this collection lesion that ultimately led to the development of CPD which was less hypertonic (5,20), and which contained additional phosphate. CPD whose formulation is shown in TABLE I has remained a primary anticoagulant for most subsequent red cell storage solutions except for those additives containing citrate (Circle PackIAAS, AS-2, AS-3), where CPD containing double thequantity of dextrose is routinely utilized (10,21,22). Recent studies on red cells drawn into CP2D and stored in AS-3 (Formulation in TABLE I) for 24 hours showed post transfusion recoveries of 91+4% confirming that there is still a significant collection lesion with manual blood collection systems (23). Recently an automated apheresis collection system has been used to harvest red cells (24,25). This system utilizes a system of metered anticoagulant addition to thewhole blood which avoids theosmotic shock of manual collection and the preliminary 35 day AS-3 red cell recoveries of 87+11$ are higher than previously reported values. Shown in Figure 1 are the post transfusion recoveriesof 42 day AS red cells collected using both automated and conventional manual blood container systems which were processed within 8 hours of phlebotomy (21). The recoveriesof the apheresis AS-3 red cells are not only better than the manual systemsbut also show improved recoveries when compared to units which had been processed to selectively harvest the lighter (Neocyte) half using a commercially available separation system (Neocel', Miles Inc., Covina, CA) (23). Although these studies were unpaired they are suggestive of improved quality of red cells as a result of avoidance of the collection lesion. Further studies willbe needed to confirm this interesting preliminary observation. Current FDA and AABB Standards specify phlebotomy of 450545 mL of whole blood and in the USA manual trip scales are generally used to control phlebotomy volume (26). However, electronic scales are increasingly utilized elsewhere with the result that donation volumes are better controlled, and if automated component separation devices are also used the final AS-RBC product has a more consistent volume (27,28), as shown in TABLE 11. Also shown are the product volumes and final red cell content of blood products collected using apheresis technology (24,25). With this device, separated packed red cells are collected by an apheresis technology which allows improved control over thedose of red cells in the finalproduct as thered cell harvest is independent of the donor's hematocrit. Since the apheresis procedure is performed with fluid replacement to maintain normovolemia it is possible to collect as much as two units of red cells percollection, and the
374
HEATON
TABLE I Additive preservative systems constituents are shown as total content in milligrams per product volume. Mannitol Svstems Anti-coagulant Preservative AS-5 CPD AS-l
Component
Sodium Citrate 2H20 1,657 Citric Acid
H,O
206
Phosphate H20 Dextrose H,O
140 1,610
Adenine Mannitol Sodium Chloride Product Volume (a) Osmolality (mOsm/Kg)
-
63
445
-
-
-
CP2D Hiah Phosuhate Svstems Anti-coagulant Preservative CP2D AS-3 1,657
588
206
42
-
-
2 ,200
900
27 750 900 100 440
140
276
3,220
1,100
-
30 525 877 100 370
30
-
410 100 300
63 582
96 RECOVERY
24HRS
' 1
component
N=8
N= -69
N=66
N =42
7
I 1 0
90
...........
............
...........
-
AS-l
AS-3
AS-S
ADDITIVE SOLUTION
Haemonetic MCS AS-3
FIGURE 1 Post transfusion recoveries of AS-1,3 & 5 red cells collected and processed in PVC-DEHP plastic containers, using manual and automated (Haemonetic MCSQ) collection systems. The values shown are the median 5 25-75 percentile (box), and range.
375
QUALITY OF RED BLOOD CELLS
TABLE I1 Red cell content and AS-RBC product constituents in units collected and separated by different methods. Anticoagulant and Additive CPD/AS-1 CPD/SAGM* CP2D/AS-3 CP2D/AS-3
*
Svstem (Reference) Manualm 363
AS-RBC Final Unit Volume mL Hematocrit(%) 5 30
Automated/Manual" 284 2 16 Automated Pheresis* 363 f: S Automated Pheresis"
714 f: 6
58 f: 4 61 f: 2 56 f: 4 55 2 3
mL of RBC in Final Unit
210 f: 17 175 f: 15 201 f: 12 396 2 22
Buffy coat depleted.
precision of harvest appears adequate to potentially allow labeling of the product with the red cell content (25). Separation and Comwnent Harvest. Post phlebotomy units are either stored at 4OC for up to 72 hours as
-
-
to preserve red cell 2,3 diphosophoglycerate or for CPDICP2D whole blood up toeight hours at 22OC ifplatelet separation is intended (29). During the eight hour 22OC hold period as much as 43% of the initial DPG is lost, though other red cell parameters such as such as Adenosine Triphosphate (ATP) and post transfusion recoveries after 42 days of storage are well maintained (30,31). In fact evaluation of post transfusion recoveriesof AS-l,AS-3 and AS-5 red cells suggests that up to eight hours of 22OC hold prior to separation and additive solution mixture with the red cells may confer a slight benefit to 42 day red cell recoveries (21). Post hold Factor VI11 preservation appears acceptable and platelet/plasma recovery are also marginally improved suggesting that apart from DPG loss a short period of 22OC is generally beneficial to component harvest (30). CPD/CP2D anticoagulated whole blood is generally centrifuged at -4100 g.min if platelet rich plasma separation is intended and at a higher speed 25,000 g-min if only plasma separation is desired (28,32,33,34). Early studies with AS-l did not show any effect of residual plasma on red cell storage characteristics and this variable is now dictated solely by the need for platelet or plasma harvest (28,32). In Europe red cell units are routinely buffy coat depleted to prevent the formation for microaggregates minimize post storage hemolysis in AS red cell units; and to maximize plasma harvest (35,36,28). Although this was originally performed manually, buffy coat depletion is now routinely performed utilizing a number of automated separation devices(37,38,39). Since thebuffy coat residue wasutilized to manufacture platelet concentrates and itwas shown that up to hours 20 of 22OC
-
hold improved platelet harvest by allowing platelet deaggregation, an extended period of 22OC hold of the CPD whole blood is now standard in many
376
HEATON
European countries (40,41,29). While thisimproves platelet and plasma yield recent studies in the US have suggested that 42 day mean AS-l post transfusion recoveriesaverage 73% which is less than thecurrent minimum FDA standard (42). The slight reduction in 42 day recoveries after a 20-24 hour hold at 22'C is similar to earlier studies which had suggested reduced recoveries if a 4% hold period extended beyond 72 hours (43). RED
CELLCONTAINERS
Early plastic containers had exhibited variable red cell recoveriesand the current generation of blood containers arenow made of polyvinyl chloride plasticized with di-2-ethyl-hexyl phthalate which makes up 30-40% of the weight of theplastic (44,45). The selection of DEHP was fortuitous in that it has subsequently been shown that red cell storage is compromised in the absence of a leachable plasticizer such as DEHP (46,47,48). DEHP dissolves in the proteins during whole blood storage at a rate of0.25 mg/ 100mL/day, and after 35 days of CPDA-1 whole blood storage levelsof 146 mg/L havebeen observed (47,49). In packed cells phthalate accumulation was significantly slower though a greater proportion of phthalate that does dissolve in the blood is found associated with red cell membranes (50,51). In spiteof many concerns over phthalate toxicity, it proved hard to replace since in its absence red cell microvesiculation increased 50% and supernatant hemoglobin accumulation was 70% greater (46). Post transfusion recoveries were also compromised such that a reduced (21 day) dating period was necessary for CPDA-1 red cells since 35day values averaged only 67% compared to control values of 76% (48). described Recently a new plasticizer butyryl-trihexyl citrate has been that is claimed to be significantly less toxic (52). This plasticizer/plastic combination known as PL-2209 (Baxter Healthcare, Deerfield, IL) has now been used to store red cells (52,53,54). Red cell osmotic fragility and hemolysis were maintained at acceptable levels following storage and post transfusion recoveries were excellent (52,53,54). As a result of the excellent 42 day post transfusion recoveries in the US studies (8421% for AS-l RBC) a limited 49 day trial was conducted which just below the acceptable limit. If demonstrated mean recoveries of 73% CPD whole blood units were held for 72 hours at 4OC prior to processing the 42 day post transfusion recoveries were marginally decreased to 7723%. All in vitro parameters were as well maintained with PVC-BTHC as withPVC-DEHP. Although it has proved hard to document significant DEHP toxicity in the clinical setting it has long been a goal to avoid leachable plasticizers and the constituents of BTHC are more physiological than previous agents (55). Unfortunately, there is little published information on rate of leaching of BTHC into blood products (45). Currently BTHC plasticized PVC containers are now routinely utilized by the American Red Cross for red cell storage, and are likely to be increasingly used worldwide.
-
-
QUALITY OF RED BLOOD CELLS
377
RED CELL ADDITIVE SOLUTIONS Red cell additive solutions systems offer the advantage that the primary anticoagulant, red cell nutrient, method of processing, and volume of solution may be optimized independently. The primary anticoagulant nutrient CPD/CP2D was originally optimized for red cell preservation and since the majority of this is removed with the plasma and platelets a number of developments have focused on modification of this to enhance Factor VI11 preservation (112 strength citrate) or platelet preservation (addition of platelet inhibitors such as Prostaglandin El, or Theophylline) (13-17.56). The volume of additive was originally established as 63mL for low hematocrit packed cells or 100 m L for high hematocrit packed cells (10,ll). Currently most solutions are 100mL since plasma harvest maximization is now routine and hematocrits in the range 55 to 60% offer flow properties that are indistinguishable from whole blood (11,32). Shown in TABLE I are theformulations of the currently licensed red cell anticoagulant/preservatives and additive solutions. The primary nutrient is glucose which is metabolized by glycolysis during storage resulting in lactate as the metabolic end product. Since dextrose containing solutions must be prepared at a low pH, -5.5, to avoid glucose caramelization during steam autoclave sterilization, the red cells have an initial pH of 6.9 to 7.0 prior to storage. During storage the conversion of glucose to lactic decreases the pH to a point that glycolysis is inhibited, red cell ATP falls and post transfusion viability is compromised (21,57). Shown in Figure 2 is the relationship between post storage red cell ATP, storage duration, and post transfusion recoveries for currently licensed additive solutions (21). During the first two weeksDPG is rapidly lost at low pH and DPG metabolism is associated with chloride-ion influx to maintain electrical neutrality (58,59,60). Inhibition of the Na/K pump through lackof ATP allows loss of intracellular potassium, red cell osmotic fragility increases, and hemolysis occurs (61). Originally, it had been assumed that hemolysis was the result of cell lysis but recent studies have suggested that as much as 70% of the supernatant hemoglobin is not free but in microvesicles (62). These workers have also shown that microvesiculation was increased in the presence of leukocytes in the red cell product and that filtration decreased microvesiculation to a greaterextent than buffy coat depletion (63). Although glucose serves as an effective nutrient it was not until adenine was included in red cell storage media that acceptable recoveries could be sustained beyond 21 days (64). Adenine is rapidly taken up by red cells and converted to ATP in the presence of glucose and phosphate. Phosphate is included both to stimulate glycolysis as well asto act as a buffer and is included in many red cell preservative solutions. When red cells werestored as whole blood post storage hemolysis was not a significant problem but with advent of packed red cells, a significant increase in hemolysis was observed (6-9). Early studies by Hogman suggested that enzymes released by deteriorating might account for the hemolysis since leukocyte depletion by buffy coat removal or the addition of enzyme inhibitors
378
HEATON
A
A
A
0
0 ,---Q
Days P C
W 28.0 0 35.0 A 42.0
'* 50
/
l
T 49.0
A A
I
I
I
I
2
3
4
5
ATP (Irmoleslg Hb)
FIQURE 2 The figure shows the relationship between single label (%r) post transfusion recoveries and the final red cell ATP values for 258 studies using AS-1,3 ,E 5 additive solutions. The symbols show the number of daysof storage at 4OC, and the line is a logarithmic least squares regression plot.
prevented post storage hemolysis (36,65). With the advent of additive solutions it proved necessary to either leave significant quantities of plasma with the packed red cells prior to ASaddition or to remove the buffy As a coat since in the absence of plasma hemolysis exceeded 1% (65,66). result, SAG which is the precursor of AS-l and AS-5, required buffy coat depletion of the red cells to ensure hemolysis remained
379
QUALITY OF RED BLOOD CELLS TABLE I11 In vitro and in vivo parameters of additive suspended red cell after 42 days of 4°C storage.
Additive Solution AS-3
AS-l AS-5 Storage Duration (Days) Post-transfusion Recovery Red Cell ATP % Original Hemolysis % Glucose mg/dl PH
42 70 2 62 2 0.32 2 555 2 6.6 2
42 %
concentrates
6 10 0.08
60 0.05
42 00 2 7 67 5 15
79 2 7 69 2 15
0.7 5 4 522 5 62 6.7 2 0.2
0.59 2 0.46 325 2 54 6.49 2 0.06
(mean 2 S.O.)
additional phosphate instead of mannitol. Post storage hemolysis is acceptable though this is generally greater and more variable than with Mannitol based additives (22,69). Surprisingly even very high levels of Iglunit failed to prevent excessive hemolysis when red cells Mannitol were stored in PVC containers that contained a non leachable plasticizer
-
-
(70).
None of thecurrently licensed additive solutions support prolonged DPG preservation sincein all casestheglucose is included with the anticoagulants or nutrients and a low pH of -5.5 is necessary for production purposes. Early studies had shown rapid regeneration of OPG post transfusion for whole blood <21 days of storage in first generation preservatives (71,72). Later studies have shown comparable OPG regeneration with CPDA-1, AS-l, and AS-3 red cells though AS-3 RBC which have significantly higher phosphate levels exhibited a maximal rate of regeneration that was double that of CPOA-1. DPG values reached -50% of final values after seven hours, and normal values were achieved 48 hours post infusion (69). Also of interest was the rapid and complete recovery of red cell ATP post infusion which is shown in F i g u r e 3; levels increased 44% in the first hour. Since between 20-25% of the red cells might be expected to be rapidly removed post transfusion, the rapid recovery of those remaining in the circulation suggests that a goal of future additive development should be orientated toward increasing the fraction of the original unit that is recovered since those red cells which were not removed recovered so completely. Shown in F i g u r e 4 is the relationship between storage duration and post transfusion recovery for AS-l, AS-3,AS-5 red cell units. After 35 days of 4OC storage recoveries fall rapidly and extended dating is clearly not a possibility with this generation of additive solutions.
-
380
HEATON 7
r
6 -
5 4 -
3 -
2 1 -
0
I
I
I
I
l
I
I
I
Q
1
2
3
4
5
6
7
Time Post-Transfusion (hr.) FIGURE 3 Mean ATP regeneration in transfused red cells during thefirst three days post transfusion. Mean values are reported from five donors who each donated units stored as CPDA-1 AS-l H, and AS-3 A. There were no significant differences in recipient characteristics.
78
K V Q)
7
7 4 1
n=39
70
n=10
Days of Storage FIGURE 4 The figure shows the post transfusion recoveries of AS-l8AS-3 and AS-5 red cells after various periods of 4'C storage. The studies performed on day 1 and 7 utilized AS-3 red cells separated by centrifugation to allow harvest of Neocytes - the upper half of hard spun units.
381
QUALITY OF RED BLOOD CELLS
Recent DeveloDments inRed Cell Storase. recent interesting developments in red Summarized in TABLE IV are some cell storage that have resulted from the work of Meryman who has been evaluating the interactive effects ofhypotonic storage media, ammonium based additives and low chloride solutions (73,74,75). Originally these studies sought to evaluate whether storage of red cells in a hypotonic solution would cause sufficient red cell swelling to prevent microvesiculation. Previous studies had shown thatosmotically fragile stored red cells were removed at an accelerated rate following transfusion and that the rate of removal was associated with thepost transfusion loss of %r labeled stored cells (60). Initially ammonium chloride was used for ionic support as a penetrating solution and this was shown to associated be with excellent preservation of post transfusion recoveries 46-86% after 84-131 days of storage (73). Later studies showed the effect of ammonium to be enhancement of ATP preservation through relief of ATP induced inhibition of phosphofructokinase (74). The hypotonicity was shown to be critical to theimproved morphology since addition of Mannitol to the hypotonic medium obviated the effect of preserving red cell morphology (75). An additional effect of washing red cells with a chloride free medium was elevation of intracellular pH as a result of the Donnan Equilibrium (76). As chloride leaves the cells to establish a Donnan Equilibrium, electrical neutrality is maintained by an influx of O H resulting in an intracellular pH that is higher than the extracellular pH. The combination of a hypotonic medium and washing the red cells with a low chloride medium that also has a high initial pH (7.4) greatly increased red cell intracellular pH and enhanced DPG preservation throughout the period of subsequent storage (75). The formulation of this solution ARC-8 is shownin TABLE V and the in vitrofin vivo resultsof trial in TABLE VI. Although the method of processing was complex requiring a 1.5 L wash of each unit on a Cobe 2991 cell washer, post transfusion recoveries were better thanwith AS-l and 42 day DPG values significantly better than any other red cell preservative (75). Others havedeveloped an ammonium chloride based additive solution along similar lines but with high phosphate concentrations in a system that involves conventional processing methods and 100 mL of solution EAS-2 (74,75). Summarized in TABLE VI are the in vivoand in vitro resultswith EAS-2. Post transfusion recoveriesaveraged 8057% with a hypotonic solution containing 20 mM NH,C1 after 63 days of storage which is animprovement over
-
-
current systems. Since ammonium is an undesirable additive subsequent development has focused on the use high of pH low chloride hypotonic storage solutions (79). In these studies it was necessary to separate the glucose from the rest of the constituents since it must be autoclaved at a low pH. The acid glucose solutionis mixed with the alkaline portion of the additive solution immediately prior to processing. This solution known as RAS-2 incorporates the hypotonic, high phosphate, low chloride concepts in an additive system with 112 strength citrate primary anticoagulant (18,19,79). The reduced citrate content of the primary anticoagulant is necessary to support DPG preservation during the hold period (17) and to keep the total citrate load equivalent to current red cell additives (22). In recent
382
HEATON TABLE I V
A summary of recent approaches taken to improve red cell preservation in additive solutions. Agent Act ion Concentration Solution Propertv Increase Hypotonicity Cellular Volume 210 m Osm RAS-2/ARC-6 to prevent 165 m Osm EAS-2 Microvesiculation 132 m Osm ARC-8
Elevate Intracellular pH to Support Glycolysis
PH
PO4 and
pH, >7.6 pHi >7.0
ARC-8 RAS-2
pH, -7.4
EAS-2
Buffer pH to
3OmM
EAS-2
promote DPG Formation accelerate Glycolysis
20mM 15mM
RAS-2 ARC-8
Relieve ATP-Induced
5OmM
ARC- 6
Inhibition of PFK
2OmM
EAS-2
TABLE V
Formulation of third generation additive solutions. mg/dL CSM ARC-8 RAS-2 447 980 735 Na Citrate.2H20 Citric Acid +Na Phosphate.H20
51 -136
170140
Dextrose Adenine Mannitol NaCL KCL CaCL,
700 27
2,500 28
MgS02 Na Bicarbonate Osmolality PH ( Na2HP04/NaH,P04) +
285169 818 20 728
645 38 25 20 300 290-305 7.4
132 7.4
212 7.3
QUALITY OF RED BLOOD CELLS
383
TABLE VI Post storage in vivo and in vitro results with third generation Red Cell Additives.
CSM Days stored (N) 24 Hour Recovery Survival (days) ATP (uM/g Hb) DPG (uM/g Hb) Hemolysis (5)
(%)
42 (5) *86 t 5
ARC-8 42 (6) 87 2 6
RAS-2 49 (10) 79 2 7
93 f- 29
99 5 17
2.5 & 0.5 0.3 & 0.2
4.1 f- 0.25 6 5 2.2
2.8 f- 0.4 1.6 5 0.9
0.32 5 0.13
0.32 & 0.29
0.23 2 0.09
*9AhTc/''Cr 78 5 4 Paired Control 84 f- 27 days Paired Control 118 2 10 days
clinical studies the intracellular pH was higher than extracellular pH for four weeks during which time DPG was well maintained (76). Although initial glucose levels in RAS-2 were similar to SAGM red cells, by 49 days RAS-2 levels were on average 54% of the SAGM levels confirming improved energy metabolism. This was associated with post storage hemolysis levels that were approximately half that of the SAGM controls. The in vivo and in vitro results are summarized in TABLE VI which confirm good preservation of in vitro parameters and acceptable 49 day post transfusion recoveries (19). An alternative approach toward red cell preservation developed from work on platelet additive solutions. This concept involved generation of a physiologic salt solution, not dissimilar to the electrolyte content of plasma with added nutrients glucose and adenine, and bicarbonate as a buffer (77,78). This solution, CSM, which could be used to store both platelets and red cells contains conventional red cell nutrients as well as KCl, CaC1, and MgSO,. The formulation is shown in TABLE V and in vivo results in TABLE VI. In the absence of any osmotic stabilizer red cell hemolysis was excellent and post transfusion recovery was comparable to the other third generation additives TABLE VI. It is interesting to note that for the studies with ARC8 where recoveries were higher than AS-1, red cell survival was shorter; whereas with CSM where recoveries were less, red cell survival was longer. When total red cell availability is calculated (the area under the curve of the decay scheme) there is no significant difference between test and controls. Possibly the enhanced post transfusion recovery represents a relatively short lived fraction of the original unit (perhaps a population of chronologically older red cells in the unit) such that preservation of this population results in reduced mean survival of the total population. Nevertheless, both ARC-8 and RAS-2 appear to offer the potential for prolonged red cell dating albeit with the inconvenience of additional
384
HEATON
processing steps required for component manufacture. Since all the newer red cell additives involve higher pH solutions it will either be necessary to sterilize the glucose separate from the other red cell constituents, or to take advantage of a new approach developed in Japan which allows autoclave sterilization ofglucose in an oxygen free environment to prevent caramelization at a high pH. If this method can be implemented on a routine basis, it will allow avoidance of the additional processing steps that are necessary to ensure consistent mixing of the additive constituents prior use. SPECIAL UNIT PROCESSING As described earlier there evidence is that the red cell storageinduced microvesiculation is accelerated in the presence of leukocytes and that buffy coat depletion prevents much of the storageinduced hemolysis (36,63). Other studies of granulocyte functional sensitivity to temperature and temperature induced morphologic changes suggest that there are rapid effects of phlebotomy on granulocytes which might result in degranulation, aggregation and possibly generation of interleukins and cytokines (82,83). Some granulocyte effects appear to be beneficial in that bacterial growth is slower in deliberately spiked units that contain leukocytes than in those that do not (84) and when spiked units are buffy coat depleted bacterial growth is observed in the buffy coat rather than in the BC-poor red cells ( 8 5 , 8 6 ) . Similarly when spiked units arefiltered there is slower growth in fewer units where theunit was subjected to prestorage leukodepletion (8789). If the bacterial load is high enough, all units exhibit growth such that this is a relativebenefit at best. However, evidence increasingly suggests that early leukodepletion removal prior to storage prevents the build-up of interleukins and leukokines It is not clear what level of leukodepletion is during storage (83). necessary to avoid these effects since there are comparatively few studies relating leukocyte related sequelae such as immunization to thequantity of residual leukocytes in a red cell unit (90). The AAEE standard (84.240) for avoidance of immunization now specifies less than 5 x lo6 leukocytes per product, and there are no specifics relating to the timeof filtration or levels of fragmentation that may have occurred prior to storage. Recent studies have shown that current 3+ generation leukodepletion filters can remove themajority of intact white cells from AS red cells thoughfragments cannot be removed (91,92). Although leukocyte fragments may not cause primary immunization (93) it seems prudent to remove leukocytes as soon as possible. In fact, an increasing proportion of red cells (-20% in the USA) are now filtered at the bedside using leukodepletion filters, though there is little quality control of filter performance and hence inadequate knowledge of the effectiveness of the performance of those filters with blood of varying storage durations. Prestorage leukocyte depletion was originally described in 1981 b u t t h e filter technology was inadequate to remove more than 85-99% of leukocytes (12). Nevertheless, it was clear that microaggregates were prevented and red
to
QUALITY OF RED BLOOD CELLS
385
cell in vitro studies suggested improved storage characteristics (94,95). Subsequent improvements in filter efficiency have resulted leukodepleted Current prestorage units that consistently meet the 5 X lo6 Standard. leukodepletion systems (Leukotrap-RC, Miles Inc., Covina, CA) with an in line RC-300 filter (Pall Corp., Glen Cove, NY) achieve residual leukocyte loads within hours of of 4 x 10’ per unit if the blood was filtered at 22% collection and 3 x lo4 when filtered at 4% 16-24 hours following phlebotomy. None exceeded 1 x lo6 per product (96). 42 day post transfusion recoveries exhibited a small (3%) but significant improvement in post transfusion which should largely offset the filtration induced loss. Mean post storage hemolysis was reduced by approximately two thirds (0.18 versus 0.54%). Red cell lactate production, pH fall, osmotic fragility and potassium leakage were significantly reduced suggesting that the absence leukocyte of enzymes may have reduced the storage associated membrane lesion (96). Although similar levels of leukodepletion can be achieved with bedside filters it is very much harder to achieve theconsistency of usenecessary to ensure high efficiency leukoreduction (97). Recently a number of studies have been undertaken to establish whether the degree of leukoreduction with 3+ generation filters is adequate to prevent transfusion transmitted viral diseases of viruses known to be leukocyte associated, such as CMV (98,99). The most recent reports havesuggested that TA-CMV is avoided by the useof these filters and as a result it is likely that use of leukodepleted blood products may increase rapidly. Within the last few years there have been at least two reportsof graft versus host disease following transfusion of blood filtered with 3+ generation filters (100,101). Consequently, it is now the standard of practice to irradiate red cells where there is likely to be a high degree of HLA homology between donor and recipient (102) or where the recipient is sufficiently immuno-compromised that they might not be able to clear donor lymphocytes. The US Food and Drug Administration has issued guidelines describing btandards and now limits red cell dating to 28 days following irradiation (97). In view of the significant numbers of directed donations and continuing public anxiety over transfusion transmitted viral disease the issue of irradiated red cell quality is likely to become increasingly an important issue. Early studies focused on theeffect of irradiation immediately prior to transfusion and showed that radiation doses in the 5,000-10,000 cGy range had little effect on in vivo and in vitro quality (104). Larger doses (20,000 cGy) were, however, associated with -50% increase in supernatant potassium levels. Later studies showed a significantly accelerated storage lesion 68.5% at following 3,000 cGy on day 1 with AS-l RBC mean recoveries of only 42 days from compared to control values of 78.4% (105). Subsequently, a number of studies have been undertaken to define the radiation dose needed to produce inhibition of T cell activity and block mixed lymphocyte culture activity (106,107). Based on these studies a dose of 2500 cGy was selected as effectively inhibiting lymphocytes and a series of in vivo studies were performed to document red cell quality under various storage conditions (108). Assuming irradiation on day 1, day 28 recoveries
386
HEATON
averaged 79% compared to 84% for controls (108). However, if irradiation occurred after day 1 post transfusion recoveries were more adversely affected. Specifically, if units were irradiated on day 14 and infused on 82% -but if these units were infused on day 28, recoveries wereacceptable day 42 recoveries averaged 70% which is well below theacceptable minimum. These resultssuggest that whilea 28 day dating may be acceptable if units are irradiated on day 1, (as currently recommended by the FDA), dating should be reduced to 14 daysfor irradiation after day 1 (103,108).
-
SUMMARY
In summary there have been a number of developments in red cell acquisition, separation, preservation, purification and packaging which have improved the consistency and bio availability of the final product. The use of automated collection scales with flow monitoring devices or ofred cell apheresis devices allows theacquisition of a more consistent initial dose of red cells. Automated separation with prestorage leukodepletion allows preparation of a more pure product with less undesirableleukokines, better quality red cells and fewer microaggregates. High pH, low chloride, low osmolality additives improve both DPG preservation and the shelf life of the product. Finally storage in a PVC container plasticized with BTHC reduces the plasticizer load of the blood product and the potentialtoxicity. Taken together each of these small steps should result in a more consistent product of improved quality. REFERENCES 1.
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GROWTH
FACTORS
AND
CORD
BLOOD
STEM
AND
PROGENITOR
CELLS
Hal E. Broxmeyer Departments of Medicine (Hematology/Oncology) and Microbiology/Immunology and the Walther Oncology Center Indiana University School of Medicine Indianapolis, Indiana46202-5121
ABSTRACT
This article reviews recent information on the proliferation kinetics of hematopoietic progenitor cells in patients on clinics1 trial with growth factors, and the use of umbilical cord blood as a source of transplantab stem and progenitor cells. INTRODUCTION Most cells circulating in the blood have limited life spans. These
cells are replenished by a finely tuned network of interacting cells. This includes hematopoietic stem and progenitor cells and the cell-derived biomolecules, termed cytokines, which regulate the proliferation, renewal
and
Stem
differentiation
cells
are
of
the
multipotential
stem cells
and
self-
progenitor cells (1,Z).
with
the
capacity
toof make
more
themselves (self-renew). It is believed that a hierarchy exists within this compartment, with the earliest cells having the greatest capacity self- for renewal, and the more mature cells within this category having decreased capacity for self-renewal. The marrow repopulating cells reside
in the
earlier, more immature population of stem cells. Stem cells differentiate into the next broad category of cells, the progenitors, which include multipotential as wellas more lineage-restricted cells. There is also a hierarchy within the progenitor compartments with the more immature progenitors within both the multipotential and lineage-restricted subsets having the greatest capacity for proliferation and the more mature cells having less of this capacity. Table 1 lists the different assays currently
391
392
BROXMEYER Table I Assays for Hematopoietic Stem and Progenitor Cells
Name
Abbreviation
‘Abbreviations: CFU, colony forming unit; GEMM, granulocyte, erythroid, G, granulocyte; M, macrophage; BFU, burst forming macrophage, megakaryocyte; unit; E, erythroid; MK, megakaryocyte.
used to detect subsets of hematopoietic stem and progenitor cells. More information on these assays can be found elsewhere (1,2). Since stem and progenitor cells are rare populations
of
cells
present
in blood forming tissues at frequencies S 1/1000, proof for direct acting effects of cytokines on these populations requires the physical and immunological purification of these cells (3). More rigorous proof for direct acting effects requires studies at the single cell level by sorting single purified stem or progenitor cells into single in wells the presence of cytokines(3-5). Presently, there are over 40 biochemically defined cytokines with known activity, either direct or indirect,
on hematopoietic stem or
progenitor cells (1,2,6). The genes for most of these cytokines have been cloned and recombinant molecules are available for preclinical and evaluation of their efficacy of action. Some of these cytokines act as growth stimulating molecules such as the colony stimulating (CSFs) factors
clinical
BLOOD CORD
393
STEM AND PROGENITOR CELLS
which include: Granulocyte Macrophage (GM)-CSF,G-CSF, M-CSF (also called CSF-l), multi-CSF (also termed interleukin(1L)-3),
IL-5 (an eosinophilic
CSF), and erythropoietin (Epo), the erythroid humoral regulator. The CSFs have been shown
to have direct acting effects
on various subsets of
progenitor cells, but they can also trigger the release of other cytokines with
either
CSF-activity,
co-stimulating
activity,
or
suppressing
activity.
Hence, many cytokines have apparent pleiotrophic activity. Some of these effects are due to the induction
of the production/release
of other
cytokines. CO-stimulating cytokines include steel factor (SLF, also termed stem cell factor, mast cell growth factor, c - k i t ligand)(7)), the
newly
identified Flt3/Flk-2 ligand ( 8 ) , IL-4, IL-9, IL-11. Suppressing cytokines include members of the chemokine family such as macrophage inflammatory protein(M1P)-la,
MIP-2a, IL-8, plateletfactor
4
(PF4),monocyte
chemotactic and activating factor (MCAF), and interferon inducible protein molecular weight 10 KD (IP-10) (9,lO). Other suppressor molecules include H-ferritin, lactoferrin, transforming growthfactor-8, the interferons (a,
p, S
) , and the tumor necrosis factors
stimulating
and
suppressing
(a,
molecules
8)(6).
can
The effects of co-
also
be on stem/progenitor direct
cells or mediated indirectly through actions on accessory cells. Kinetic ResDonses of Proeenitor Cells In Vivo to CSFs The
CSFs
have
shown
efficacy
of
action
when
administered
to
patients
during phase I to I11 clinical trials (11,12). Thus, GM-CSF, G-CSF, IL-3 and Epo have accelerated blood cell production of selected hematopoietic cells in the absence or presence of other treatment modalities such chemotherapy
or
bone
marrow
or
blood
stem/progenitor
cell
as
transplantation.
The CSFs have also been used to mobilize stem/progenitor cells into the peripheral
blood
for
their
use
in
autologous
and
allogeneic
transplantat
An understanding of the kineticsof progenitor cell proliferations could enhance the capacity to utilize more intensive chemotherapy. To this end, such events have been evaluated for GM-CSF, G-CSF, and the genetically engineered PIXY321 (a GM-CSF/IL-3 fusion protein). Administration ofGM-CSF to patients increases cycling rates of CFUGEMM, BFU-E and CFU-GM in the bone marrow (13-16). However, within 1 day after discontinuation of GM-CSF to patients, progenitors are in a slow- or non-cycling
state,
information
is
usually
being
used
below ofthat the to
attempt
pretreatmentvalues(17). This dose-intensification of chemotherapy
by
decreasing the interval time between chemotherapy dosing To (17). determine if similar kinetics of response were apparent with other CSFs, G-CSF (18) and
PIXY321
(19)
were
evaluated
under
similar
conditions.
Administration
of
394
BROXMEYER
G-CSF resulted in enhanced proliferation CFU-GEMM, of BFU-E, and CFU-GM in the marrow. In contrast to patients receiving GM-CSF, however, progenitor cells from patients off G-CSF treatment for 2 to 4 days were still rapidly cycling (18). The use of PIXY321 demonstrated similarities and differences to that ofGM-CSF (19). Administration of PIXY321 at 125 to 500 pg/m2/day to patients at least doubled cycling rates of marrow CFU-GEMM, BFU-E and CFU-GM.
Also,the cycling rates of progenitors in the blood were increased
from a slow or non-cycling state to rapid cell cycle. Within1 to 2 days after cessation of PIXY321 infusion, progenitors were either in a slow or non-cycling state below that of pre-values, or were back to background cycling levels. These effects were similar to those noted for progenitors of patients on clinical trial with GM-CSF. In contrast, higher dosages of PIXY321, especially 1000 pg/m2/day increased cycling of marrow and blood progenitors early during treatment, but cycling rates of these cells decreased while patients were still being administered PIXY321; decreased cycling was maintained after cessation of PIXY321. The above noted differences
in
proliferation
kinetics
of
progenitor
cells
may
be
of
use
design of clinical trials to more efficaciously utilize these growth factors.
Whether
modulation
on
decreases
progenitors
in or
cycling to
rates
induction
noted of
are
due
suppressor
to
receptor
cytokine
dow
mechanisms
(6,9,10) remains to be determined. Alternative Found
in
Source
of
Umbilical
Transplantable
Cord
HematoDoietic
Stem
and
Progenitor
Cell
Blood
The main sourceof cells for transplantation and engraftment of the hematopoietic system is adult bone marrow. Most recently, cells from the umbilical cord and placental blood of babies at birth have been used for transplantation purposes. Colony assays had demonstrated the presence of relatively late, more mature subsets of progenitor cells in cord blood (Reviewed in 21). 20 and However,
whether
and
repopulating
was
not
or
not
cells
evaluated
a
could
until
therapeutic ofdosage long-term be
obtained
later (20). Using
from
colony
marrow
single
assays
for
engrafting
cord
blood
collection
progenitors
and
correlating these results with reports from others which indicated the numbers of such cells in bone marrow that were associated with successful autologous and allogeneic marrow transplantation, it was suggested in that most cases single collections of cord blood should contain transplantation (20). This study demonstrated the capacity to collect
enough
cells
reasonable amounts of cord and placental blood, to cryopreserve and to retrieve these cells in viable form after thawing, and to transport these
395
CORD BLOOD STEM AND PROGENITOR CELLS
cells at room temperature with little or no loss in numbers or quality of cells ( 2 0 ) . Based on this laboratory study (20) a clinical trial was started. Results
indicated
that
cord
blood
could
be
of hematopoietic stem and progenitor cells (22).
used
as
a
transplantable
Source
A young male with Fanconi
anemia was transplanted with HLA- matched cord blood from his sister. This isnow almost 6 years
patient
cured of the The
hematological
first
patient
post
cord
blood
manifestations with
leukemia
transplant
associated to
be
that
has
responded
with
with
is
(23),
varying ofdegrees success
healthy
Fanconi
transplanted
a child with juvenile chronic myelogenous leukemia leukemias
and
anemia.
with
cord
a subset
of
to
marrow
bone
and blood
transplantation. Engraftment of this child with HLA-matched sibling cord blood was documented. The child relapsed after 9about months, but in this case
the
cord
that he could whose
cord
after
the
blood
be
transplanted
blood he had bone
transplantation with
received.
marrow
bone
The
transplant.
extended marrow
child Had
is
the
the
child's
cells still
child
from alive
not
lifeso long the
same
sibling
more yearthan
received
enough
the
a cord
blo
it is not likely he would have lived long enough to receive a marrow transplant.
A
number
of
other
cord
blood
transplants
have
been
performed
children with acute myeloid (AML) and lymphoid (ALL) leukemia without apparent relapse after two years of follow-up. At
present
there
have
been40 cord over
blood
transplants
performed
to
treat a variety of disorders for which bone marrow transplantation is currently used( 2 2 - 2 8 ) . These diseases include the malignant disorders AML, ALL, juvenile CML, pH+-CML and neuroblastoma, and the non-malignant
disord
Fanconi anemia, severe aplastic anemia, inborn errors of metabolism, Wiskott-Aldrich
syndrome,
beta-thalassemiax-linked and lymphoproliferative
syndrome. Most of the transplants have been done using HLA-matched sibling cord
blood
cells
and
cells, more
some
recently
have three
utilized 1- ,2-,or 3- antigen cord
blood
mismatched
transplants
have
sibling been
completed
Duke University, using HLA-matched or l-antigen mismatched unrelated cord blood cells that had been stored frozen in the cord blood bank New at the York Blood Center. Thus far the results have been very encouraging. The patients engrafted with little or no GVHD, including the complete HLAmatchedorl-antigenmismatchedsiblingandunrelatedcordblood transplants. A cord blood transplant registry was established and dispense the information regarding cord blood transplants (24). Estimating
the
reconstituting
contents of cordblood requires
attention to the number of stem/progenitor cells present,
to
not
coordinate only
but also the
quality of these cells. Re-evaluation of the numberof progenitor cells in
396
BROXMEYER
single
collections
of
cord
blood,
using
the
potent
co-stimulating
cytokine
SLF, made it clear that we had grossly underestimated the frequency of the earliest subsets of progenitor cells compared to that in bone marrow (29). It was suggested, based on this re-assessment, that there should be enough stem/progenitors in single collections
of cord blood to engraft and
repopulate the hematopoietic system of adults (29). The
following
studies
regarding
quantity
and
quality
of
cells
blood confirm this estimate. It was shown that cord blood CFU-GE" extensive GE"
capacity
for
being
replated
with
the
in
cord
had
formation of secondary CFU-
colonies at least as large as the primary colonies from which they
were obtained (30,31). The capacity was enhanced by a factor or factors present
incordblood plasma (31). While CFU-GE"-colonies frombone marrow
could also be replated, most of the secondary colonies were CFU-GEMM, not but were more restricted, e.g.CFU-GM and BFU-E. In cord blood however, CFU-Gm-colonies gave rise mainly to secondary
CFU-GEM-colonies (31).
Moreover,
replatedCFU-GEM"co1ony primary
many
more
secondary
colonies
per
were apparent using cord blood compared with bone (31). marrow In another study, it
was
found
CD34-antigens (CD34-)
that
cord
blood
cells
expressing
the
highest of
density
can give rise as single-sorted and -isolated cells
in the presence of a combination of growth factors to a high percentage colonies derived from HPP-CFC (3). These HPP-CFC-coloniesthat derived from a
single
cell in
resultant
asingle
well
could
be
replated
into
secondary
dishes
of
with
secondary HPP-CFC colonies (3). Moreover, the extensive replating
capability of these cells was documented by their capacity to be replated from Z 0 to 3O dishes, from 3O to 4 O dishes, and from 4 ' to So dishes. A direct comparison with single CD34+++ sorted bone marrow cells demonstrated 8-fold fewer HPP-CFC in
bone
marrow
than
Others have documented the quality cells. for
Cord
adult
blood bone
progenitors
in
cord (3). blood
of cord blood stem/progenitor
maintained CFU-GM for
16
weeks
versus 9 weeks
marrow in along term culture system (32). Using CD34%45RA10
CD71b cells and cytokine supplemented serum-free cultures the total number of myeloid progenitor cells in culture from adult bone marrow remained relatively constant over time (33). In contrast, in cultures initiated
with
cord blood the progenitors increased 2 100-fold over the same period. The fraction of responding cells and their ability to CD34' produce progenitor cells
decreased
markedly
in
adult
bone
marrow
when
compared
with
cord
(33). Using optimal culture conditions in which the transforming growth factor (TGF)-beta1 inhibitory loop was interrupted by of antisense use TGFbeta
1
oligodeoxynucleotides
or
TGF-beta
1
antibodies,
it
was
estimated
the CD34+CD38- cells from a typical cord blood sample contained equivalent
bl
STEM BLOOD CORD
AND PROGENITOR CELLS
391
ofCFU-GEMM,two times as many CFU-GM and three times as many BFU-E
numbers
as the same population froman average bone marrow sample usedin adult transplantation (34).
Also, as noted by us (29), the colonies from cord
blood cells were larger than from bone marrow(34). cells While a
there
are
no
assays
yet
for
long-term
marrow
repopulating
cell
numberof animal models are available that might allow for assessment of
the
growthof these
early
human
cells
in
sheep
or
in
SCID
mice.
The
ability
of human cord blood to reconstitute sublethally irradiated SCID mice demonstrated high levels contrast
to
previous
In
of multilineage human cell engraftment. studies
with
human
bone
marrow
(36),
treatment
of
mice
with human cytokines (e.g. SLF and PIXY321, a GM-CSF/IL-3 fusion protein) was
not
these
required
mice
with
to cord
establish blood
high
cells.
level Human
human
hematopoieticin repopulation
hematopoiesis
wasinmaintained the
mice inoculated with cord blood cells for at 4 least months (35). The
capacity
to
expand
stem
and
progenitor
cells
offers
possibilities
for increasing the utility of cord blood. First, if numbers of these cells are low because cord blood collections are small and not enough cells are present
for
adult
transplantation,
it
would
be
possible
to
use
the
expanded
cells as supplement. Second, the capacity to utilize cells from a single collection of cord blood for multiple donors would improve the costeffectiveness of cordbloodbanks.Thus,thecapacitytoexpand hematopoietic stem and progenitor cells has practical implications and has been studied by a number of groups (32,34,37-40) including our
own
(29,41,42). Assays for the earliest human stem cells, including long-term marrow repopulating cells, do not appear to be available. Our readout of the output to input cell types relies mainly on the use of assays that probably
detect
CFU-GM, etc.).
later
cells
(e.g. LTC-IC, S-cells,HPP-CFC,CFU-GEMM,BFU-E,
Therefore, we cannot yet be sure that we are expanding or
maintaining the earliest stem cells. It is likely that new cytokines and combinations of these cytoklnes, as well as technological advances in culture
systems,
such
as
the
use
of
bioreactors (43), will enhance
expansion
of earlier subsets of stem and progenitor cells. However, until we can be sure that the early hematopoietic cells are being expanded, or at least maintained, it is not likely that patients will be transplanted only with expanded Use
cells.
ofCord Blood Stem and Proeenitor Cells as a Vehicle for Gene Therapv. Gene therapy is currently being evaluated as a possible option in
patients for treatmentof certain inherited diseases (44-46).It has been demonstrated that cord blood-progenitors and -LTC-IC are more efficiently
BROXMEYER
398
transduced by retroviral-mediated gene transfer than are the same types of cells from adult bone marrow (47).
With the likelihood that cord blood
stem/ progenitors will be used as vehicles for gene therapy to correct genetic disorders, it was found that populations of CD34- cord HPPblood CFC, Cm-GEMM, BFU-E and CFU-GM could be retrovirally neo gene at very high efficiency (48).
transduced
with TKa
This was accomplished also at the
single isolated CD34- cell level, with the gene stably integrated into cells
with
high
replating
capacity.
Adeno-associatedvirus(AAV)-vectorshavemorerecentlybeen considered for gene transduction, Recombinant AAV-mediated gene transduction into rapidly cycling hematopoietic progenitor cells in murine bone marrow has been demonstrated (49). Moreover, recombinant AAV were used to infect either low density, or highly enriched populations of columnseparated CD34' cord blood cells( 5 0 ) . neomycin
High frequency transduction of the slowly-cycling, CFU-GEMM,BFU-E and CFU-
resistance (mR) gene into
GM, including
those
with
high
proliferative
capacity
in
the of presence S W
with either Epo or GM-CSF was obtained. Of particular interest, this high efficiency transduction was apparent without prestimulation of the cord blood
cells
with
factors (50). This observation is important preincubation of cells with growth factors prior to infection with retroviruses
growth
could
potentially
lead
to
differentiation
of
because
these
cells
pri
to transplantation. In fact, while gene transduction has been accomplished with
high
efficiency
in
clongenic
cells
from
mice
and
in
apparent
long
marrow repopulating cells in mice, transduction of the earlier cells in other
mammals
observation
is
has not
not
been
yet
as
clear,
successful (51). Although the reason for this it
could
possibly
reflect
the
differentiati
of these earlier cells during the growth factor preincubation phase. Attempts
at
utilizing cordblood stem/progenitor
cells
as
vehicles
for
gene therapy have already begun
(52). Three children with adenosine deaminase (ADA)-deficient SCID were transplanted with autologous columnseparated CD34' cord
blood
cells
that
had
been
manipulated anto ADA-place
gene into these cells with retroviral vectors. While it is still too early to determine the efficacy of this treatment, similar clinical studies will no doubt be in the offeringin the near future. CONCLUSION The successful translation of information from the basic science laboratory to the clinical setting and the continuing interactions between the laboratory and clinic are exemplified by the rapid progress that has been made in the use of growth factors to accelerate hematopoiesis and
BLOOD CORD
STEM AND PROGENITOR CELLS
399
expand cells ex vivo and also in the characterization and utilization of cord
blood
hematopoietic
cells
for
transplantation
purposes.
ACKNOWLEDGEMENT Many of the studies reviewed herein were supported byU.S. Public Health Service Grants R37 CA36464, R01 HL46549 and R01 HL49202 from the National Cancer Institute and the National Institutes of Health and by National Institutes of Health Training Grant Linda Cheung for typing the manuscript.
Dk07519
to
the author. I thank
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Radiation
BROXMEYER
402
52.
D.B. Kohn, Shaw, M.E. Elder, T. Muller and
K. I. Weinberg, R. Parkman, C. Lenarsky, G.M. Crooks, K. Hanley, K. Lawrence, G. Annett, J . S . Brooks, D. Wara, M. Bowen, M.S. Hershfield, R.I. Berenson, R.C. Moen, C.A. M. Blaese, Blood 8 2 , 315a (abstract) (1993).
THE DEVELOPMENT AND USE OF OXYGEN-CARRYING BLOOD SUBSTITUTES Robert A. Dracker, M.D.
S U N Y - Health Science Center at Syracuse, Syracuse, NY 13210
INTRODUCTION The search for a clinically useful blood substitute continues to be stimulated by the inherent limitations of our homologous blood system with regards to its sufficiency, safety and costs. The goal of developing a useful blood substitute has been pursued by numerous researches over the years and currently, by many corporate ventures. It has been estimated that such a product may represent an annual market in the billions of dollars. However,
the
inherent complexity of "blood" was originally remarked upon by W. R. Amberson in 1937 as the "most complicated fluid in animals"'. Surely, an attempt to formulate a blood
substitute is misguided, in that it represents a complex mixture of fluids, cells, salts and molecules having varied functions and characteristics. The hemorrhage or loss of blood from the circulatory system results foremost in a hypovolemic state and ultimate depletionof oxygen delivery capacity. In the acute, life sustaining setting, the abilities of blood to maintain intravascular volume, electrolyte balance and cellular gaseous exchange, are the most critical. For these reasons, efforts in developing a blood substitute have focussed upon a compound or solution which can fulfill these essential requirements, while remaining biocompatible and non-toxic. BLOOD SUBSTITUTE USE Alternatives to blood for infusion have been investigated for hundreds of years. various fluids which have been intravenously administered include: d e , Urine, Opium, scammony (a plant resin), milk, animal blood and more "traditional" fluids such as serum, plasma and crystalloid. Xenogeneic transfusions into humans, generally using lamb blood, had been attempted prior to the early 1900's Only with the development of blood anticoagulation and storage methods and typing and compatibility testing, did homologous blood transfusions become a reliable practice. 403
the use of
404
DRACKER
The applications of a blood substitute are numerous and include: -Intravascular resuscitation following trauma
-A medium for hemodilution on the elective surgery patient -A transfusion alternative for patients with red cell incompatibilities -For patients with ischemic vascular diseaseor requiring coronary angioplasty Other potential applications include a varietyof non-traditional uses such as a solution for extracorporeal organ perfusion, cell and tissue culture media, hematopoietic stimulation, tumor therapy and in research. BLOOD SUBSTITUTE FORMULATIONS
A characteristic of a substitute for blood should focus upon its ability to maintain intravascular volute and solute equilibrium. This issue essentially involves the relative merits of crystalloid versus colloid utilization. For the majority of adult patients experiencing less than 750 ml. of acute blood loss, either crystalloid or colloid volume replenishment may suffice for hemodynamic recovery. Hemodilution of greater than 25% of estimated blood volume with a variety of colloid or crystalloid solutions may however have varied results. Animal studies have demonstrated that isovolemic hemodilution with different oncotic solutions such as albumin, hetastarch and pentastarch, induces variable hemostatic, oncotic and rheologic changes, only partially attributed to dilution, suggesting a direct influence of the volume expander itsel?. Controversy
as to the relative merits of either crystalloid or
colloid volume resuscitation is ongoing3s4. Suffice it to say that a suitable blood substitute should be isotonic while having suitable oncotic characteristics.
The obvious purpose of any blood substitute formulationis its ability to effectively deliver oxygen to the tissues, while removing carbon dioxide and maintaining acid-base balance. Hemoglobin is elegantly suited to not only maximize oxygen delivery and
CO2
removal, but to modify its binding capacity depending upon the cellular milieu, as demonstrated by shifts in its oxygen dissociation curve. Insufficient oxygen delivery to the tissues for their metabolic requirements represents the essence of anemia. Anemia is a dynamic, relative diagnosis determined by the capacity or lack thereof for sufficient oxygen delivery. The efficiency of oxygen delivery is determined by the total blood flow and volume, oxygen content, red cell or hemoglobin mass, oxygen affinity and the rate of oxygen consumption. The relationships between oxygen content, delivery and utilization are best exemplified by the Fick Equation. It is therefore apparent, that a blood substitute which can carry and deliver a maximal amount of oxygen per unit volume, while maintaining excellent rheologic characteristics would be ideal. Efforts to develop an oxygen-carrying blood substitute have taken many venues, utilizing a variety of technical approaches, from biochemical reactions to recombinant
405
OXYGEN-CARRYING BLOOD SUBSTITUTES
technology. The major categories of blood substitutes include the following: -Perfluorocarbon solutions -Lyophilized or "instant" red cells -Hemoglobin-based compounds -Nee-hematocytes
Each of these approaches will be briefly discussed after which a summary of the current status and progress of the major contenders will be reviewed. PERFLUOROCARBON SOLUTIONS The perfluorocarbons are water insoluble, halogenated compounds which exhibit great solubility for oxygen. Oxygen can be dissolved into these solutions in high concentrations, usually requiring elevated partial pressures.
The advantages of the perfluorocarbons is that
they are synthetic and pose no infectious risks, they can be easily made in large quantities and require no pretesting of the recipient prior to administration'.
The limitations of these
compounds include the facts that they must be emulsified in order to improve their aqueous miscibility and require a high inspired FIOz in order to carry sufficient oxygen at a maximal solution concentration in the blood of 20%. The emulsified particle size of the solution is also critical in that if it is too large, capillary plugging may occur. If the particle size is too small, an increase in blood viscosity and subsequent microvascular sludging could develop. There is also variable clearance of these compoundsdue to uptake throughout the body's reticuloendothelial system, particularly in the lung and liver. Currently, there are three perfluorocarbon solutions which are in various stages of development. Fluosol-DA, marketed in the United States by Alpha Therapeutics, has the distinction of being the only FDA approved blood substitute available. This product, which utilizes perfluoroddin as its primary perfluorocarbon, can be used for select patients undergoing percutaneous coronary angioplasty andon a compassionate basis for individuals who are unaccepting of human blood products because of religious beliefs. Oxygent, manufactured by the Alliance Pharmaceutical Group, utilizes a bromated perfluorocarbon called perfluorooctylbromide. This solution demonstrates improved oxygen solubility, requires no additive solutionprior to administration and hasso far demonstrated only minimal toxicity. Recently the Sanguine Corporation has filed patents for its product, PHER-02 which carries three to four times the oxygen per unit volume as compared to blood, while being capable of prolonged storage at m m temperatures. "INSTANT" RED CELLS Maintaining hemoglobin in an intracellular environment affords many advantages. Hemoglobin remains stable in tetrameric form, optimizing cooperative binding of oxygen.
DUCKER
406
The availability of 2,3-DPG results in improved oxygen unloading when
required. The red
cell cytosol also contains protective superoxide radical scavengers. And finally, varying the hemoglobin concentration within the cell has no effect upon plasma oncotic pressure. Unfortunately, preserving red cells is a science unto itself, which despite significant improvements in transfusable red cell products, still remains limited to refrigerated storage constraints of 42 days. The ability to store red cells in a solid state, subsequently liquefied when need would Seem ideal. To that end, attempts to lyophilize or freeze-dry red cells have been ongoing through the work of two companies, both of which have received support from the NAVY Blood Research Program. The Life Cell Corporation utilizes a rapid freezing technique in microdroplet form. Cryopharm utilizes a non-toxic chemical cryopresewative and improved cooling and evaporation procedures.
To date however, these products continue
to demonstrate impaired efficacy and shortened reconstituted red cell life span. HEMOGLOBIN-BASED COMPOUNDS Hemoglobin remains the model oxygen transporting molecule.
The most useful and
effective blood substitute will most likely be based upon some modification of this unique protein. All of the attempts to utilize hemoglobin as the oxygen transporting medium have centered around prolonging the intravascular survival of the extracorporeal form and modifying the structure to provide useful oxygen dissociation characteristics. The first step in using the hemoglobin molecule as the major component of any substitute solution is to decide upon a reagent source. The simplest source of hemoglobin is from outdated or otherwise discarded blood. In the case of outdated blood, the homologous blood supply represents a tremendous reservoir of reagent hemoglobin. However, animal sources, particularly bovine hemoglobin which has a high degree of structural homology with human hemoglobin, also represent another alternative. Regardless of the source, the hemoglobin must be rendered red cell stroma-free, purified and sterilized prior to use, while insuring the minimization of met-hemoglobin formation. One might question why stromafree hemoglobin itself does not represent a viable oxygen carrying substitute for whole
blood? In the extracorpuscular state, hemoglobin does not remain stable in tetrameric form and is cleared by the kidneys. Additionally, raising the free hemoglobin concentration in the blood would result in significant elevations in oncotic pressure.
The infusion of free
hemoglobin has also been noted to be associated with elevations in systemic and pulmonary vascular resistance and reduced cardiac output6s7. This effect is thought to be related to the avid binding of intravascular nitric oxide with free hemoglobin. Reagent hemoglobin can also be derived from non-traditional means, utilizing in-vivo and in-vitro recombinant techniques.
The DNX Corporation has demonstrated the ability to
produce significant quantities of human hemoglobin from transgenic pigs*. The harvested
SUBSTITUTES OXYGEN-CARRYING BLOOD
407
hemoglobin is subsequently purified by ion-exchange chromatography. Recently however, the company has decided to curtail its research in this area. Somatogen’s product, rHb 1.1 is derived from the in-vitro fermentation of DNA-modified Escherichia coli. In
order to
harvest the produced human hemoglobin, the bacteria are lysed and the hemoglobin is subsequently purified by column chromatography. Intramolecular modifications involving stabilization and improved oxygen dissociation characteristics are also possible using these recombinant techniques. Modifications of the hemoglobin molecule have essentially two priorities. The first involves an increase in the size of the oxygen transporting molecule, thereby raisingthe oxygen binding density while minimizing the effect on the oncotic pressure of the solution. The simplest approach is to stabilize the tetrameric form utilizing a variety of di-acids,
sugars and poly-enes’.
The stabilized tetrameric hemoglobin demonstrates improved
intravascular survival. Hemoglobin can also be polymerized, either intermolecularly forming long, branching chains or by attaching the hemoglobin molecules to synthetic starches such as hetastach, amylopectin polymers such as dextran or other polymers such as polyethylene glycol. Baxter Healthcare Corporation has completed a segment of Phase I testing of its diaspirin crosslinked hemoglobin product (DCLHb), derived from outdated blood.
This
product is being tested as a treatment for hypovolemic, hemorrhagic shock. OR poly-Hb (Hemolink), made by the hemosol corporation is another stabilized hemoglobin compound, utilizing purified human hemoglobin cross linked with oxidized raffinose, resulting in both inter and intramolecular bonds. This product has demonstrated no vasoconstrictive effects following administration. Hemosol has also been investigating and alternative chemical modificationutilizing[timesoyltris(methyphosphate)].
This procedure apparently results
in simple protein modification with high yields. Biopure Corporation is also performing Phase I testing of its stabilized bovine hemoglobin product called Hemopure. This product has demonstrated functional similarities between bovine and human hemoglobin.
the structural and
Finally, BioTime Inc. has
been developing a variety of polymer based blood substitutes such as Hextend and Dextend
as well as a product called Zero Plus which can be utilized at less than normal body temperatures. These products are still undergoing preclinical trials. NEO-HEMATOCYTES Neo-hematocytes can be defined as surrogate red cells, made of a variety of materials, from nylon polymers to liposomes, all of which contain hemoglobin as the oxygen wrying vehicle in addition to other useful molecules such as 2,3 DPG, dismutases and oxygen radical scavengers. Previous work with nylon microspheres containing hemoglobin
ct
408
DRACKER
TABLE I Oxygen-Carrying Blood Substitute Development- 1994 A. Perfluorocarbon Compounds Company
Product
Primary Perfluorocarbon
Status
Alliance Pharm. Group
Oxygent
Perfluorooctylbromide
Phase I
Alpha Therapeutics
FlUOWl-DA
Perfluoroddin
Approved
B. RecombinanVHybrid Hemoglobin Compounds Company
Product
Hemoglobin Type
status
Somatogen Inc.
rHBl. 1
Recombinant (E. coli)
Phase I
............................................................. DNX Corp.
" " "
-
Discontinued Hgb. Transgenic-pig
I Phase Hemopure Modified Corp. Biopure Hgb. Bovine
C. Modified Hemoglobin Compounds Product
Company
Hgb. Modification
status
" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " ~ " ~ " " " " " " " " " " " " " " " " " " ~
Baxter Healthcare Corp. Northfield labs
DCLHb " " "
Diaspirin crosslinkage Crosslinked human Phase Hgb.
Phase I I
crosslinkage Pre-clin. Raffinose Hemolink Corp. Hemosol TMA-Hb Trimesoyl
tri methy PO,
Pre-clin
D. Polymer Compounds Company
status
Biotime
Preclinical
.................................................... Pre-clinical Dextend Pre-clinical Zero-Plus
"_
OXYGEN-CARRYING BLOOD SUBSTITUTES
409
demonstrated that gaseous exchange couldbe achieved, however, wide variation in microsphere size and prolonged retention in the reticuloendothelial system have proven to be problematic. Liposome-encapsulated hemoglobin solutions have demonstrated good rheologic properties, acceptable hydrodynamic stability and little met-hemoglobin formation". Other work by Deshpande and Beissinger demonstrated that liposome encapsulated hemoglobin can reach concentrations of 15.5 gms.1100 ml".
These solutions were found to be isotonic and
isooncotic and having slightly higher viscosity as compared to whole blood. They found that survival could be supported in rats bled to hematocrits of less than 5 % . Usuba et al demonstrated that liposome encapsulated red cells can potentially bind 2 to 4 times the amount of oxygen as compared to red blood cells'*. Following administration of these neohematocytes, it appears as if the majority are removed by the reticuloendothelial systemsof the liver, bone marrow and ~pleen'~.Very little accumulation was noted in the kidneys. SUMMARY The major criteria by which any blood substitute product willbe evaluated prior to commercial usefulness will include evaluationsof its: -oxygen transport characteristics -purity and physical properties -potential for and modality of toxicities -efficacy in various clinical settings -biologic half life -metabolism -immunogenicity Although a thorough review of these aspects are laborious, they are necessary prior to the performance of clinical trials. The promise and availability of a clinically useful oxygen-carrying blood substitute has been long in coming, however we fortunately enjoy the continued supply of the safest blood products ever available. Further improvements in our homologous blood system continue, while we strive for the development of a blood substitute. The availability of a safe, multi-use oxygen-transporting product could significantly contribute and improve upon the care of acutely ill patients. Table I reviews the current status of the various categories of blood substitutes. We look forward to the introduction of one or more of these products in the near future. References: 1. Winslow R M. Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore and London, First Edition, 1992
410
DRACKER
2. Amin H, O’Leary C, Dracker R. Camporesi E, Hakim T. Hematologic changes during isovolemic hemodilution with human albumin, hetastarch or pentastarch in domestic pigs, The FASEB Journal, Vol 8, No 5 , March 18, 1994, pA9022 3. Pollack C V. Prehospital fluid resuscitation of the trauma patient. An update on the controversies.Emerg MedClinNorthAmer 1993, Feb;ll (1) : 61-70 4. Donner M, De Wachter P, Cauchois G, Gentils M, Kurtz M, Laxenaire MC, Stoltz JF. In vivo effects of plasma substitutes on the rheologic properties of blcwd, J Mal Vasc 1993; 18(2):126-133 5. Biro GP. Perfluorocarbon-based red blood cell substitutes, Transbs MedRev April; 7(2): 84-95
1993
6. Winslow RM. Vasoconstriction and the efficacy of hemoglobin-based blood substitutes, Transfus Clin Biol 1994; l(1): 9-14 7. Hess JR. MacDonald VW, Brinkley WW. Systemic and pulmonary hypertension after resuscitation with cell-freehernoglobin,JApplPhysiol1993April;74(4):1769-78 8. O’Donnell JK, Martin MJ, Logan JS, Kumar R. Production of human hemoglobin in transgenic swine: an approach to a blood substitute,CancerDetectPrev1993;17(2):307312
9. Takahashi T, Iwaski K, Malchesky PS, Harasaki H, Matsuchita M, Nose Y, Rolin H 3d, Hall PM. Renal effects of multiple infusions of pyridoxylated-hemoglobin-plyethylene conjugate (PHP) solution in dogs,ArtifOrgans1993 Mar; 17(3): 153-163 10. Zheng S. Zheng Y, Beissinger RL, Wasan DT, McCormick DL. Hemoglobin multiple emulsion as an oxygen delivery system, Biochim Biophys Acta 1993 Aug 20; 1158(1): 6574 J 1. Deshpande SV, Beissinger RL. Liposome-encapsulated hemoglobin using film hydration processing to form artificial red blood cells, Biomater Artif Cells Immobilization Biotechnol 1993: 21(2): 135-151
12. Usuba A, Miyazawa M, Motoki R, Sakaguchi K, Suzuki K, Kamitani T, Takahashi A. Oxygen transport capacity and hemodynamic effect of newly developed artificial lood “New Red Cells(NRC),Int J ArtifOrgans 1993 Jul; 16(7) : 551-556 13. Rudolph AS, Cliff RO, Klipper R, Goins B, Phillips WT. Circulation persistence and hidistribution of lyophilized liposome-encapsulated hemoglobin: an oxygen-carrying resuscitative fluid, Crit Care Med 1994Jan;22(1) : 142-150
NOVELCELLULAR
THERAPIES
Harvey G. Klein, M.D. Chief,
Department
of
Transfusion
Medicine
Warren G. Magnuson Clinical Center National Institutesof Health
INTRODUCTION Almost
years
25
have
passed
since
automated
blood
cell
separators
enabled
(1). clinical investigators to explore novel cellular immunotherapies
Although Kirkpatrick and his colleagues at the National Institutes of Health reported that allogeneic lymphocyte transfusions improved chronic mucocutaneous candidiasis in a patient with a congenital cellular immune deficiency,
more
than
a
decade
passed
before
further
efforts
to
infuse
allogeneic lymphocytes as a means of cellular immunotherapy were reported in human subjects ( 2 ) . recurrent
Two of these trials, one involving three women with
spontaneous
abortion
and
a
second
involving
patients
with I
type
diabetes mellitus, used small numbers of allogeneic cells, quantities easily collected without automated apheresis techniques,an in effort to induce tolerance immunity
in
conditions
(3.4).
believed
to
be
mediated by in disordered part
cellular
However, several seemingly unrelated developments stimulated
renewed interest in high-dose cellular immunotherapy: 1) The appearance of the acquired immunodeficiency syndrome ( A I D S ) provided a disease model of lethal 2)
progressive
cellular
immune
deficiency nowith known therapy.
Advances in basic and clinical immunology, especially in the field of tumor
immunology, suggested a role for the cellular immunotherapy of cancer. 3) The availability of recombinant
human
cytokine
growth
factors
presented
a
new
way
to manipulate cellular immunity ex vivo as well as in vivo. Selected examples of novel cellular therapies that have evolved from the confluence of new
technology
and
advances
in
basic
immunobiology
(Table I).
411
will
be
described
below
KLEIN
412
Table I HUMAN
MONONUCLEAR Selected
Immunotherapy
for
Kirkpatrick
CELL
THERAPY
Examples
Chronic
Mucocutaneous
Candidiasis
1 9 7 0( 2 )
Cellular Immunotherapy of Spontaneous Abortion Taylor
1 9 8 1 (41
Cellular Immunotherapy of AIDS Lane
1 9 8 4( 5 )
Cellular Immunotherapy of Type 1 Diabetes Mellitus Pozzilli
1985 (3)
-
Adoptive Immunotherapy of CancerLAK Rosenberg
Cells
and
TIL
1 9 8 5 - 1 9 8 6( 1 2 . 1 4 )
Cellular Immunotherapyof Leukemia Kolb
1 9 9 0 (23)
Somatic
Cell
Blaise
Gene
Therapy of SCID-ADA
1 9 9 0( 2 7 )
Immunotherapy ofE-B Virus Papadopoulos
AIDS
and AIDS
Lymphoproliferative
Disease
1994 ( 2 5 )
Immunotherapy is
a
protean
illness
resulting
in
severe
immune
suppression
and
death as a result of opportunistic infection or neoplasia. Destruction of the CD4+
subset of lymphocytes appears to be a critical event underlying the
severe immunodeficiency. TheCD4 membrane antigen is a high affinity receptor for the human immunodeficiency virus (HIV), although other receptors may exist. Monocytes, macrophages, glial cells and possibly bone marrow progenitors
may
also
Before HIV-1 had
harbor been
the
virus.
recognized
as
the
major
agent
responsible
for
AIDS,
investigators from several institutes at the National Institutes of Health developed
a
therapeutic
strategy
involving
immune
reconstitution
(5). syngeneic lymphocyte transfusion and bone marrow transplantation
using
The
initial protocol involved a of setmonozygotic twins discordant €or HIV (W), infection. Both subjects were immunized with keyhole-limpet hemocyanin
and peripheral blood lymphocytes (PBL) collected from the healthy twin were transfused to the afeected sibling at monthly intervals. After two months lOlo
nucleated
bone
marrow
cells
from
the
healthy
twin
were
transplanted
without a conditioning regimen. The patient demonstrated recovery of PEL and developed both fever and a maculopapular cutaneous eruption after most subsequent lymphocyte infusions. The CD4+ cell count peaked at three months
413
NOVEL CELLULAR THERAPIES
post transplant. While the patient had no response KLH to antigen stimulation before immunotherapy, he was able to mount a substantial delayed cutaneous for months. Despite the response subsequently and this response persisted immunologic
improvement,
the
patient's
clinical
condition
deteriorated he
and
succumbed to opportunistic infections. The
next
strategy
involved
combining
treatment
with
anti-retroviral
agents to inhibit viral replication with immune reconstitution ( 6 ) . Sixteen sets of monozygotic twins were treated in a fashion similar to that previously described, but with the addition of zidovudine (AZT) in therapy half of the affected twins. Once again, transient immunologic improvement could be demonstrated, but there was no sustained immunologic improvement or significant clinical improvement in either the AZT-treated group or in the controls. Two further trials involved treatment of HIV-infected subjects with ex
vivo
expanded
and
activated
syngeneic
lymphocytes
and
with
autologous
CD8+
cytotoxic T-cells from a clone specific for HIV-1 the nef protein; in both instances, expanded and activated lymphocytes appeared to be safe, but there was little evidence of efficacy (7). Phase I studies of adoptive immunotherapy
with
CD8+ T-cells are
being
carried
out
and
(8). for the cellular immunotherapy of infectious disease
may
serve
as
a
model
Future trials of
adoptive immunotherapy for AIDS will likely combine cellular therapy with different
medications
and
PEL
will
certainly
be
gene-modified
for
specific
cellular therapy. Lymphokine-activated Killer (LAK) Cells The discoveryin 1976 of T-cell growth factor, a cytokine later designated adoptive
interleukin-2
immunotherapy
(IL-2).
models
in
proved
pivotal
in
pursuing
newly
developed
animalsinand designing the later clinical
studies. In 1982, Grimm et al. reported that human lymphocytes cultured in IL-2 acquired the ability to lyse fresh tumor cells, as well as a variety of cultured cells, but not normal autologous or allogeneicincells, an in vitro assay system (9). This relatively non-selective, MHC non-restricted cytotoxic activity, has been termed the lymphokine activated killer ( W ( ) cell phenomenon. The cells involved appear to be a heterogeneous population, predominantly natural killer (NK) cells, rather than some newly-discovered specific cytotoxic lymphocyte a3 was originally postulated(10,ll). Extensive investigation
in
a
murine
model
using
splenocytes
cultured IL-2 defined in
the
optimum conditions for treating established tumors or pulmonary and hepatic metastases withW(-IL-2 therapy. Results of these studies predicted that the dose of cells necessary to treat human tumors would be in the 1011 range of cells,
that
produce
the
not
prove
concurrent maximum
high-dose IL-2 administration would be necessary to
effect,
beneficial.
and
that
adjunctive
chemotherapy
would
probably
414 The
availability
of
recombinant
human IL-2 provided
the
opportunity
to
undertake LAK cell clinical trials of adoptive immunotherapy. Using data from to design the murine model selected
patients
with
the a
protocol,
variety
of
Rosenberg advanced
et
al.
tumors
studied
25
unresponsive
highly
to
conventional therapy. Patients receivedan initial five-day course of bolus high-dose IL-2 (loO.000 u/kg every eight hours) therapy which resulted in systemic lymphopenia followedby a rebound lymphocytosis (12). Cells were then collected daily by lymphocytapheresis for five days, and cultured for four days in IL-2. The expanded cells were then washed, and different collections
were
combined
and
infused
over
several
days
with
continued
bolus
injections ofIL-2. The investigators hypothesized that LAK cells would traffic to the site of distant tumor metastases, expand in situ under the influence of systemicIL-2, and destroy the neoplastic cells. The early success, including several prolonged complete remissions, encouraged the investigators to expand this trial. More than 230 patients have been treated since 1984. of the large number of tumors treated, renal cell carcinoma, melanoma, colorectal carcinoma, and lymphoma appear to respond best with a 10
percent
complete
response
rate
and
a
20
percent
overall
response.
Several aspectsof LAK cell therapy have discouraged its continued use. First,
the
response
method
has
been
rate
is
lower
than
predicted
found to predict
which
patients
will
in
the
respond
to
animal this
models
and
therapy.
LAK cells do not traffic to well distant sites when studied with radioisotopic labeling techniques. The most probable explanation is that only about 15 percent of the cultured cells show LAK activity, far fewer than would be necessary for optimum therapy. Second, significant toxicity has been noted including dose-related capillary vascular permeability, pulmonary edema, shock, renal insufficiency, intellectual impairment, coma, anemia and thrombocytopenia. These side effects are related to high-dose the IL-2 and are reversible. The treatment-related mortality approximates two percent. Third, controlled studies IL-2 of alone indicate that LAK cells prepared by current techniques do not add very much to the treatment. Finally, LAK cell therapy remains technically demanding and expensive (13). Tumor Infiltrating Lymphocytes (TIL) The search for immunologically active cells with higher therapeutic efficacy
resulted
in
the
description
of
T-lymphocytes a ofclass
with
more
specific antitumor activity, so-called tumor-infiltrating lymphocytes (TIL) (14). TIL are lymphocytes isolated from resected tumor by culturing single cell suspensions of minced, enzyme digested tumor for up to six IL-2. weeks in In these cultures, tumor cells fail to grow, while the lymphocytes contained in the tumors, cells theoretically "programmed" for this specific tumor, expand by several logs. In the murine mode and in vitro, TIL toproved be up to 100 times as potent as LAK cells. TIL differ from LAK cells in several
no
415
NOVEL CELLULAR THERAPIES ways.
TIL are relatively specific cytotoxic T-cells that recognize tumor
antigens in conjunction with MHC class I determinants. are CDB+, although both Finally, in effect.
cD4+
and cD8+ TIL
In the mouse, most TIL
are isolated from human tumors.
themurine model, cyclophosphamide treatment markedly enhanced TIL
This phenomenon was ascribed initially to suppression of some
blocking factor or of an inhibitory lymphocyte subset; it now appears that cyclophosphamide has a direct effect on the tumor that enhances subsequent TIL act
ion. TIL have now been generated from approximately 80 percent of more than
300 human tumors (15).
More than 100 patients have received TIL therapy
according to several protocols that are variations of the LAK protocol.
TIL
often traffic well to distant tumor sites and accumulate in tumors for at least several days after infusion (16). Gene-marked TILcan be detected at tumor sites for months (17). A 38 percent overall response rate has been reported in patients with metastatic melanoma (18). Tumor responsiveness correlates to some extent with HLA class I1 antigen expression on the tumor cell surface and in vivo responsiveness can be predicted by gamma interferon release by TIL
co-culturedwith tumor.
Adoptive immunotherapy of cancer is following several related investigative paths.
Efforts are underway to enhance tumor expression of HLA
antigens as well as tumor-specific antigens.
Initial studies attempting to
repeatedly stimulate lymphocytes in culture with tumor cells or synthetic antigens suffered from problems with bacterial contamination of cultures, but efforts to enhance both specificity and potency of TIL and
W(
are continuing.
Finally, large-scale culture technology and the ability to transduce dividing cells with a variety of genes and vectors have stimulated intense activity directed toward increasing cellular cytotoxicity, tumor recognition, and cytokine release by a variety of gene insertion methods.
Graft-versus-leukemia
(GVL)
and Lymphoproliferative Disorders
The first experimental evidence that transplanted bone marrow might play an active role
ineradicating murine leukemia appeared almost 4 0 years ago
(19). Analysis of human marrow transplantation data has provided supportive, if indirect, evidence of an immune-mediated
GVL
effect:
1) Transplanted
patients with chronic myelocytic leukemia (CML) demonstrate an inverse relation between relapse rate and degree of graft-versus-host
disease(GVHD),
suggesting a potent graft immune response is associated with eradication of leukemia ( 2 0 ) .
2) A direct correlation has been found between relapse rate
and degree of T-cell depletion (21). suggesting that a lack of allogeneic immune mediators may permit tumor persistence.
3) Higher relapse rates have
been noted when syngeneic transplants are compared with allogeneic transplants (22).
The first direct clinical evidence for the GVL effect appeared when
three patients with relapsed CML achieved cytogenetic remission after
KLEIN
416 treatment with donor mononuclear cells and alpha interferon (23) been ample confirmation of this observation. 5 0 CML
There has
Aggregate studies of more than
patients indicate a clinical response rate exceeding 8 0 Percent and
cytogenetic remission, as assessed by polymerase chain reaction analysis. in the range of75 percent.
No dose response effect has been noted for
lymphocyte doses between 0 . 3 4 and 12.3 x lo8 cell/kg.
A SUIPriSin9
observation was the relatively mild degree of GVHD in patients receiving lymphocyte or buffy coat infusions, in view of the large number of allogeneic T-cells infused. Recent experience suggests that post-transplant response rates for patients with relapsed multiple myeloma, acute leukemia, lymphoma, and myelodysplasia treated with donor leukocyte infusions are substantially lower than that for patients with CML (24). Although the mechanisms of this GVL effect are poorly understood, indirect evidence suggests that both NK
cells and cytotoxic T-cells play an important role. Allogeneic donor lymphocyte transfusions in the T-cell depleted transplant setting have recently been reported as successful therapy for Epstein-Barr virus (EBV)-associated lymphoproliferative disorders ( 2 5 ) .
Five
patients who underwent T-cell depleted allogeneic transplants developed EBVpositive immunoblastic lymphoma.
The lymphoma regressed 8 to 21 days after
treatment with approximately lo6 CD3+ T-cells/kg, and remissions were achieved within 14 to
30
days.
These remissions appear to be durable.
The success of this early report suggests several other potential applications of mononuclear cell transfusion, for example prophylactic T-cell infusions, ex Vivo expansion of EBV-specific CD8+ lymphocytes, and selective removal of the contaminating lymphocytes that mediate GVHD in patients treated with allogeneic bone marrow transplants. Cellular adoptive immunotherapy might be further extended to suppress other viruses, such as HIV, cytomegalovirus in the transplant setting, or suspected oncogenic viruses such as human papilloma virus, hepatitis B virus, or hepatitis
c
virus.
Cellular Gene Therapy cellular gene therapy is a therapeutic technique in which a functioning gene is inserted ex vivo into the somatic cells of a patient in order to correct an inborn genetic error or to provide a new function to the cell. Since gene insertion is limited intentionally to somatic cells, the inserted gene does not enter the patient's germline and thus is not transmitted to human gene pool.
the
While the concept of cellular gene therapy seems remarkably
straightforward, only within the past decade have the tools become available to characterize disorders that might be potential targets of this treatment. Ironically, these same tools have identified unanticipated problems that currently limit widespread application of this technique (26). Several conditions must be met before gene therapy can be undertaken: 1) the gene of interest must be cloned; 2) an appropriate vector must be
NOVEL CELLULAR THERAPIES
417
available to deliver the gene to the target cell;3 ) there should be preferential bindingto the target cell and the efficient delivery of the gene to the cytoplasm or the nucleus; 4 ) the gene must be expressed in a stable fashion, in an appropriate tissue, at the appropriate level; and 5 ) expression of the gene must reverse the pathologic manifestations of the disease. The last condition is particularly important. Homozygous disorders in which the heterozygous state is well managed by drug therapy, for example familial hypercholesterolemia, diseases like Gaucher disease that are "cured" by bone or incompletely marrow transplantation, and disorders that respond temporarily
to alternative therapy, such as chronic granulomatous disease, are all candidates for cellular gene therapy. A selection of candidate diseases for cellular gene therapy is listed in 11. Table Cellular gene therapy may involve a variety of different
tissues
and
different treatment strategies. Early efforts have focused on easily accessible cells such as blood cells and bone marrow, however skin fibroblasts, vascular endothelial cells and hepatocytes are additional candidate targets. The ease of obtaining such cells as lymphocytes, monocytes and
peripheral
blood
progenitor
cells
by
cytapheresis
makes
these
cells
excellent candidates for a vaxiety of cellular gene therapy protocols. The strategy employed for the first successful somatic cell gene therapy used autologous peripheral blood lymphocytes (PBL) as a temporary drug delivery system: the "drug" was the gene product, adenosine deaminase (ADA), and ADA was vivo
delivered and
by
inserting
re-infused
into
the an
immunodeficiency syndrome( 2 7 ) .
ADA
genePBLinto which
ADA-deficient
were
patient
then with
expanded severe
This model, which takes advantage of
apheresis technology and the cell expansion methods developed for LAK cell TIL
therapy,
will
be
ex
combined
discussed
in
detail
and
below.
An approach to treating infectious diseases by cellular gene therapy involves interruption of the life cycle of the infectious agent: autologous lymphocytes from patients with HIV-infection, for example, can be genemodified by insertionof custom-designed "resistance genes" (28).
Several
potential cellular gene therapy Strategies planned for the immunotherapy of cancer have been mentioned previously. Finally, permanent correction or modification of blood
disorders
can
be
achieved
by
modifying
purified
hematopoietic progenitor cells so that the gene of interest will be expressed in all hematopoietic progeny (29). The first gene therapy trials
were
undertaken
at
the
National
Institutes
of Health in September1990 (27). The model selected, severe combined immunodeficiency syndrome(SCID) resulting from inherited ADA deficiency demonstrates many of the basic principles of cellular gene therapy. ADA deficiency
is
a
single
gene
defect
Tesulting
in
an
autosomal
recessive
disorder easily recognized early i n life. About one thirdof all SCID patients have an ADA defect. There is a quantitative assay for the deficient
418
KLEIN TABLE I1
CELLULAR GENE THERAPIES IN DEVELOPMENT Genetic Diseases Adenosine deaminase deficiency Gaucher disease Hunter/Hurler mucopolysaccharidosis Chronic granulomatous disease Fanconi anemia Hypercholesterolemia Hemoglobinopathies
Hemophilia8
Infectious Disease AIDS CMV
Neoplastic Disease CML
Melanoma Breast cancer Ovarian cancer Lymphoma
enzyme and the ADA gene has been cloned.
The course of the disease involves
recurrent infections, and death occurs ordinarily during childhood or adolescence.
Although ADA is deficient in every tissue, the target cell is
the T-lymphocyte. untreated patients experience gradual deterioration of the immune system with decreasing T-lymphocytes, and eventually B-lymphocytes, should the patient survive long enough.
The phenotype can be reversed by bone
marrow transplantation and in selected patients by the infusion of ADA bound
.
to polyethylene glycol (PEG-ADA)
The pathophysiology of this disease apparently involves toxic purine metabolites (deoxyadenosine, adenosine) that accumulate in the absence of ADA. The
T-lymphocyteis particularly vulnerable since this cell has the highest
concentration of kinases that convert adenosine to the toxic phosphorylated derivatives.
As little as five to ten percent of normal ADA is sufficient to
maintain some subjects with ADA deficiency in good health and increases of ADA
419
NOVEL CELLULAR THERAPIES 30-50 times normal do not appear to be toxic.
The ADA gene inserted and
expressed in lymphocytes collected from SCID patients confers a gxowth advantage on such cells in culture and gene-corrected cells circulate and express ADA for months in animal models.
A l l of these characteristics
suggested that ADA deficiency would be an appropriate disorder for cellular gene therapy.
Two children at NIH had PBL removed by apheresis, transduced with a modified MolOney retroviral vector containing the gene coding for ADA, and reinfused after ex vivo expansion. of corrected cells.
Both children received monthly infusions
After eight infusions in ten months, the first patient
increased the number of circulating T-cells in the normal range, and ADA levels increased from 2% to 20% of normal.
Gene coxrected cells cleaxly
enjoyed the same in vivo survival advantage as had been observed in vitro. Measurements of immune function such as skin test reactivity, isohemagglutinin titers, and T-cell cytotoxicity improved significantly.
Both children are now
in school, growing and developing normally, and showing no stigmata of SCID. Both children will receive gene-corrected CD34+ progenitor cells collected by apheresis in an attempt to effect permanent cure. Cellular gene therapy remains highly investigational, difficult, and expensive.
However, the early results are promising.
Alternative strategies
will certainly develop from the initial clinical experience and improved methods for each step of this complicated pxoceduxe are already under development.
Cellular gene therapy will likely play a major role in the
investigation and treatment of a wide range of diseases during the next several years and cytapheresis will certainly be involved in many of these studies.
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Taylor and W.P.
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H.J. Kolb, T. de Witte, J. Mittermueller, et al. The American Society of Hematology 35th Annual Meeting Abstracts, (1993) p. 214a.
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PART V: TRANSF'USION STRATEGIES
This Page Intentionally Left Blank
TRANSFUSION STRATEGIES: OPPORTUNITIES FOR IMPROVEMENT J. E. Menitove Hoxworth Blood Center and Department of Internal Medicine University of Cincinnati Collegeof Medicine Cincinnati, OH 45267-0055 ABSTRACT
Opportunities for improving transfusion practice that involve patient care, technological advancements, and technology changes in the clinical arena are discussed. Patient care should be enhanced by optimizing transfusion therapy and the source of donors of platelet concentrates, i.e., single donor platelets (obtained by plateletapheresis from a single donor) or to random donor platele (containing a pool of six to eight platelet concentrates separated fromwholeblooddonations).Theavailabilityof"thirdgeneration" leukocyte-reduction filters provides the technology for significantly and consistently providing leukocyte-reduced blood components. The potential benefits of using these filters is presented. Platelet crossmatching represents a technology change involving clinical practice. Suggestions for incorporating this test into platelet transfusion algorithms are included. INTRODUCTION
Optimizing the risk:benefit ratio for transfusion is a longstanding goal of transfusion medicine specialists. Previously, guidelines for transfusion of fresh frozen plasma (FFP), platelets, and red blood cells (RBCs) were discussed and published as Consensus Conference Reports and practice parameters.(" Currently, opportunities for improvement remain. This discussion will emphasize several areas for investigation and consideration.Itisnotanexclusivelist,butservesto highlight approaches to patient care, technological advancements, and technology changes in the clinical arena. Equally compelling issues that are outside the purview of the current discussion include the use of pharmacologic agents for avoiding transfusion, such as recombinant human erythropoietin, other hematopoietic growth factors, and cytokine/peripheral blood stem cell therapy; pharmacologic agents that promote hemostasis, such as aprotinin an 423
424
MENITOVE
desmopressin; and oxygen-carrying substitutes, such perfluorochemical or hemoglobin solutions. APPROACHES TO PATIENT Appropriate use of transfusion
as
CARE
Compliance with recommendations urging appropriate use of transfusion therapy is audited by transfusion committees and transfusionservicedirectors.Fortunately,thecriteriaare reevaluated and updated periodically. For example, the transfusion-trigger for red cell transfusions, once considered sacrosanct at a hemoglobin concentration of 10g/dL and a hematoc of 3 0 % ("10/30 rule"), was replaced by 117/2711 and 1'8/2411 triggers severalyearsago.Todaymanyregardtheconceptofa lltransfusion-triggerll as obsolete. Instead, clinical judgement forms the basis for deciding when to order transfusions rather a pre-set hemoglobin/hematocrit value.(6-7' Clinical judgement involves an understanding of the physiologicmechanismsthatcompensateforanemiarelated, diminishedtissueoxygendelivery as welland an accurate assessment of the patient's clinical status. Compensatory mechanisms include increasing cardiac output by raising the heart rate or stroke volume, enhancing oxygen off-loading as a result of elevated 2,3 diphosphoglycerate (2,3 DPG) levels or the Bohr effect, increasing coronary artery blood flow, shunting blood from areas of lower oxygen need to regions with higher demand, and increasing respiratory rate. When the compensatory mechanisms of cerebral ischemia, such as syncope fail, patients develop signs and transient ischemic attacks; or signs and symptoms of cardiac hypoxia, such as tachycardia, angina, and postural hypotension. Dyspnea also develops. These clinical events signal a need for additional oxygen-carrying capacity, i.e., red cell transfusion. Counterbalancingthebenefitsoftransfusionarethe associated risks that include transfusion reactions, transmission of infectious agents, immunomodulatory aspects, and other adverse events . Although transfusions are ordered when the hemoglobin is less than 8g/dL and the hematocrit is less than 24%., the ultimate judgement for deciding to transfuse relies on clinical judgement. The clinician should evaluate whether the anemia is acute or chronic and whether bleeding, ifany, is on-going. In addition, the level of fitness and activities, the age, and the level of monitoring should be considered."'
TRANSFUSION STRATEGIES
425
Eliminating the lltransfusion trigger" concept requires astute clinicalskills.Notwithstanding,cardiacischemiamayoccur silently. For example, patients undergoing peripheral vascular bypass surgery demonstrated EKG evidence of ischemia in the absen of a change in heart rate when the hematocrit was less 28%.than From a pragmatic viewpoint, clinicians will continue to rely on a "transfusion trigger". An opportunity for improvement in transfusion strategy awaits better methods for assessing tissue hypoxia, especially those sufficiently flexible to be of value in wide-ranging settings such as acute gastrointestinal bleeding or sepsis with pulmonary insufficiency. Platelet
transfusions
The I1transfusion triggere1 for prophylactic platelet transfusions has been reassessed also. The sentinel work by Gaydos et al., and its reevaluation by Freireich etal.,'l2) in ZO,OOO/pl platelet count as the predictive the 1960's established a point for thrombocytopenic hemorrhage. Although these conclusions were confounded by a subsequent realization that many of the patients in the above cited study received aspirin, few seriously challenged the 8120,000/p1platelet trigger" until recently. An opportunity for improvement rests with a greater acceptance and a of the clinical settings in which better understanding thrombocytopenia hemorrhage occurs. Solvent/detersent
fresh
frozen Dlasma
The availability of virally inactivated fresh frozen plasma (FFP), i.e., solvent/detergent treated FFP(S/D FFP), presents a potential challenge to the concept of transfusion avoidance." Assumingtheviralinactivationprocess is effectiveand breakthrough infections are restricted to less dread complications, such as hepatitis A and parovirus infection, will this new produc receive wide-spread acceptance? Will the uses of S/D FFP be similar to those currently in place for FFP or will they be expanded since the product is llsafell?The answers to these questions should provide the background for future transfusion strategies and set the stage for acceptance of other technological achievements. Source of donors
Another patient care issue involves the source of donors for platelet transfusions: multiple random donors providing a pool of six to eight platelet concentrates (RDP, random donor platelets) or
426
MENITOVE
a single donor providing an equivalent dose of platelets obtained by plateletpheresis (SDP, single donor platelets). There is a paucity of information to support the selection of SDP. RDP versus Intuitively, SDPs present less risk of disease transmission if one assumes the risk is linear per donor exposure, i.e., the risk of transfusion-transmitted infection is eight-fold higher if one RDPs compared to anSDP. The exposure to receives eight units of foreign antigens is decreased leading to a concomitant diminution intherate of alloimmunizationoroccurrenceoffebrile, transfusion reactions. In addition, if platelet destruction occurs on the basis of an “innocent bystander” reaction, it is more lik RDP that an donor is contained in pool a of eight than an SDP.(14’ However, proof is lacking that platelets are consumed by an “innocent bystander” process following transfusion of “incompatible” platelets. It is equally intuitive to suggest that a “compatib1e1l platelet concentrate is more likely to be SDP present in a pool of platelets from eight donors anrather than from a single donor. Nonetheless, SDP usage increased markedly during the past several years. An additional opportunity for transfusion improvement lies in an assessment of the relative benefit of selectingSDPs or RDPs for transfusion. TECHNOLOGICAL ADVANCES Leukocyte-reduction filters Technologicaladvancements also contributetoimproved
transfusionstrategies.Thirdgenerationleukocyte-reduction filters provide a consistent method for producing components with 5X106 leukocytes, a level considered sufficient to less than stimulate an alloimmune response in transfusion recipients. Proposed benefits of “third generation” leukocyte-reduction filters include: (a) reduction in the rate of alloimmunization to HLAantigens, (b) prevention of cytomegalovirustransmission through transfusion, (c) prevention of recurrent “chill-feverrl or febrile, non-hemolytic transfusion reactions, and (d) amelioration of immunosuppressive effects of transfusion. (l5’ However, enthusiasm for wide-scale use of these filters has been dampened because of cost considerations and concern about efficacy in fulfilling the purported benefits. Several, recently published reports provide strong support f those advocating expanded use of these filters. Most published studies indicate that patients receiving leukocyte-reduced red c and platelet components have lower rates of HLA alloimmunization than patients receiving non-leukocyte-reduced components.
TRANSFUSION
427
Unfortunately, the methods for achieving leukocyte-reduction were not similar, the number of patients in each study was relatively small, and some of the reports compared present results with historic data. In contrast, animal model studies address this issue in a more defined manner. One found that blood subjected to pre-storageleukocyte-reductionwasassociatedwithalower incidence of platelet refractoriness (approximately 30%) than observed when leukocyte-reduction was performed after storage In followup studies, platelet (approximately 67%) . (16s17) refractoriness was decreased in rabbits given leukocyte-reduced and plasma-reduced red cell transfusions (0%) compared to red cells subjected to leukocyte-reduction alone.'17) These two studies, conducted in the same laboratory, suggest that alloimmunization is induced by contaminating leukocytes, but that factors present in the supernatant plasma such as soluble HLA antigens or microparticles may be involved also. While provocative, it is clear that further investigation, especially studies involving humans, is required for understanding the optimal use and timing for filtration to prevent alloimmunization. The Trial to Prevent Alloimmunization to Platelets (TRAP), a multi-center, National Heart, Lung, and Blood Institute sponsored study, is currently in progressandshouldprovideinformationabouttheroleof leukocyte-reduction in preventing alloimmunization to platelets. Another benefit of leukocyte-reduction involves interdiction of transmission of intracellular viruses. The result of a large clinical trial indicates third generation filters are equivalent to serological testing prevent to transfusion associatedcytomegalovirus infection. The levels of several cytokines, including tumor necrosis factor-a, interleukin-l, interleukin-6, and interleukin-8 increase ("' in the plasma of platelet concentrates during storage. Recipients of platelet concentrates with relatively high cytokine levels develop febrile, non-hemolytic transfusion reactions. Of note, cytokine levels do not increase in platelet concentrates if the leukocytes are removed prior to storage. Again, the benefit of pre-storage versus post-storage leukocyte reduction is contrasted, thereby presenting a potential opportunity for improved transfusio strategy. Investigations of immunomodulatory effects of blood transfusion currently focus on the etiologic role of contaminating leukocytes. For example, a higher incidence of post-operative infection was reported in recipients of non-leukocyte-reduced bloo
MENITOVE
428
than to those given leukocyte-reducedor blood no transfusion (23%, 2%, 2% respectively) . (''l Leukocyte infusion is implicated as a potential cause of latent virus activation. Addition of allogeneic mononuclear cells to HIV-infected mononuclear cells resulted in release of HIV p24 antigen. Activation did not occur when cells were incubated with non-leukocyte containing red cells, platelets, or plasma. (") Another immunomodulatory effect of transfusion, decreased cancer-free survival of patients transfused during tumor resection surgery, has been investigated recently in a series of animal m experiments. It appears that leukocyte-reduced blood ameliorates transfusion-induced enhanced tumor growth."" Preliminary data suggest that metastases develop when leukocyte-reduction was performed following storage, but was abrogated if leukocytes were removed at the time of blood collection.(24' This tantalizing observation provides further support for studying the optimal time for removing white cells from blood components as a strategy for improving transfusion practice. TECHNOLOGY CHANGES IN THE CLINICAL ARENA Platelet crossmatchinq
The final issue for discussion involves technology changes in the clinical arena. Platelet crossmatch techniques have been under discussionfordecades.TheapparentsuccessofHLA-matched platelet transfusions, in part, decreased enthusiasm for platelet crossmatching.Also,mostplateletcrossmatchstudieswere to provide sufficient, positive predictive retrospective and failed or negativepredictivevaluesforendorsingthetechnique. Investigators used fluorescence microscopy, FACS scanning, radioisotope tagging, and enzyme immunosorbent assays. Platelets were mixed with patient sera and platelet-associated immunoglobul was measured. Utilization of platelet crossmatching increased after the solid phase red cell adherence assay became available commerciall One group of investigators used the assay to find serologically compatible R D P s foralloimmunizedpatients.If an RDPwere associatedwithareasonableposttransfusionplateletcount increment, the donor was recalled for plateletpheresis. Overall 3% to 13% of platelet concentrates were compatible; and the majority of crossmatch negative platelet concentrates provided acceptable (251 Recently, it has been posttransfusion platelet count increments. shown that approximately 50% of crossmatch compatible S D P s provide adequate posttransfusion platelet count increments compared to no
TRANSFUSION STRATEGIES
429
increment if crossmatch incompatible SDPs are given.(26' If50% a success rate is compared to the usual expectation for red cell compatibilitytesting,plateletcrossmatchingfarespoorly. However,thebestalternativetoplateletcrossmatchingis selectionofSDPdonors on thebasisof HLA matching. Unfortunately, only 60% of HLA-matched platelet concentrates provide adequate posttransfusion count increments. As such, the success rate for crossmatching compares favorably with the current "gold standard," HLA-matched platelet transfusions. Hence, this technology provides an opportunity for improving transfusion practice andis likely to find an appropriate niche in transfusion strategy. CONCLUSION
Inconclusion,theareasthatwerediscussedprovide opportunities for improving transfusion therapy. Each was chosen Their to highlight an approach to patient care in the 1990's. implementationrequirestransfusionmedicinespecialiststo appreciate the benefits and to encourage clinicians to integrate them into the transfusion strategy decision making process. REFERENCES 1.
JAMA, 2 5 3 , 551-553 (1985).
2.
JAMA, 2 5 7 , 1777-1780 (1987).
3.
JAMA, 2 6 0 , 2700-2703 (1988).
4.
JAMA,
5.
M. Contreras, F.A . Ala, M. Greaves, et al., Trans Med, 2,
6.
H. G. Welch,K. R. Meehan,L. T. Goodnough, Annal Intern Med,
271,
777-781 (1994).
57-63 (1992).
116, 393-402 (1992). 7. 8. 9.
Annal Intern Med,116, 403-406 (1992). R. J . Faust, Mayo Clin Proc,a, 512-514 (1993). A . H. Nelson, L. A. Fleisher, S . H. Rosenbaum, Crit Care Med, 2 l ,
10.
860-866 (1993).
E. Beutler, Blood, 8 l , 1411-1413 (1993). L. A. Gaydos, E.J . Freireich, N. Mantel, New EnglJ Med,
11.12.
266,
13. 14.
905-909 (1962).
E. J . Freireich, Transfusion, 6 , 50-54 (1966). B. Horowitz, R. Bonomo, A. M. Prince, etal., Blood,7 9 , 826831 (1992).
15.
R. H. Herzig, G. P. Herzig, M. I. Bull, et al., Blood, 46, 743-750
.
(1975)
430
MENITOVE
16.
H. G. Klein, Blood, 8 , 1865-1868 (1992).
17.
M. A . Blajchman, L. Bardossy,
R.A . Carmen, etal., Blood, 2,
1371-1375 (1992). 18.
J. 0. Borden, L. Bardossy, M. A. Blajchman, Transfusion,3 3 ,
19.
N. M. Heddle, L.N. Klama, L. Griffith, et al., Transfusion,
798-801 (1993). 33,
20.
794-797 (1993).
L. Muylle,M. Joos, E. Wouters, et al., Transfusion,33 195-
.
199 (1993) 21 * 22. 23. 24.
G. Stack, E. L. Snyder, Transfusion,3 4 , 20-25 (1994). L. S. Jensen, A. J. Andersen, P. M. Christiansen, et al., Brit J Surg, 7 9 , 513-16, (1992). M. P . Busch, T. Lee, J. Heitman, Blood,EO, 2128-2135 (1992). M. A. Blajchman, L. Bardossy, R. Carmen, et al., Blood,8 1 , 1880-1882 (1993).
26.
J. 0. Bordin, L. Bardossy, D. P. Singal, M. A . Blajchman, Blood G, Suppl 1, 392a, (1993). B . A . O’Connell, E. J. Lee, K. Rothko, et al., Blood, 7 9 , 527-
27.
R. C. Friedberg, S. F. Donnelly, J. C. Boyd, et al., Blood,
25.
531 (1992).
81, 3428-3434
(1993).
TRANSFUSION-ASSOCIATED IRRADIATION
GRAFT-VERSUS-HOST OF
BLOOD
DISEASE
AND
THE
COMPONENTS
Richard J. Davey, M.D. National Institutes of Health Bethesda, Maryland
Abstract Transfusion-associated
graft-versus-host
disease
(TA-GVHD)
is
a rare but lethal disorder caused when viable donor lymphocytes
engraft and proliferate ina susceptible transfusion recipient. Patients with immune deficiency disorders, hematologic malignanci and bone marrow transplants are at risk to TA-GVHD, as are prematurenewbornsandtransfusionrecipientswhoareHLA heterozygous for an HLA-haplotype that is shared with an HLA homozygous donor. Irradiation of blood components with 2 5 0 0 cGy will inactivate donor lymphocytes and prevent TA-GVHD. Platelets and granulocytes are not functionally impaired by this radiation dose, but red cells sustain detectible damage. Red cell units irradiated and stored for 4 2 days have significantly higher supernatant recovery of chromium-51 labeled cells is sub-optimal. Based on these data, the maximum permissible storage time for irradiated red cells has been reduced 2 8 days. to Text of Manuscript
(TA-GVHD) is a rare but often Transfusion-associated graft-versus-host disease lethal complicationof transfusion caused when immunocompetent lymphocytes in donor blood engraft, proliferate, and mount an immunologic reaction against a transfusion recipient. Most casesof
TA-GVHD occur soon after the implicated
transfusion (median8 days; range 3-30 days). The illness usually follows a fulminant course with fever, skin and gastrointestinal involvement, and bone
TA-GVHD are usually fatal(>go%)), marrow hypoplasia. While reported cases of milder formsof the syndrome may not be recognized or reported. Exposing blood components to an appropriate dose of gamma-irradiation will inactive donor lymphocytes and prevent TA-GVHD.
Irradiated blood components
are indicated for defined patient groups in which TA-GVHD has been documented. (1) (Table I )
Most cases of
TA-GVHD have occurred in
immunocompromised patients. Cases have also been reported in immunocompetent 43 1
DAVEY
432
TABLE I Clinical Indicationsfor Blood Component Irradiation Clearly Indicated Recipients of Allogeneic or Autologous Marrow, or Peripheral Blood Stem Cell Transplants Congenital Immune Deficiency Syndromes Intrauterine Transfusions Hodgkin’s Disease Directed Donations from Blood Relatives Probably Indicated Acute Leukemia Non-Hodgkin’s Lymphoma Premature Infants (
Not Necessary
-
Term Infants
AIDS Aplastic Anemia Most Solid Tumors
recipients who have received transfusions from donors who are homozygous for an
HLA haplotype shared with the HLA heterozygous recipient.(2,3) Directed donations from family members have a greater likelihood for similarity in the
HLA system(4) and shouldbe routinely irradiated. The precise number of donor lymphocytes that can cause TA-GVHD in a susceptible recipient is not known. However, children with congenital immunodeficiency
TA-GVHD after transfusionof fresh, unfrozen plasma syndromes have developed containing approximately 1.5 x lo5 lymphocytes per unit.(5) Leukocyte-reduction of leukocytes to below1.0 x IO5 cells per filters can now reduce the number of leukocyte-reduction may not always be achieved. unit, although this degree
Leukocyte reduction may be effective, therefore, in preventing TA-GVHD for many susceptible patients. However, the small numberof lymphocytes remaining in some filtered units may place some patients at risk. Until further data are available regarding the efficacy of leukocyte-reduction in preventing
TA-GVHD,
blood component irradiation remains the procedure of choice for patients at risk.
A radiation doseof 500 Lymphocytes are very sensitive to gamma-irradiation. cGy (500 rad) will abolish trmphocyte activity in the mixed-lymphocyte culture
433
GRAFT-VERSUS-HOST DISEASE
TABLE I1 21 Days” IR NI 40.7
”
3.6
Plasma hgb (mg/dL) 32.2 Potassium (meq/L) 36.0 63.4 1.9 hgb) ATP (uM/g 3.2 RBC recovery (%) 90.4 82.7 NI = Not Irradiated IR = Irradiated
28 Days” NI IR IR 71.2 76.0 47.7 69.4 2.1 3.8 3.5 85.0 80.7
35 Days” NI IR 174.5 314.0 428.6 623.1 50.0 68.3 42.6 78.1
81.8
78.0
42 Days” NI
78.4 68.5
(MLC) assay, while a dose of 2500 cGy will completely abrogate lymphocyte clonal proliferation in limiting-dilution studies.(6) 2500 cGy is now the recommended dose for blood component irradiation. Small cesium-l37 irradiators can deliver a dose of 2500 cGy within 1.5 to 3.0 minutes. These irradiators, while simple to operate, are very expensive and require accurate calibration and periodic quality control procedures to insure safe and accurate operation. (7) Irradiation injures redcells, perhaps by damage to membrane proteins and lipids from radiation-generated oxygenfree radicals (Table 11). Although this injury takes place at the moment of irradiation, several days to weeks may pass before such damage i s evident. The stress of long-term storage in artificial media may accentuate the effectsof irradiation-induced injury. Irradiated units have higher supernatant hemoglobin and potassium levels, and lower red cell ATP than non-irradiated controls. Althoughthe elevated potassium in irradiated units is of little consequence to most recipients,(8) transfusion of stored, irradiated red cells should be avoided in neonates.(9) We have demonstrated a significant decrease in 24-hour posttransfusion recovery of red cells irradiated with 3000 cGy and storedfor 42 days, compared to paired non-irradiatedcontrols.( 10) Friedman and colleagues( 11) and Mintz and Anderson(l2) have shown reduced, but acceptable, red cell recovery after irradiation and subsequent storagefor 21, 28 and 35 days. Red cells can be irradiated and subsequentlyfrozen, however, with no detectable progression of irradiation-induced damage during the period of frozen storage.(l3) Based on these data, the Blood Product Advisory Committee of the Food and Drug Administration has recommended that irradiated red cells should not be stored for more than 28 days from the timeof irradiation. Irradiation of platelet concentrates after collection results in no detectable damage following storage. Read and colleagues demonstrated no adverse effects
434
DAVEY
on platelet recovery and survival after 3000 cGy irradiation followed by five days of storage.(l4) Most studies suggest that granulocytes do not demonstrate
(15) significant functional damage from standard doses of irradiation. In summary, irradiation of blood components that contain viable lymphocytes
TA-GVHD. with 2500 cGy will prevent
The indications for irradiated blood
components are increasing as additional cases TA-GVHD of are recognized. Although most cases of TA-GVHD have occured in immunocompromised patients, the disease has occured in immunocompetent patients as well. Irradiation damages red cells, which should be transfused within 28 days following irradiation. Platelets and granulocytes do not demonstrate functional damage following irradiation and subsequent storage.
REFERENCES
2.
S.F. Leitman, in SDecial Considerations in Transfusinq the ImmunocomDromised Patient,D.M. Smith and A.J. Silvergleid, eds, American Associationof Blood Banks, Arlington, VA, (1985) pp. 15-37. M. Thaler, A. Shamiss, S. Orgad, et al. New Engl J Med, U, 25-8,
3.
T. Juli, K. Takahashi, Y. Shabita, et al. New Engl J Med,
1.
(1989).
4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15.
3 2 1 , 56, (1989). L.D. Petz, L. Calhoun, P. Yam, et al. Transfusion, 33, 742-5, (1993). B.H. Park, R.A. Good, J. Gate, B. Burke. Transplant Proc, 6, 385-7, (1974). M. Pelszynski, G. Moroff, N. Luban, B. Taylor, R. Quinones. Blood, 18, 275a, (1991). G.A. Anderson, in Irradiation of Blood ComPonents. M.L. Baldwin and L. Jeffries, eds. American Association of Blood Banks, Bethesda, MD, (1992) pp. 63-76. R.G. Strauss. Transfusion, 3 , 675-77, (1990). T.L. Hall, A. Barnes, J.R. Miller, D.M. Bethencourt, L. Nestor. Transfusion, 33, 606-9, 1993. R.J. Davey, N.C. McCoy, M. Yu, J.A. Sullivan, D.M. Spiegel, S.F. Lei tman Transfusion,. 3 _2 ., 525-8. (19921. K.D. Friedman, W.C. McDoiough; D.F.Cimino. Transfusion, 3 , 50S, (1991). P.D. Mintz, G. Anderson. Ann Clin Lab Sci, 2 3 , 216-20, (1993). B.A. Suda, S.F. Leitman, R.J. Davey. Transfusion, 3 3 , 389-92, (1993). E.J. Read, C. Kodis, C.S. Carter, S.F. Leitman. Transfusion, 2 8 , 446-50, (1988). D.T. Eastlund, T.T. Charbonneau. Transfusion, 2 8 , 368-70, (1988).
.
AUTOLOGOUS BLOOD TRANSFUSION: EVALUATION OF AN ALTERNATIVE STRATEGY IN REDUCING EXPOSURE TO ALLOGENEIC BLOOD TRANSFUSION S. Qutaishat
The Ernest Witebsky Center for Immunology, Department of Microbiology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214.
ABSTRACT
Theperceivedriskoftransfusion-transmitteddiseaseledtotherejuvenationof autologous blood transfusion(ABT). ABT, a process in which the blood donor and recipientarethesame, is increasingly becoming an integral component of the elective surgical protocol in many institutions. Various methods of ABT are being utilized. These include: preoperative blood donation, in which the patient donates blood prior to surgery and the blood is stored for an expected need during or after surgery; acute normovolemic hemodilution,in which blood is collected immediately prior to surgery and replaced with cell free fluids and then returned to the patient upon need; intraoperativeblood salvage in which blood is collected from the surgical field and is reinfused after being washed and finally, postoperative blood salvage in whichcollectedshedbloodfromsurgicaldrainsisreinfusedtothepatient. Although ABT is known to reduce the risk of allogeneic blood transfusion, it is not risk free and should be evaluated in relation to the patient's clinical picture. The combination ofvarious methods of ABTin addition to the proper utilization blood of may consequently lead to the elimination of patients' exposure to allogeneic blood transfusion in many surgical procedures. INTRODUCTION
In spite ofmajorimprovements
in thesafetyof
blood supplies in the Western
hemisphere, there is still a great fear amongst patients as well as physicians of transfusion-transmitted disease vectors, especially human immunodeficiency virus (HIV). The introduction of HIV and Hepatitis C virus (HCV) testing were part of a major effort by the blood banking establishment to improve the safety of the blood 435
436
QUTAISHAT
supply. The risksoftransfusion-transmittedHIVandHCVwereestimatedat
'
1/225,00 and 1/3,300 respectively, per unit blood. Other viral risks associated with allogeneic blood transfusion include the transfusion transmission of Hepatitis B virus H),
I and II (HTLV-I and
(HBV), Human T-cell Lymphotropic virus types
cytomegalovirus (CMV), Epstein-Barr virus (EBV), Human Parvovirus (HPV) B19 and HumanHerpes virus4
*.
Bacterial and parasitic infections are also known
transmitted in associationwith blood transfusion.Therisksofallogeneic
to be blood
transfusion are not limited to transmission of disease but also include the following: alloimmunization, hemolytic, febrile, allergic and graftvs.host transfusion reactions. These complications are virtually absent in ABT. Great efforts have been made
to reduce the risk of allogeneic
blood transfusion.
These include technical improvements in testing methodology and enhanced quality control and quality assurance procedures to ensure the effectiveness of new testing procedures. Most importantly, mechanisms were introduced to monitor transfusion practice
'.
Thesemechanismsencouragephysicians
to base the transfusion
decision on the overall clinical need of the patient and not solely
on established
laboratory values. Concurrent with these technical advancesin eliminating infected units of blood y& improved donor screening and testing, transfusion practice has undergone a major change. These efforts reducepatientexposure
to improve transfusion practice and to
to allogeneicbloodtransfusionwereaugmentedbya
noticeable growth in ABT. ABT became the transfusion method of choice in various surgicalproceduresespeciallycardiacandorthopedicsurgery thismethodofbloodtransfusionisbeingusedincreasingly procedures
46.
Furthermore,
in othersurgical
'S*.
PREOPERATIVE AUTOLOGOUS BLOOD DONATION
Commonlyknownaspredepositautologousdonation(PAD),this
isthemost
common and widely used type of ABT. The collection and reinfusion blood of to the same patient is an old concept which has shown significant growth and utilizedwidelyinvarioussurgicalprocedures.Thisstrategyhasbecome
is being the
standard of carein elective surgery in the nineties. Despite its underutilization in the past ', more patients and physicians are presently aware of the benefit of PAD. This increasing awareness has led to a remarkable increase in the collection of PAD in thepast12years
lo.
Severalmethodshavebeenimplemented
to enhance
AUTOLOGOUS BLOOD TRANSFUSION
437
autologous blood donations. Erythropoietin has been shown to increase the volume ofPADaswellas
the preoperative hematocrit
”,’*.
Blood substitutes combined
with autologous blood transfusion have been shown to increase the amount of blooddonatedinananimalmodelandmight numberofunitsof
be ofbenefit
in humans
13.
The
PAD collectedattheBuffaloGeneralHospitalshowed
an
increasing pattern in the past five years (Table l.). In order to avoid overcollection of PAD, a maximum surgicalblood order schedule(MSSOS) or guidelines similarto it, needs to
be established to determine the number of units required for each
procedure, as outlined previously
14.
Over-transfusion of PAD should be monitored
using the same criteria used for allogeneic blood transfusion.
In addition, quality
assurance monitors shouldbe used to evaluate the ratio of collected to transfused blood ( C m and a threshold should be established. This threshold may be similar to that used for allogeneic blood transfusion i.e., crossmatched to transfused (Cm ratio.Sinceabout
50% ofPADunits
collectedarediscarded,thedilemmaof
wastage in PAD willcontinuetohauntbloodbankersandisperceivedasan additional cost. The question ofwho is going to pay for this procedurein a health care environment that is scarce in dollars continues to be unanswered. The risks associated withPAD are two-fold; the first is inherent in the donation process itself, the second is associated with the reinfusion process. Moderate to severe reactions during autologous blood donationswere shown to be often similar to those of the allogeneic donations
’I.
Clerical errors and some of the other adverse effects of
transfusion are also possible in PAD. Adverse effects in PAD transfusions that were reportedattheBuffaloGeneralHospitalforexample,includedhypotension. Mistransfusions due to clerical errors were also reported (unpublished data),
It is
believed that there is still underreporting of such transfusion reactions in PAD due to a misconception about the lack of adverse effects associated with the procedure. One of the reported risks associated with the reinfusion process includes bacterial contamination
”.
Controlledstudiesareneededtoevaluatetheprevalence
of
various adverse effects in PAD. ACUTE NORMOVOLEMIC HEMODILUTION (ANH) ANH represents an effective method of ABT that can replace
or at least augment
predeposited bloodas a means of decreasing patient exposure to allogeneic blood.
438
QUTAISHAT
TABLE I The Increase inthe Number of Predeposit Autologous Donor (PAD) Blood Units Collected at the Buffalo General Hospital Between 1989 and 1993 ~
tologous Predeposit Allogeneic Year Number
7197
%
1989 171
1990
161
1991
15875
1992 15800
1993
In thismethodbloodiswithdrawnfromapatientandisreplacedwithcell-free crystalloidand/orcolloidsubstitute
",
Bloodiscollectedfromthepatient
immediately before and shortly after the induction of anesthesia, volume is replaced with cell-free substitute to maintainnormovolemia.As
the patient bleeds during
surgery, more of theblood substitute is infused to counteract volumeloss and once hemostasis is restored, or sooner if needed, the autologous blood collected earlier is reinfused. In ANH, fewer red blood cells are lost during the surgical procedure. Theviscosityof
bloodis
lowered,improvingtheflow
of blood,andthat
in
conjunctionwithlowerhematocritvaluesmayenhanceoxygendelivery.Not surprisingly, since blood is collected and reinfused within a few hours and is stored at room temperature it is considered to be fresh with all coagulation proteins and platelets intact. Clerical errors are eliminated when using ANH because
blood is
stored in the operating room in close proximity to the patient. Blood collected using this method does not require any laboratory testing which reduces the cost of the procedure compared to other autologous transfusion methods. Ness
&t 4. ",
in
a prospective controlled study, demonstrated that ANH is beneficial in eliminating the use of allogeneic blood. However, the risks associated with this method in certain groups of patients are not to be ignored and should be Considered when a patient is evaluated for ANH. Gillon
*'
recommended that this technique be limited to a
certaingroupofpatientsandproperpatientscreeningshouldbeimplemented before the procedure is performed.
AUTOLOGOUS BLOOD TRANSFUSION
439
INTRAOPERATIVE AND POSTOPERATIVEBLOOD SALVAGE Intraoperativeandpostoperative
blood salvageproceduresareoftenused
in
conjunctionwithotherautologousbloodtransfusionprocedures.Collectionand reinfusion ofblood recovered from the operative site or from an extracorporeal circuit is called intraoperative blood salvage. This procedure is mainly used vascularandorthopedicsurgery
4-J*21.
in cardiac,
Its usehasalsowitnessedaremarkable
growth in the past ten years. Blood is collected, washed, centrifuged, filtered and eventually reinfused to the patient. This procedure has its share of risks because of the nature of the blood collected and the instrumentation used. Reports on adverse effects due to intraoperative blood salvage include fatalities dueto introduction of airembolismsduringthereinfusion
z. Anotherconsiderationinchoosingthis
procedure involves cost: the procedure involves having additional trained personnel and equipment in the operating room to perform and monitor the reinfusion. Postoperative blood transfusion(PBT), the collection and reinfusion of shed
blood
fromsurgicaldrains,hasbeensuccessfullyusedbutwasmainlylimited mediastinal and chest drainage following cardiopulmonary surgery.
to It appears to
be rapidly gaining popularityin orthopedic surgery. The blood is collectedin sterile plastic containers and reinfused, usually unwashed, within6 hours after collection. Although earlier studies suggested that the use of shed blood is safe 4*5, it is still not clear if these techniques are effective. The concern over the presence of fibrinogen degradation products and tissuetype plasminogen activator stimulating activity in shed blood is still a major controversial issue in the use of this method. Recently, it was demonstrated that there is acorrelation between the presence of these factors
in the shedblood and the amount of postoperative blood loss suggesting that these factors mayenhance bleeding m. Most recently Ulmas
B a. 24 showed that the
routine useof postoperative blood drainage systemsin orthopedic patients does not appear to be justified. In a study of31 patients undergoing hip replacement surgery and 20 patients undergoing knee replacement surgery, they showed a mean red blood cell loss of 55 -+: 29 and 121 -+: 50 ml respectively. They concluded that the red blood cell content of this drainage system does not justify the cost and they further recommended that this method of blood conservation should be notused as aroutineprocedure
on allpatients.Inaddition,thisstudydemonstratedthat
postoperative blood salvage in joint replacement surgery would not substantially affect the need for allogeneic blood transfusion. The need to evaluate the adverse
440
QUTAISHAT
effects of this method further is important in view of the increasing number of reports in theliteratureontheadverseandfatalcomplicationsofintraoperativeblood salvage. CONCLUSIONS
Autologous blood can supply all or part of patients’ blood needs in most elective surgicalprocedures.Thesuccessofthisapproachisdependent comprehensive,wellplanned,
on usinga
presurgical approachand by strongcoordination
betweenthevariousdepartmentsinvolved
in patientcare.Thepresenceof
an
autologous blood coordinatorwho is responsible for organizing a programlike this is strongly recommended. Furthermore, continuous monitoring of ABT and how
it
meets patients’ needsis essential. More studies are neededto evaluate the efficacy of thevarious methods of ABT and various combinations of it.In addition, evaluation of the adverse effects of these methods and of their riswbenefit implications
is
necessary for the proper utilization of ABT.
Commentary
in terminologytoreflectcurrent
Recently,theAABBhasimplementedchanges
a descriptionoftransfusionfromone scientificunderstanding:homoloqousas person to another has been replaced with alloaeneic Thus autoloqous transfusion- transfusion of blood to oneself would more correctly be replaced with autoaeneic transfusion. REFERENCES 1. R.Dodd, N. Engl. J. Med., 327, 419-420 (1992).
2. J. C. Pehta, Laboratory Medicine,
2 5 ,
102-105 (1994).
3. L. E. Silberstein, M. S. Kruskall, L. C. Stehling, M. F. M. Johnston, R.C. Rutman,C. T. Samia, G. Ramseyand R. S. Eisenstaedt, G. DLundeberg (section editor), JAMA, 2 6 2 , 1993-1997 (1989). 4. A. J.. McMahon and J. S. McCormick, J. Royal College of Surgeons of Edinburgh, 3 8 , 71-74 (1993).
5. D. G. Seltzer, M. D. Brown, J.S. Tompkins, I. Enger and J. Spinal Disorders,G, 412-421 (1993).
F. P Cammisa Jr.,
AUTOLOGOUS BLOOD TRANSFUSION
441
6. D. H. Biesma, C. E. van Iperen, R. J. Kraaijenhagen, J. J. M. Marx, H. B. M. van de Wiel and A. van de Wiel. Vox Sanguinis, 60, 270-275 (1994). 7. L. T. Goodnough, J. Riddell IV., E. K w h and M. I. Resnick, urology, g, 201205 (1 992). 8. L.T. Goodnough, P. Saha, N. V. Hirschler and 63, 96-101 (1992). t 9. P. T. C. Y. Toy g
a.,N. Eng. J. Med.,
316,
10. M. A. Papovsky, Laboratory Medicine, 2
5 ,
11. L. T. Goodnough, Seminars in Oncology,
R. Yomtovian, Vox Sanguinis,
517-520 (1987).
106-109 (1994).
B,19-24 (1992).
12. J. Hayashi, K. Kumon, S. Takamashi, Y. Kawashima, S. Eguchi, F. Takaku and H. Yamamura, Transfusion, 34, 142-146 (1994). 13. P. J. Slanetz, R. Lee, R. Page, E. E. Jacobs Jr., R. J. Laraia and G. J. Vlahakes, Surgery, 115, 246-254 (1994). 14. F. B. Axelrod, S. H. Pepkowitz and D. Goldfinger, Transfusion, 3,677-688 (1 989).
S. Hoag, D. Polan, S. Skettino, L. C. Stehling, 15. P. A. McVay, A. Andrews, M. G. Strauss and P. T. C. Y. Toy, Transfusion, 5 9 , 70-72 (1990). 16. C. Richards, J. Kolins and C. D. Trindade, JAMA,
268,
R.
1541-1542 (1992).
17. L. Stehling, H. L. Zauder, Transfusion, 3 1 , 857-868 (1991). 1 8.P. M. Ness, D.
L. Bourke and P. C. Walsh, Transfusion,
19. L. Stehling, H. L. Zauder, Transfusion,
3 2 ,
226-230 (1992).
34, 265-268 (1994).
20. J. Gillon, Transfusion, 34, 269-271 (1994). 21 R. L. Lennon, M. P. Hosking, J. R. Gray, R. A. Klassen, M. A. Papovsky and M. A. Warner, Mayo Clin. Proc., g,1090-1094 (1987) I
2 2 . J. V. Linden, Blood Bank Association of New York StateQuarterly,B, 2 (1 994). 23. J. de Haan, J. Schonberger, J. Haan, W. van Oevenren and A. Eijgelaar, J. Thoracic & Cardiac Surgery, 106, 1017-1023 (1993). 24. J. Ulmas, R. R. Foster, S. A. Dalal, S. M. OLeary, L. Garcia and M.G. Kruskall, Transfusion, 34, 402406, 1994. 25. F. K. Widmann, e d . , Standards for Blood Banks and Transfusion Services, 15th edit., American Association of Blood Banks, Bethesda, MD. (1993) p. iii.
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HEMATOPOIETIC STEM CELLS "FORM - METHOD - CHARACTERISTICS" Robert A. Dracker, M.D. SUNY - Health Science Center at Syracuse Syracuse, New York
INTRODUCTION This review of hematopoietic stem cells provides an overview ofthe therapeutic concepts of stem cell use; the various sourcesof stem cells includingtheiruniquecharacteristics, advantagesanddisadvantages;collection,processingandstoragemethods;andthe
future
directions in this exciting field. Adiscussion
of hematopoietic stem cells is somewhatdependentuponabasic
understanding of antineoplastic and myeloablative therapy. In all cases of stem cell use, there is the precedent use of either chemotherapy and or radiotherapy, intended to eradicate residual disease or dysfunctional cells and to "prepare" the marrow for the receipt o f pluripotential stem cells from which the engrafted red blood cells, white blood cells and platelets will be derived.
In the case of allogeneictransplantation, there is the obvious need torender the recipient immuno-tolerant to the transplanted cells in order for successful engraftment to be possible. The majority of treatment modalities for neoplastic diseases can be divided into induction, consolidation and maintenance phases. The induction phase of treatment attempts to remove the majority of the "naive" tumor burden, whereas consolidation therapy is intended to minimize or eradicate any residual disease. Maintenance therapy serves the dual purpose of eradicating residual disease whilealso providing a surveillance function for limiting recurrence. Traditional therapies for the somewhat arbitrary separation of treatmentphasesmentionedabovehave included various combinations of chemotherapy, surgery and radiotherapy. Newer therapies however, including immunotherapy with cytokines and hematopoietic stem cell infusions have demonstratednovelandveryusefulexamplesofcombinedandeven
alternative treatment
approaches. This discussion focuseson the utilization of pluripotential stem cell therapyat the present and in the near future. We will concentrate on the most common source of stem cells, that is,
443
DRACKER
444
bone marrow; proceedingwith the increasingly utilizedalternate source, specifically, peripheral blood stem cells; and finally reviewthe future uses and benefitsof a truly unique and potentially unlimited stem cell resource, cord blood stem cells. HEMATOPOIETIC STEM CELL ASSAYS Although there is no consensus among transplanters and stem cell bankers as to the most reliable method for assessing the quality and quantity of a stem cell product, there is a variety of assays thatare routinely performedby most centers. Many laboratories perform combinations of these testsand studies, unfortunately utilizing a variety of different methods. There has been a long-standing need for technical consensus and procedural standardization in this area, however we must yet decide how one could identify the most critical cell population in sampleandthecriticalnumberneeded Currentlyutilized
for successfulengraftmentand
surrogate assays for theassessment
the stem cell
in vivo expansion.
of astemcellproductinclude:
mononuclear cell enumeration with viability; CD34 marker studies performed by flow cytometry in conjunction with other lymphocyte antigen markers, providing data as a percent or absolute count of the mononuclear fraction; and colony forming unit (CFU) assays and long-term colony
the sufficiency of any stem cell product is the in vivo demonstration of the rate, completeness and endurance of its engraftment.
culture (LTCC) assays. Probably the most definitive assay of
STEM CELL PROCESSING AND STORAGE Regardless of the source or origin of the stem cell product, i.e. bone marrow, peripheral blood or cord blood, the cell suspension must be processed, cryoprepared, cryopreserved and stored in a timely manner. Stem cell processing and storage represent areas where there is also great variation in methods and technologies in stem cell laboratories. The processing of the stem cellproductgenerallyinvolvesthepurificationofmononuclearcells;however
CD34+
enrichment by either solid phase immunological methods involving affinity columns or magnetic bead separation, or gel separation, is also utilized with increasing frequency. At this point in the processing of the product,purging of thesuspension, with the intentionof eliminating contaminating malignantcells, may also be performed. Purging of stem
cell suspensions, especially inthe case of autologous products, can be performed with the use of pharmacological agents, radioisotopes, cytokines, photo-activated dyes
or combinations of
these various methods.
The actual processing of the stem cell product can be performed either manually in a clean environment suchas a biohazard hood,or by the use ofa semi-automated apheresis device. The use ofan
apheresisdevice
or other automatedmethod
affords a decreased risk of
contamination, less overall procedure time and a more consistent product. Once the product has been purified of contaminating materialssuch as bone spiculesand fat, especially in the case of
EM
HEMATOPOIETIC
445
bone marrow, it can be prepared for cryopreservation. Generally, the product is admixed with a colloidal solution, either in the form of plasma, albumin or a polymer such as Pentastarch. Pre-diluteddimethylsulfoxide(DMSO)
is thenadded
5 % finalconcentration
to reacha
immediately prior to the start of cryopreservation. (Prolonged exposure to DMSO in liquid media is toxic to mononuclear cells.) The stem cell suspension is then cryopreserved utilizing either a controlled-ratefreezing deviceor a simplified manual freezing method, especially when the cryoprotectant consists of a combination of DMSO and starch.
Some researchers feel that
there is no significant difference in freezing efficiency and cell survival with either method. Once frozen, the product may be stored ineither liquidor vapor phase nitrogen.If a mechanical freezing device is utilized for storage, it is essential that a backup system be available in case of failure. This backup system is usuallyin the form of liquid nitrogen. All be both locally and remotely alarmed and monitored in an
freezers should
area which is staffed twenty-four
hours a day. STEM CELL ADMINISTRATION The stem cell product should never be removed from
its cryopreserved state until it is
intendedto be used for infusion. Any unnecessary thawing and refreezing potentiallyresult
in adecrease
in cellviability.Whenaproduct
of a product can
is requestedby
the
transplantation service for patient use, the product is transported to the patient’s bedside in the frozen state. Prior to release, the identification of the unit is confirmed and a gross inspection of each is performed to insure the integrity of the stored product. At
the patient’s bedside,
“matching” of the patient and the stem cell units is performedin a fashion similar to that used for bloodunits prior totransfusion.Oncepositiveidentification plunge-thawedina
is obtained, the units are
37°C. water bathand the cell suspensions are infused,generallywithin
twenty minutes. Multiple units of stem cell suspensions
are typically administered to patients
per stem cell infusion. BONE MARROW AND PERIPHERAL BLOOD STEM CELLS Bone Marrow Stem Cells represent the traditional source of stem cells for hematopoietic reconstitution. Hematopoietic transplantation represents one of the fastest growing and most
one billion dollars are spent annually on transplantation in the United States alone. Additionally, there are over 100 donor centers and 65 transplant centers nationwide performing these procedures for an ever-expanding list of
important forms ofmedicaltherapy.Over
indications. Some of the newer indications include genetic disorders, autoimmune diseases and
as adjunctive therapy as a form of stem cell rescue for a variety of solid tumors such as breast and ovarian carcinomas.
DUCKER
446
Although the use of bone marrow stem cells has resulted inthe prolongation of thousands of lives, there havebeen
inherentlimitations in theiruse.
diseased marrow, the use of autogeneic stem
In the casesofpatientshaving
cells is generally limited, although attempts to
purge the marrow of residual tumor cells prior to usehavebeenmade.
For the allogeneic
patient population, thereis the continuing shortage of suitably matched donors, especially inthe
case of minority recipients. The National Marrow Donor Program (NMDP) has been a valuable and worthwhile resource in this regard, although the search for a suitable donor is a costly and complex process, resulting in an average waiting period of six months. The NMDP also suffers fromalack
of ethnicdiversityamongpotentialdonorsand
the relatively high budgetary
requirements for the program. Peripheral Blood Stem Cells (PBSC’s) represent an alternative stem cell source when there is: documented marrow involvement with malignancy;damage to the posterior iliac crests due to prior irradiation, fibrosis or infection which interferes with marrow collection; and an inability of the patient to undergo the anesthesia typically required for bone marrow collection.
The use ofperipheralbloodstemcells
is basedupon the recognitionofthepluripotential
hematopoietic potential of small numbers of circulating stem cells. These
cells are typically
collected by repeated leukapheresis procedures which attempt to concentrate the mononuclear fraction of the white cell product. Within this mononuclear fraction reside the more immature toti-andpluri-potentialstem cells, necessary for marrowreconstitution.Variousmedical maneuvers can be employed in conjunction with the repeated leukapheresis procedures, having the intent of increasing the circulating population of stem cells. These therapies have included transient pre-collection myelosuppression of the patient with chemotherapy such as cytoxan, timedwiththemononuclearcollectionsuchthat
the circulatingstem cells are at increased
concentrations during the post-treatment marrow recovery phase. Additionally, cytokines such as Granulocyte-ColonyStimulating
Factor (G-CSF)andGranulocyte-MacrophageColony
Stimulating Factor (GM-CSF) can be administered to a patient in addition to myelosuppressive chemotherapy or by as by itself, which has been shown effectively
to increase the circulating
stem cell population. Peripheral blood stem cells have demonstrated multiple advantages in patients requiring hematopoietic reconstitution or transplantation. The use of PBSC’s as a source of stem cells results in equivalent or actually improved engraftmentas compared to marrow. It is thought that a PBSCinfusionprovidesa
larger totalnumberofmononuclear
cells, withan
increased
concentration of committed progenitors, thereby resulting in earlier marrow recovery. If a program of peripheral blood stem cell collection and utilization is planned for an institution, a number of concerns and prioritiesmust be considered.Withregards
to the
Transfusion Service, despitethe fact that peripheralblood stem cell use may translateinto more rapid engraftment and therefore fewer transfusion episodes per patient,
there is a significant
HEMATOPOIETIC STEM CELLS
447
workload placed upon the apheresis staff for the collection procedures andupon the staff of the stemcelllaboratory
for stem cellproductpreparationandcryopreservation.
The PBSC
collection devices and techniques must also be determined, based upon financial and staffing needs. Particular to collection devices, there is a variety ofeither continuous or discontinuous
apheresis devices available for stem cell collections, each with a variety of advantages and limitations. Attention to vascular access methods; procedural specification with regards
to the
frequency,timinglengthandnumberofprocedures;andthemonitoringandanticipated treatment of adverse effects must also be considered. In the case of bone marrow collectionfor subsequent transplantation, typicallyone to two liters of marrow are harvested from both posterior iliac crests of the patient or donor. When it comes to the collection of peripheral blood stem cells, both anticipatory and concurrent review of the collectionprocedures mustbe
considered. A numberof
factors willinfluence the
sufficiency ofeachPBSCcollection.Someofthese factors include how the patient was "prepared" prior to collection with regards to chemotherapy and or cytokine administration, the general health of the patient, the patient's disease status, the prior myelo-reductive therapy the patient may have received and how recent the last exposure has been. Generally, autogeneic hematopoietic transplantationwith PBsC's alone utilize between 3
-
10 x 108 mononuclearcellslkg.bodyweight.
lO*cells/kg. If used in conjunction with bone marrow,
The current average is between 5 - 7 x the dose can be lowered to 2 - 5 x
108cells/kg. Increasingly, hematopoietic stemcells are being recognized as a valuable resource as a "rescue" therapy for high dose chemotherapy, particularly in cases of breast cancer and ovarian wcinoma. In this setting, the autogeneic stem cells provide an exogenous means of marrow replenishment following exposure to high dose chemotherapy. in addition to cytokinestimulation,reducestheseverity
The use of these cells
and duration of theresultant
pancytopenia, In such a situation, multiple pre-therapyPBSC collections can be performed and
the cells subsequently cryopreserved. Since a lower number
of the stem cells is required for
infusion as compared to a transplantation procedure, the stored PBSC collections can be used for multiple cyclesof therapy. In such a scenariowhich I consider tobe "Therapy-Synchronized Stem Cell Rescue",
1
-2x
l@ celldkg. are usually administered one to two days after the
completion of chemotherapy. CORD BLOOD STEM CELLS Some believe that the full potential of transplantation therapy is not being reached on account of a chronic shortage of transplantable stem cells. Generally stem cell sources
are
limited to either bone marrow or peripheral blood, both of which involve time-consuming and costlyprocedures.Additionally,fewer suitable match within their own family.
than 30% of allogeneictransplantcandidatesfind
a
448
DUCKER
It has been demonstrated that blood collected from the umbilical cord shortlyafter birth contains a pool of uncommitted progenitor cells. Sequential stimulation of these cells results in synergy between a variety of cytokines and
the development of differentiated cell colonies in
virro. Cord blood has also demonstrated significantly greater capacity for long term culture growth as compared to bone marrow cells, averaging longer than 6 weeksin culture. Cord blood stem cells demonstrate high proliferative potential and self renewal capacity and exist at a higher concentration and at an earlier stage of development as compared to other sources of stemcells.Atthetimeofbirth, CD34+ stem cells mayexceed 20% of themononuclear fraction of cordblood,however
the numbersofcirculatingstem
cells rapidlyfallinthe
newborn within 24 hours. The collection of cord blood at the time of birth therefore represents a unique opportunity
to collect a unique stem cell resource which may
be immunologically
immature and relatively naive with regards to infectious agents. Cord blood stem cells have also demonstrated greater immune-tolerance to HLA nonidentical lymphocytes in culture, possibly representing a decreased propensity for graft versus hostreactivity in theallogeneicrecipient.Potentialdisadvantagesto
the useof
these cells
include the risk of maternal T cell contamination, risks of microbial contamination at the time of collection, and a reduced number of available progenitor cells for the recipient sincethe total number depends on the cell concentration in the collected cord blood volume. Once collected, the cord blood is usually processed as whole blood and is cryopreserved in a manner identical with that performedfor either bone marrowor peripheral blood stemcells. Testing of thecordbloodusuallyinvolvescellenumeration,viabilitydetermination,CD34 enumeration, bacteriologicand fungal cultures and colony forming unit assays. Since fetal blood carries the risk of disease transplacentally from the mother, testing of the mother for certain infectious diseases must also be performed. Up until the present, over 50 cord blood stem cell transplantations have been performed for avarietyofdiseases
in children,includingmalignancies,inborn
errors ofmetabolism,
congenital immunodeficiency states, aplastic anemia as and a vehiclefor retroviral mediated gene transfer. Research in this exciting
area is accelerating as the number of successes increases.
Dr. Pablo Rubinstein of the New York Blood Center has developed an allogeneic cord blood bank with the intent of addressing the long standing shortage of suitable hematopoieticstem cell sources for minority populations. The Biocyte Corporation of Stamford, Connecticut has also introduced an autologous cord blood service at the McGee Womens Hospital in Pittsburgh. The obvious goal of cord blood stem cell research isto develop a histocompatibilitylibrary of stem cells, available to patients inneedof
hematopoietic therapy. With the
future success of cell
expansion technologies, cordblood may evolve into a resource which will be useful to many for a variety of diseases.
M
HEMATOPOIETIC
CELLS
449
CONCLUSIONS Hematopoietic transplantation isone of the fastest growing and most important formsof medicaltherapytoday.However,the
full potentialoftransplantationtherapyhas
not been
reached due to the chronic shortage of transplantable stem cells, especially in the allogeneic setting. We will see an increase in the use of stem cell for non-traditional diseases, particularly non-neoplastic conditions. Peripheral blood
stem cells represent a useful alternative to bone
marrow stem cells, especially in the autologous setting. Although not yet routine, we also will see
an increase in the use of allogeneic peripheral blood stem celluse without the use of bone marrow.Althoughstillin its infancy,cordbloodstemcellcollectionand use willplay an expanding role in a variety of disease treatments, substantially reducing the limitations associated withboth bonemarrowandperipheralbloodstemcells. expansion,CD-markerselection
The fieldsofhematopoietic
cell
of cells and theuse of combinationcytokinetherapies
all
represent exciting areas of development for future cellular therapies. REFERENCES AND RECOMMENDED READING Gee A. Bone Marrow Processing and Purging, A Practical Guide, CRC Press, Inc., 1991 Golde D. The Stem Cell, Scientific American, December, 1991, Vol 265, No. 6 pp.86-93 de Magalhaes-Silverman M, Donnenberg A, Pincus S , Ball E. Bone Marrow A Review, Cell Transplantation, Vol 2, pp. 75-98, 1993
Transplantation,
Armitage J. Bone Marrow Transplantation,New England Journal of Medicine, March 24,1994, V01 330, NO. 12 ,pp.827-838 Areman E, Sacher R, DeegH. Cryopreservation and Storage ofHumanBoneMarrow: A Survey of Current Practices, Bone Marrow Purging and Processing, Alan R Liss, Inc, 1990 pp. 523-529 Aird W, Labopin M, Gorin NC, Antin JH. Long-Term Cryopreservation of Human Stem Cells, Bone Marrow Transplantation, 1992, Vol 9, pp. 487-490 Broxmeyer H, Hangoc G, Cooper S, Ribeiro R, Graves V, Yoder M, Wagner J, Vadhan-Raj S, Benninger L, RubinsteinP,Broun R. GrowthCharacteristicsandExpansionofHuman Umbilical Cord Blood and Estimation of its Potential for Transplantation in Adults, Proc. Natl. Acad. Sci., Vol 89, pp. 4109-4113, May 1992 Wagner J. Umbilical Cord Blood Stem Cell Transplantation, Amer. J. Ped. HemlOnc., Vol 15 NO. 2, pp. 169-174,1993 Moritz T, Keller D, Williams D. Human Cord Blood as Targets for Gene Transfer: Potential Use in Genetic Therapies of Severe Combined Immunodeficiency Disease, J. Exp. Med., Vol 178, pp 529-536, August 1993 Bifano E, Dracker R, Lorah K, Palit A. Collection and Storage ofHuman Placental Blood, Pediatric Research, Vol 36, No. 1, pp. 90-94, 1994
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USES OF INTRAVENOUS GAMMAGLOBULIN IN IMMUNEHEMATOLOGIC DISEASE
James B. Bussel and Ted P. Szatrowski New York Hospital--Cornell University Medical Center New York, NY 10021
INTRODUCTION Intravenous gammaglobulin was first developed in the latter part of the 1970's. This required removal of IgG aggregates from previously available preparations of gammaglobulin
for intramuscular use without adversely impactingthe structure and function of IgG. This has subsequently been achieved by a number of different techniques.
The development of intravenous gammaglobulin was intendedfor patients with primary humoral immuno deficiency: hypogammaglobulinemia or antibody deficiency. The possibility of other usages included secondary immunodeficiency states such
as premature newborns or
following bone marrow transplantation. Hematologic use of "gammaglobulin" had taken place in two settings connected with thrombocytopenia. First in the late 1950's and easily
1960's there were several studies of the
infusion of fresh frozen plasma the in treatment of thrombocytopenia. At that timeit was thought that what was being infused was "thrombopoietin";
in retrospect, it appears that the doses of
plasma required i.e. 30 mllkg, were in line with current dosing of IVIG. The second setting involved the use in intramuscular gammaglobulin in the treatment of thrombocytopenia. In this setting it waspresumedthatthegammaglobulinusagewasprimarilyantiviralandthat increases seen in
theplateletcountsinvolvedviralneutralization.Lowerdoses
the
were
administered in these studies; serum IgG levels were not reported(1). The usage of IVIG specifically for the treatment of thrombocytopenia followed infusion of gammaglobulin in
two immunodeficient viremic children who
were incidentally
thrombocytopenic. The dramatic increase in the platelet count in these two patients lead the investigators, Drs. Paul Imbach and Silvio Barandun, to treat a seriesof children with ITP with very good results(2). These studies marked the beginning of the usage ofIV gammaglobulin in the hematologic disorders. 45 I
452
BUSSEL AND SZATROWSKI
EARLY STUDIES IN ITP In addition tothe initial Imbacharticle which documented dramatic rapid increases inthe platelet count following the infusion of gammaglobulin(2), severalother important points were made both in this article and a coincidentally publishedarticle in Acta Helvetia Pediatrica. First, in patients with chronic ITP it was clear that infusion of gammaglobulin was not immediately curative inthatpatientscontinuedtorequireretreatmentin
order to maintainanincreased
platelet count. Second, the use of a pepsin treated IVIG preparation that did not havean intact Fc portion ofthe IgG, was ineffective. The investigators suggested that the mechanism of action of IV gammaglobulin included Fc receptor blockade.
Third, several patients with aplastic or
hypoplastic thrombocytopenia were treated and their lack of response to gammaglobulinfurther reinforced the notion that gammaglobulin was not stimulating platelet production. Subsequently, in 1982 a study by Fehr specifically demonstrated that intravenous gammaglobulin resulted in Fc receptor blockadein a fashion that was temporally associated withthe increase in the platelet count(3). Therefore by 1982 many of the basic tenetshad already been discovered aboutthe use of IVIG in ITP. ACUTE ITP IN CHILDHOOD Anumberofstudiesdemonstratedthatthe
great majority of children with ITP in
childhoodwouldhavearapidanddramaticresponsetointravenousgammaglobulinin
the
platelet count. Since many of these children would achieve complete remission in any event, it was unclear that the effect was curative. One trial by Imbach
et al. compared 50 children
receiving IVIG to 50 children receiving prednisone(4). It showed a faster and more permanent response of the platelet count amongthe children receiving IVIG,but there was an insufficient number of patients to demonstrate any significant changes in the
cure rate. A recent smaller
controlled trial in acute ITP was unable to show a significant advantageof gammaglobulin over prednisone, but both were clearly superior tono treatment in the rate of increase of the platelet count. Questions arose as to the dosage. The original studies used 400mg/kg for 5 consecutive days (total 2 gm/kg). Subsequently, studies using essentiallyequal
lgm/kg for one to three days demonstrated
efficacy. Reanalysis of the controlledtrialcomparinggammaglobulinto
prednisone suggested that one could divide the patients, with two thirds in a "rapid response group" in whom two days of treatment with 400 or 500mg/kg would be sufficient and who would very like wouldnot need treatmentagain.
The exactbestdosagescheduleremains
uncertain as does the optimal dose in followup treatments. ADULTS WITH ITP The greatest difference in treatmentof ITP with IVIG (or any other therapy) reflectsthe marked tendency to spontaneous remission in children (80% within 6-12 months) unlike adults
INTRAVENOUS
453
who have no such tendency. Multiple studies ofIVIG treatment of ITP, summarized in a review published in 1987(5), documented the dramatic ability of intravenous gammaglobulin to provide a sizable platelet increase in adults with ITP. The average platelet increase was greater than 1OO,OOO/p1within o x week and often within 72 hours. Patients over 60 may have responded slightly less well as compared to younger adults, and children responded slightly better than adults.Of
note,adultsbeforeand
after splenectomyrespondedequally,suggestingthat
intravenous gammaglobulin is a useful therapy for adults with ITP at any stage in need of an emergent platelet increase. Again the exact optimal doseis difficult to ascertain. We performed a small, crossover study comparing two days of 0.5 gm/kg to two days of 1.0 gm/kg using the New York Blood Centerpreparation
of IVIG.
The plateletincreasefollowinginfusion
of 1 gm/kg was
approximately twice as great (160k) as the platelet increase following 0.5 gm/kg (73k) but the duration of effect was the same. These results are similar to those found in an alternating dose trial in the maintenance of patients with low platelets. In these adults, single treatments were infused of 0.5 gm/kg in alternation with 1.O gm/kg. Again, the peak platelet increase with the higher dose was greater but the duration of effect was similar (14 versus 16 days)(6). In general, the most economic treatment would be to use 400 or 500 mglkglday, until a set plateletincrease, i.e. 30-60k,isachieved.Alternatively,ifhavinga
greater platelet
increase sooneris important, theuse of 1 gm/kg/day would be more appropriate. Finally a study by Bierling implied thatthe acute repeated use of IVIG might havecurative effects even inadults with ITP(7). If true, then a lower dose of IVIG might be equally effective with a higher one in the short term in achieving and maintaining hemostasis but might jeopardize the possibility of long term cure. CHRONIC ITP
The use of gammaglobulininchildrenwith
chronic ITP has been activelystudied
although no controlledtrials have been attempted. Two large studies suggested that somewhere between 50 and 67% of children with chronic ITP treated with IVIG for up to 2 years by repeated single infusions could ultimately avoid splenectomy.
A medical decision analysis(8)
suggested that this wouldbe cost effective in children < 6-10 years of age, depending upon the costs especially the cost of IVIG per gram. In the largest study of maintenance treatment of 30 already splenectomized adults,39% were eventually able to discontinue treatment including several who had been unresponsive to at least 5 other treatments in addition to failing splenectomy. However, the average dose of WIG required was 660 grams per patients and some patients became unresponsive to IVIG following initial response(9).
SZATROWSKI
454
BUSSEL AND
Therefore, in contrast to children, maintenance treatment with repeated IVIG infusions in adults with chronic ITP has generally not been actively pursued because:
1)
the high cost of treatment.Sincethedose
ofgammaglobulin
is basedonbody
weight, the cost per treatment in adults is substantially higher than in children. 2)
adults with ITP have a less benign course than do children in that there is a lesser tendency for them to improve spontaneously with time.
Therefore they tend to
continue to require treatment indefinitely whereas children may spontaneously improve more readily and not continue to require treatment.
PREPARATIONS OF WIG InEuropeand
the UnitedStates,anumberofpreparationsofIVIG
were so-called
"modified" because the processingto eliminate aggregatesof IgG modified the monomeric IgG in the preparations. The review of studies from1987 clearly documented via a metaanalysis that patients treated with :mdified preparations of IVIG had lesser platelet increases following IVIG infusionthandid
the patientstreated withunmodifiedpreparations(5).Recently,modified
preparations, at least in the United States have been discontinued but two preparations have becomeavailablethat are viralinactivatedbysolventdetergenttreatment. preparation,VeinoglobulinmanufacturedbyAlphaTherapeutics,wecompared
For alicensed a seriesof
patients treated with their previous preparation to a series treated with their virally-inactivated product. In both series, the mean platelet increase was18O,OOO/pland when controlled for acute and chronic patients the results were still identical. As illustrated in Figure 1, when we compared separate pilot studies performed with a number of different preparations of WIG, there were no significant differences seen with any of the preparations. Note:not
all of the treatment protocols were identicaland the studies
comprisedapproximatelytwentypatientseach
and werenotprospectivelyintended
for
comparison.
VIRAL TRANSMISSION BY IVIG Solvent detergent and other viral inactivation steps have become importantfor IVIG as a result of the recent transmission of hepatitis C in a number of cases by IVIG. This Seems to be a paradoxical result of screening plasma for hepatitis C and eliminating hepatitisC antibody units. Apparently the absence of antibody in the preparations allows trace amountsof hepatitis C to transmit disease. This finding has been primarily with one preparation that had no viral
inactivation steps but other preparations may be implicated as well. This recent transmission is particularlyinterestingbecause:
1) it doesnotprovethat
the hepatitis C antibody is
455
INTRAVENOUS GAMMAGLOBULIN
250
A
COMBINED
IGlV Treatment
FIGURE 1 Plateletincreasesobtained with different IVIG preparations. The Y axis is the mean peak platelet increase. The X axis represents 7 different single arm pilot studies with 7 different preparations of IVIG. Different treatment protocols were used for the different preparations. There were no statistically significant differencesin the platelet increases seen with any of the different gammaglobulin products ( P > 0.20 ).
neutralizing; its presence or absence in the source plasma could affect which plasma fraction the hepatitis C virus separates into (cryosupernatantor cryoprecipitate); and 2) it is one of the first times that the use of a screening test actually increased transmission of
the screened disease.
In summary, at present, viral inactivation by solvent detergent methodology Seems optimal means of ensuring that there
is no viral transmission of hepatitis
to be the
B or C (as well as
HTLV-1 and HIV-1).
SUMMARY
In summary, for intravenous gammaglobulin use ofITP in children and adults,it is clear that intravenous gamr:laglobulinis an effective way to increase the platelet count acutely and this will be faster than or as fast as any other therapy. However, there is no proven curative effect of IV gammaglobulin. Its use in situations requiringa rapid increase in the platelet count seems secure as does its use in children with chronic ITP.
The latter however and the treatment of
456
BUSSEL AND SZATROWSKI
HIV-ITP mayfind WIG treatment largely replaced in the
future byIVAnti-D(10)which
is
currently experimental. The use of a viral inactivated form of IVIGcurrently seems mandatory to avoid post-transmission hepatitis.
REFERENCES 1.
Bussel, J. B.: Intravenousimmunoglobulintherapy for the treatment of idiopathic thrombocytopenic purpura. Progress in Hemostasis Thromb. 8: 108-126, 1987.
2.
Imbach, P., Barandun, S., d’Apuzzo,V., Baumgartner,C., Hirt, A., Morell,A., Rossi, E., Schoni,M.,Vest, M., and Wagner, H.P.: High-doseintravenous gammaglobulin for idiopathic thrombocytopenic purpura childhood, in Lancet 1(8232):1228-31,1981.
3.
Fehr, J., Hofmann, V., and Kappeler, U.: Transient reversal of thrombocytopenia in idiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin, New Eng. J. Med. 306(21):1254-8,1982.
4.
Imbach, P., Wagner, H.P., Berchtold, W., Gaedicke, G., Hirt, A., Joller, P., MuellerE., andBarandun, S.: Intravenousimmunoglobulin EckhardtC.,Muller,B.,Rossi, versus oral corticosteroids in acute immune thrombocytopenic purpura in childhood, Lancet 2(8453) 464-8, 1985.
5.
Bussel, J. B. and Pham, L. C.: Intravenous treatmentwith gammaglobulin in adults with immune thrombocytopenic purpura: Review of the literature. VoxSang. 52:206-211, 1987.
6.
Bussel, J. B., Fitzgerald-Pedersen, J., Feldman, C.: Alternation of two doses of intravenousgammaglobulinin the maintenancetreatmentofpatientswithimmune thrombocytopenic purpura, Amer. J. Hem. 33:184-188, 1990.
7.
GodeauB.,Lesage, S., Divine,M.,Wirquin,V., Farcet, J.P., andBierling,P.: Treatment of adult chronic autoimmune thrombocytopenic purpurawith repeated highdose intravenous immunoglobulin,Blood 82(5): 1415-21, 1993.
8.
Hollenberg, P. J., Subak, L. L., Ferry, J. J., and Bussel, J. B.: Cost-effectiveness of splenectomyversusintravenousgammaglobulinin the treatment of chronic immune thrombocytopenic purpura in childhood. J. Pediatrics 112530-9, 1988.
9.
Bussel, J., Pham, L. C., Aledort, L., and Nachman, R.: Maintenance treatmentof adults with chronic refractory immune thrombocytopenic purpura using repeated intravenous infusions of gammaglobulin,Blood 72(1):121-127, 1988.
10.
Bussel, J. B., Graziano, J. N., Kimberly, R. P., Pahwa, S., Aledort, L.: V anti-D treatment of immunethrombocytopenicpurpura:Analysis of efficacy,toxicity,and mechanism of effect. Blood 77:1884-1893,1991.
AUTHOR INDEX A
G
Adams, L E.,233 Anstee, D. J., 187
Garratty, G., 213 Greenwalt, T.J., 3 B H
Balakrishnan, K, 233 Bianco, C., 155 Blajchman, M. A., 163,
Heaton, A., 353 Heaton, W.A. L,371 Hemming, N. J., 187 Holme, S., 353 Homburg, C. H. E.,245
311
Bordin, J. O., 311 Bowden, R. A., 117 Broxmeyer, H. E.,391 Busch, M. P., 147 Bussel, J. B., 451
K Klein, H. G.,
411
D Daniels, G., 199 Davenport, R. D., 319 Davey, R. J., 431 de Haas, M., 245 Dodd, R. Y.,25 Dracker, R. A., 403, 443 Dzik, W.,95
L Lambrecht, B., 73 M Menitove, J. E.,423 Meryman, H.T.,303 Mincheff, M. S., 303 Mohr, H.,73
F Friedman, L L, 49
457
458
AUTHOR INDEX
P Perkins, H.,289 Piertersz, R. N. I., 87
Strauss, R. G., 341 Stromberg, R. R., 49 Sweeney, J. D.,353 Szatrowski, T. P., 451
Q T
Qutaishat, S., 435
R
Tanner, M.J. A., 187 Tamer, P. I., 277 Tippett, P., 173
Reed, E.,273 Reesink, H.W.,87 ROOS,D.,245
V S
Sazama, K., 131 Selz, A., 73 Snyder, E. L., 333 Steneker, I., 87
van der Schoot, C. E.,245 von dem Borne, A.E. G. Kr., 245 W
Wagner, S. J., 49
SUBJECTINDEX anergy 278 anestheticagents 278 antibodies 155, 333 199 Aantigen antibody dependent cellular cytotoxicity ABC antigens 246 assay 248 ABH phenotypes 202,215 anticoagulant 4 AB0 group 199,213,297,324 antigenpresentingcell (APC) 235 abortion 277 anti-HBc 131, 291 acidcitratedextrose 372 anti-HCV 131 acquired immunodeficiency syndrome anti-immunoglobulin idiotype antibod(AIDS) 134, 137,156,164,289, ies 234 292,411,412 anti-inflammatorycytokines 323 activatedB cells 233 antineoplastictherapy 443 acutenormovolemichemodilution 435, antinuclearantibodies 234 437 anti-PP,pk 217 acuteT-cellleukemia 292 autoimmune neutropenia (AINP) of infanadeno-associated virus (AAV) veccy 249 tors 398 autoimmunity 233 adenosinedeaminase (ADA) 417 autologousblood 278 adhesionmolecules 226, 233, 333, 336 autologousbloodtransfusion (ABT) 435 AE-1 SA0 187 AE-1 187,188,224 alanineaminotransferase (ALT) 30, 38, 156,291 B A-likeantigens 216 alloantibodies 297 199 Bantigen allogeneicbloodtransfusion (AB”) 311, B cells 234,240,262 435 B19 parvovirus 31,32,49,224,294, allogeneicdonorleukocytes 95 436 allogeneicdonorlymphocyte 416 Bacillus cereus 167 allogeneic lymphocytes 411 bacterialovergrowth 95,289 allogeneicmononuclear cells 428 leukocytefiltration for 96 allogeneictransplant 447 bacterialremoval 95 alloimmunization 8, 311 biologicalresponsemodifier (BRM) 333 American Association of Blood Banks bloodbanks 4, 132,289,295,395 (AABB) 290,373,440 A
459
460
SUBJECI' INDEX
blood contamination bacteria 49,87 parasites 49 viruses 25,49, 87, 131 bloodgroupantigens173,213 bloodgroupgenes 199 bloodgroups7, 199 bloodinfectivityreduction25,49 blood stem/progenitor cell transplantation 393 bloodsubstitutesxiii,403 blood supply confidential unit exclusion (CUE) 36, 148 donor selection 34, 49, 147, 155, 169, 425 laboratorytesting37,49, 131 viralinactivation 40,454 bloodtestinglimitations135 calculationerrors140 effects ofphysicalagents 140 falsenegatives137 falsepositives 136 performanceerrors 140 sampleidentificationerrors 139 seroversion 139 bonemarrowstem cells 443,445 bone marrow transplantation (BMT) 239,293,393,412,447 bloodstorage303
C C8 bindingprotein255 cancerrecurrence283 CD4+ cells 236,412 CD8+ cells 239,416 CD28 236 CD34+ 444,448 CD99 183 CDP 372,373 cellularimmunotherapies411 cellular therapies 411 Centers for Disease Control 137, 150,290 CFU-GEMM 396
Ch/Rg antigens224
Chagasdisease289,295 chemokines 323 chemotherapy393,443 chill-feverreactions295,426 chronicmyelocyticleukemia ( C m ) 415 class I1 antigen236 coagulation 214 collagen type I1 234 colonyassays394 colony forming unit (CFU) assays 444 colonystimulatingfactor(CSF)392, 393 colorectalcancer 280,283 complementfragments 333 complementreceptor 1(CR1)225 complementsystem225 congenital immunodeficiency syndromes 432 cord blood stem cell transplantations 448 444,447 cord blood stem cells Crohn'sdisease277,285, 311 crossmatching 297 CTLA4Ig 236 cytokine growth factors411 cytokines239,319, 333, 391,427 cytomegalovirus (CMV) 31, 32,49,117, 118, 123, 131, 289, 293, 416, 426, 436 bloodproduct risk 118 populationsat risk 119 second CMV straininfection124,125 seropositivepatient 124
D
(WC)135,
decayacceleratingfactor (DAF) 173, 225 delayedcutaneoushypersensitivity278 delayed-type hypersensitivity reaction (DTHR)219,236 dendriticcells233,236 detectionmethods 133 Di'antigen 190 dialysis277,280
SuBJECrINDEx
461
dimethyl sulfoxide (DMSO) 445 DNA detection 150 Duffy blood group antigens 220
E endothelial leukocyte adhesion molecule-l (ELAM-1) 319, 321 enzyme immunoassay (EIA) 138, 147, 159 enzyme-linked immunosorbent assay (ELISA) 30,38, 133, 158, 159, 175, 191 Epstein-Barr virus (EBV) 416, 436 ERIK antigen 206 erythropoietin (EPO) 341 Escherichia coli 222, 283
glycolysis 5 glycophorins C/D (GPC/D) 191 graft-versus-host disease (GVHD) 239, 298,311,385,415 graft-versus-leukemia (GVL) 415 granulocytecolony stimulating factor (G-CSF) 446 granulocyte-macrophage colony stimulating factor (GM-CSF) 446 group I 246 group 0 blood 201 growth factors 391
H H antigen 199 Haernophilusinfluenzae
213,223 hemodilution 404 hemoglobin 3 F hemoglobin-based blood substitutes 406, 424 factor Vn 330 hemolytic anemia 3, 8, 294 factor VIII 294, 372 hemolytic transfusion reactions 319 Fanconi anemia 395 hemophilia 294 Fcr polymorphisms 261 Helicobactre pylori 213, 223 and disease susceptibility 262 hemorrhage 403 FcRIII 256 hepatitis A virus (HAV) 31, 32, 49, FcRIII deficiency 256 291,294 febrile nonhemolytic transfusion reaction hepatitis B virus (HBV) 26, 29, 32, (FNHTR) 165,333,426,427 49,155, 157,291,416,436,455 Food and Drug Administration hepatitis B virus core antibody (FDA) 131, 134, 140, 156, 166, (HBcAb) 156 250,290,296,373,385 hepatitis B virus surface antibody fresh frozen plasma (FFP) 423, 425 (HBsAb) 157 hepatitis B virus surface antigen (HBsAg) 29,38,131, 155, 157 G hepatitis C virus (HCV) 26, 29, 32, 134, 136, 155, 159, 292, 416, gamma irradiation 431 454 gammaglobulin 451 hepatitis C virus core antibody gastrointestinal bleeding 278 (HBcAb) 155 gene therapy 397, 416 hepatitis D virus (HDV) 158 genetic disorders 398 herpes virus4 436 GIFI' 248 HIV seroconversion window 149 glyceraldehyde phosphate dehydrogeHLA 224,235,246,284,273,385,426 nase 5 HLA anti-idiotypes 273
157, 34,
49, 435,
462
SUBJECT INDEX
homing-associated cell adhesion molecule (HCAM, CD44) 226 homologousblood278, 282,403 host-versus-graftdisease(HVGD)239 human immunodeficiency (HIV) 25,26, 33, 34, 49, 62, 120, 131, 135, 147, 224,290,412,417,428,435 human papillomavirus416 human T-lymphotropic retrovirus ("'LW 26,28,32,131,138,289, 292,436,454
I IgG 248,249,451 IgM 248,249 immunehematologicdisease451 immunoblasticlymphoma416 immunocompetentpatient431 imrnunocompromisedhost117, 120,434 immunologicallyimportantproteins213 immunomodulation299,303, 311 immunosuppression312,412,426 incompatibilityreactions297 infection214,281 inflammatoryboweldisease312 insulindependentdiabetesmellitus 285, 411 intercellular adhesion molecule-l (ICAM-l) 319,321 interleukin-l (IG1) 320,334 interleukin-lP 319 interleukin-l receptor antagonist (IGlra) 319,323,324,328 interleukin-2 QL2) 313,413 interleukin-6 (IL6) 320 interleukin-8 (IG8) 319, 323, 324, 334 intraoperativebloodsalvage435,439 iron 344 irradiation385,431,433 ITP 451,452 acutechildhood452 adult 452 chronic 453 M G 451,452 preparations 454 viraltransmissionby454
K keyhole-limpethemocyanin 297 Knopssystem174,225
0;o
412
Klebsiella
H Le antigens246 leukemia 395 leukocyte adhesion deficiency typeI1 (LADII) 246 leukocytedepletedbloodproducts117, 123 leukocyte depletion filters 88, 95, 334 column 88 flat bed 88 leukocyte filtration 87, 96, 99, 102, 126, 423,426 leukocytefiltrationmechanisms 88 adhesion89,105 cell-cell interactions (indirect adhesion) 89 effect of filter material 90, 107 effect of flowtime 91 effect of plasmaproteins91, 103 effect of platelets 91 effectofstorage91, 102 effect oftemperature 91 mechanicalsieving 89 leukocytesxiii, 50 leukotrienes 283 leukotropic 95 long-term colony culture (LTCC)assays 444 lymphocyte subsets 279 lymphocytes233,278 lymphokine-activated killer (LAK) cells 413,414 lyophilizedredcells405 M McCoybloodgroupantigens 225 macrophage inflammatory protein (MIP-l) 336
SUBJEm INDEX
463
macrophages 233,283 neutrophil antigens 245,246 major histocompatibility complex neutrophil chemiluminescence test (MHC) 233,313 (NCLT) 248 malaria 220 neutrophil cytotoxic test (NW 248 malignant melanoma 312, 415 neutrophil immunofluorescence test malignant tumors 214 (NW 248 MART antigens 258 neutrophil membrane glycoproteins 245 membrane inhibitor of reactive lysis nomenclature 8 (m)225,255 non-A, non-B hepatitis (NANBH) 29, meningococcal disease 262 156, 159, 291 microchimerism 241 non-A,non-B, non-C hepatitis 132 mixed-lymphocyte culture @KC) 432 nosocomial infections 282 MNS blood group system 199, 203 monoclonal antibodies 8, 175, 188, 224, 246 0 monoclonal antibody-specific immobilization of erythrocyte antigen OND antigens 258 (MAIEA) 173, 174 oxygen-carrying blood substitutes 403, monocyte chemoattractant protein 424 (Mm)323 myeloablative therapy 443 P N NA antigens 256 NA system 247
P blood group antigens 222,246 P. falciparum 220, 295 P. knowlesi 220 P. v i v u 213, 220, 295
paroxysmal nocturnal hemoglobinuria National Institutes of Health (NIH) 290, PNH) 225,255 412,471 peffluorocarbon solutions 405, 424 National Marrow Donor Program peripheral blood lymphocyte (PBL) 412, (NMDP) 446 417 natural killer (NK) cells 239, 248, 254, peripheral blood stem cell (PBSC) 444, 313,413 445 natural killer cytotoxicity 280 phosphatidyl-inositol glycan (PIG) 255 NB system antigens 252 phosphofructokinase (PFK) 3,5 N B 1 252 photoinactivation 73 NC antigens 258 dye concentration 81 ND antigens 258 influence on plasma component activineohematocytes 407 ty 74 neonatal alloimmune neutropenia light intensity 81 ( N A I N P ) 249 placental blood 394 neonatal anemia 341 plasma processing 51 neonatal isoimmune neutropenia 256 affinity column 52 neoplastic diseases 443 gamma irradiation 53 neutrophil agglutination test (NAT) 248 iodine 53 neutrophil alloantigens 249 methylene blue 54 neutrophil antibodies 248 microporous membrane filtration 52
464
[plasma processing continued] miscellaneous approaches 55 pasteurization 53 photoinactivation 73 solvent detergent 53 plasma 50 Plasmodiummalariae 295 Plasmodiumovale 295 platelet additive solutions 383
platelet allotypes xiii platelet concentrate contamination 97 platelet concentrates 96,97,163,168, 353,433
quality 353 platelet crossmatching 428 platelet potency 361 platelet processing 60 collection, transportation, and preparation 353 containers 357 end processing 361 invitro storage 356 liquid environment 357 miscellaneous approaches 60 photochemical approaches 61 physical conditions 357 preparation 354 shipping 361 storage 361 platelet rich plasma (PRP) 354 platelet transfusions 425 plateletpheresis 428 platelets 423 polymerase chain reaction (Pa) 140, 151,202
postoperative blood salvage 435,439 postoperative blood transfusion
(PBT) 439 postoperative infections 277,427 posttransfusion hepatitis 291 predeposit autologous donation
( P A D ) 436 preoperative blood donation 435 pretransfusion testing 163 progenitor cells 391,393 prostaglandins 283,334 protozoa 295
R radiotherapy 443 random-donor platelets O P ) 423,425, 428
recombinant EPO 344,423 recombinant immunoblot assay (RIBA) 138, 159
recurrent spontaneous abortion 284,311, 411 red blood cells xiii, 3, 50,213,423 cytoskeleton 11, 173
glycosyl-phosphatidylinositol protein anchors 15 lipid bilayer 13 membrane 10, 187, 199 surface 173 transport mechanisms 15 red cell additive solutions 377 red cell antigens 199 red cell concentrate contamination 98 red cell concentrates 87,95, 98, 163 red cell processing 55 buffy coat depletion 384 chemical approaches 58 collection 372 containers 376 component separation 372,375 extended storage 58, 381 leukodepletion 56,297,311,336, 384 photochemical approaches 59 platelet depletion 57 temperature elevation 58 washing 57 reperfusion injury 338 Rh antigens 9, 177 Rh blood group system 208 Rh complex 3, 8
Wd
208
S
selectins 226,246 self-antigens 234 self-proteins 234 seronegative blood products 117, 121
SUBJECT INDEX
465
severe combined immunodeficiency syndrome (SCID) 417 Sialyl-Tn antigen 218 single-donor platelets (SDP) 423,426,
tumor markers 213 tumor necrosis factor 0319,320, 325
429
U
solid organ transplants 240,293,295 solvent detergent 454 South-east Asian ovalocytosis
umbilical cord blood 391,394 urinary tract infection 223
(SAO)
187,188 splenectomy 453 St'antigen 204 Staphylococcus aureus 167 Staphylococcus epidermidis 95,97,102, 106, 110, 167 Staphylococcus xylosus 109 stem cell assays 444 stem cell therapy 443 stem cells 391,443 administration 445
V virus removal and inactivation 51,73, 78, 147
processing and storage 444 syngeneic lymphocyte transfusion 412 syphilis 294
methylene blue 73, 78 phenothiazine dyes 73 photoinactivation 73, 78 viruses 25,49, 169 blood distribution 32 W
T T antigen 218 T cell receptor (TCR) 233 T cells 234, 262 T lymphocytes 414 thromboxane 283 Tn antigen 218 transforming growth factor (TGF) 396 transfusion-associated graft-versus-host disease (TA-GVHD) 431 transfusion-associated sepsis ("AS) 163,
World Health Organization (WHO) wound healing 285 Wr' antigen 189
X xenogenic transfusions 403
Xg' 182
166
transfusion induced acute lung injury
Y
WI) 250,333,337 transfusion reactions 250,289,311,319, 325
transfusions xiii, 25,32,49,87,95,
117,
123, 147, 199,277,284,303,423
transplantation 233,236,273 Trypanosoma Cruzi 295 tumor antigens 215 tumor growth 284,311,312 tumor immunology 411 tumor infiltrating lymphocyte (TU.,)
Yersinia enterocolitica 95,98,102,103, 106,110, 164,296 parasites 220 York blood group antigens 225
2 414
zidovudine (AZT) 413
134
