Mollison’s Blood Transfusion in Clinical Medicine Mollison’s Blood Transfusion in Clinical Medicine 11TH EDITION Harvey G Klein MD President of the American Association of Blood Banks; Chief, Department of Transfusion Medicine Warren G Magnuson Clinical Center National Institutes of Health Bethesda Maryland, USA; Adjunct Professor of Medicine and Pathology, The Johns Hopkins School of Medicine and David J Anstee PhD FRCPath FMedSci Director, Bristol Institute for Transfusion Science National Blood Service Bristol, UK; Honorary Professor of Transfusion Science, University of Bristol A revision of the 10th edition written by P.L Mollison, C.P Engelfriet and Marcela Contreras © 1951, 1956, 1961, 1967, 1972, 1979, 1983, 1987, 1993, 1997 by Blackwell Science Ltd © 2005 by Blackwell Publishing Ltd Blackwell Publishing, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USA Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988 All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher First published 1951 Second edition 1956 Third edition 1961 Reprinted 1963, 1964 Fourth edition 1967 Reprinted 1967 Fifth edition 1972 Reprinted 1974 Sixth edition 1979 Seventh edition 1983 Eighth edition 1987 Reprinted 1988 Ninth edition 1993 Reprinted 1994 (twice) Tenth edition 1997 Eleventh edition 2005 Library of Congress Cataloging-in-Publication Data Klein, Harvey G Mollison’s blood transfusion in clinical medicine – 11th ed / Harvey Klein and David Anstee p ; cm Rev ed of: Blood transfusion in clinical medicine / P.L Mollison, C.P Engelfriet, Marcela Contreras 10th ed c1997 Includes bibliographical references and index ISBN-13: 978-0-632-06454-0 ISBN-10: 0-632-06454-4 Blood Transfusion Blood groups I Mollison, P L (Patrick Loudon) II Anstee, David J III Mollison, P L (Patrick Loudon) Blood transfusion in clinical medicine IV Title V Title: Blood transfusion in clinical medicine [DNLM: Blood Transfusion WB 356 K64m 2005] RM171.M6 2005 615′.39–dc22 2005012644 A catalogue record for this title is available from the British Library Set in 9/11.5pt Sabon by Graphicraft Limited, Hong Kong Printed and bound in the United Kingdom by TJ International Ltd, Padstow, Cornwall Development Editor: Rebecca Huxley Commissioning Editor: Maria Khan Production Controller: Kate Charman For further information on Blackwell Publishing, visit our website: http://www.blackwellpublishing.com The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp processed using acid-free and elementary chlorine-free practices Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards Contents Foreword, vii Preface to eleventh edition, ix Preface to first edition, xi Abbreviations, xiii Blood donors and the withdrawal of blood, Transfusion of blood, blood components and plasma alternatives in oligaemia, 19 Immunology of red cells, 48 ABO, Lewis and P groups and Ii antigens, 114 The Rh blood group system (and LW), 163 Other red cell antigens, 209 Red cell antibodies against self-antigens, bound antigens and induced antigens, 253 Blood grouping techniques, 299 The transfusion of red cells, 352 10 Red cell incompatibility in vivo, 406 11 Haemolytic transfusion reactions, 455 12 Haemolytic disease of the fetus and the newborn, 496 13 Immunology of leucocytes, platelets and plasma components, 546 14 The transfusion of platelets, leucocytes, haematopoietic cells and plasma components, 611 15 Some unfavourable effects of transfusion, 666 16 Infectious agents transmitted by transfusion, 701 17 Haemapheresis, 774 18 Alternatives to blood transfusion, 810 Appendices, 845 Index, 865 Plate section between pages 528 and 529 v Foreword The first edition of Blood Transfusion in Clinical Medicine was published in 1951, at a time when the subject was, if not in its infancy, certainly in its very early childhood Transfusions were given for the treatment of acute blood loss or for the relief of chronic anaemia Platelet and leucocyte transfusions were not attempted and plasma fractions were not available Only a few red cell antigen systems were recognized and leucocyte or platelet antigens were unknown It was understood that syphilis and malaria could be transmitted by transfusion, and it had recently been discovered that hepatitis could also be transmitted although no test for the virus was available Evidently, attempting to summarize knowledge of the whole subject in 1951 was not a very daunting task My own position in the years that followed made it possible for me to devote a great deal of time to trying to keep the book up to date in successive editions as the subject expanded in all directions However, by the time that the eighth edition was being planned I recognized that, even with a great deal of help from others, I could not give an adequate account of leucocyte and platelet antigens or of diseases transmitted by transfusion Professors Paul Engelfriet and Marcela Contreras, who had immense experience of these and many other aspects of the subject, became joint authors, greatly strengthening the scientific background of the book After the tenth edition was published in 1997, the three of us decided not to continue, but Blackwell Publishing felt that as the book was an established text in the field of transfusion they would like to commission a new edition I must point out that, although we are clearly responsible for such parts of the text of the tenth edition as have been retained, credit for the revision is due entirely to the new authors, who have had no help of any kind from us It is fortunate that two people so highly qualified as Harvey Klein and David Anstee have been willing to undertake this task Harvey Klein is one of the very ablest practitioners of transfusion medicine in existence and has a wide understanding of all the clinical aspects of the subject David Anstee’s research has vastly increased knowledge of the molecular structure of red cell antigens; the chapters he has revised – particularly the chapter on the Rh blood group system – now provide what must be the best available source of information on this subject Both authors have dealt in an authoritative way with the still rapidly expanding specialty, and the eleventh edition of the book will be of the greatest value to all who are interested in the scientific and practical aspects of blood transfusion in clinical medicine Professor P.L Mollison 2005 vii Preface to eleventh edition The huge challenge of revising this seminal work has been both daunting and immensely rewarding Mollison’s textbook is an icon Blood Transfusion in Clinical Medicine arose from the concept of the transfusionist as both scientist and expert consultant For many years, this text provided the primary, and often the sole, reference for detailed information and practical experience in blood transfusion A generation of scientists and clinicians sought and found in its pages those fine points of immunohaematology that helped them manage their patients and satisfy their intellectual curiosity The last two decades have witnessed an explosion of scientific knowledge, the proliferation of textbooks, handbooks, systematic reviews and specialty journals, not to mention immediate access to manuscripts not yet in print via the Internet The current authors determined to distil from this mass of information the relevant biology and technology for a timely, comprehensive and clinically useful textbook – without altering the spirit and character that has made Mollison’s textbook a cherished companion Mollison’s textbook has recorded the development of blood transfusion practice and its scientific basis for more than half a century The first edition focused mainly on the recognized blood groups and their clinical implications Immunohaematology was confined largely to the red cell The marvellous complexity of blood was defined by agglutination, and subsequently by the mixed lymphocyte reaction, lymphocytotoxicity and serum protein electrophoresis Red cell survival, a tool both for investigating clinical problems and for exploring fundamental information regarding haemolytic processes and red cell pathology, was estimated ‘with the comparative precision of differential aggluti- nation’ Whole blood was still transfused by the bottle Today, tens of millions of units of blood components are transfused annually The immune response is analysed by a wide array of sophisticated techniques and the diversity of human blood is routinely examined at the molecular level Circulating cells and their survival still teach us about immunology and cellular biology, but we can now track the persistence of transfused lymphocyte subpopulations with molecular assays of microchimerism This text endeavours to continue the tradition of integrated biology, technology and clinical practice that characterized the original book and all subsequent editions Since the last edition, major changes in practice and advances in our understanding have occurred in some aspects of the field, but not in others The human genome has been sequenced Informatics and computational biology have revolutionized the approach to biodiversity Advances in DNA-based technology, from microarrays to recombinant proteins, have had a major impact on many aspects of blood transfusion practice Transfusion medicine now involves mobilization and selection of haematopoietic progenitor cells for transplantation, storage of umbilical cord blood, and manipulation of mononuclear cells by culture and gene insertion to offer potential therapies for a wide range of diseases This edition has been revised to reflect this remarkable progress Enormous advances in protein structure determination have occurred since the last edition and these too are reflected in the revised edition It is particularly satisfying to record the three-dimensional structure of the glycosyltransferase responsible for the ABO blood groups just over a century after Landsteiner’s discovery made safe blood ix IMMUNOLOGY OF RED CELLS Table 3.4 The main immunoglobulins in serum IgG Heavy chains Light chains Molecular forms Molecular weight Sedimentation coefficient Concentration in plasma (g/l) Adult* Newborn Catabolic rate, t1/2 (d)† Percentage of total which is intravascular† Transferred across placenta? Binds to Fc receptors? Usual serological behaviour as red cell antibody Serological activity after heating to 56°C for h Effect of reducing agents on serological activity IgM IgA γ κ and λ γ2κ2 γ2λ2 150 000 7S µ κ and λ (µ2κ2)5J (µ2λ2)5J 970 000 19S α κ and λ (α2κ2)1 or (α2κ2)1 or 160 000 7S, 9S and 11S 8–16 Similar 21 52 Yes Yes Incomplete antibody 0.5–2.0 Approx 0.1 74 No No Agglutinin Unaffected Reduced 1.4–4.0 Undetectable 40 No Yes Agglutinin (dimers only) Unaffected May develop agglutinating activity No longer agglutinates Partially inactivated * Roitt (1988) † Wells (1980) J = J chain subclasses in having a much longer hinge region (see Fig 3.3) Several membrane-bound receptors recognize the Fc region of antibodies FcRI, FcRII and FcRIII recognize sites on the lower hinge region (CH2) of IgG (Sondermann and Oosthuizen 2001) (Plate 3.3) Monomeric IgG1 and IgG3 bind to FcRI; polymeric IgG1 and IgG3 bind, in addition, to FcRII and FcRIII Three closely linked genes encode three different types of FcRII: FcRIIa, FcRIIb and FcRIIc FcRIIa has two variants, one binds only weakly to IgG2 but the other, present in 30% of white people, binds strongly (Warmerdam et al 1991) IgG4 binds weakly to both forms of FcRIIa Glycosylation is essential for some functions of IgG, including adherence to Fc receptors IgG is the only Ig to be transferred across the placenta from mother to fetus, a fact that explains the role of IgG antibodies in the aetiology of alloimmune cytopenias of the fetus and newborn The transfer of IgG across the placenta from mother to fetus is mediated by the neonatal Fc receptor (FcRn) (see Chapter 12 and Plate 12.1) FcRn has a different structure from the other Fc receptors and consists of a heavy chain complexed with beta2 microglobulin (Burmeister et al 1994) IgG1 and IgG3 activate complement strongly but IgG2 activates only weakly and IgG4 not at all IgG1, IgG2 and IgG4 myeloma proteins have catabolic rates that are similar to those of total IgG, i.e they have a t1/2 of about 21 days but the t1/2 of IgG3 is about days (Morell et al 1970) The foregoing estimates were derived from measurements on myeloma proteins Similar half-lives have been found for monoclonal Rh D antibodies, namely for IgG1, 21–22 days (Callaghan et al 1993; Goodrick et al 1994) and for IgG3, 10 days (Goodrick et al 1994) IgM The plasma concentration of IgM is 0.5–2.0 g / l; about 74% of total body IgM is intravascular; the plasma 61 CHAPTER t1/2 is days IgM molecules are held together by a J (joining) chain (see Fig 3.3) IgM is more effective than IgG in activating complement The difference is due to the fact that C1 is activated only when it is attached by two of its C1q heads to the activating molecule Thus, whereas a single bound IgM molecule can activate C1, at least two IgG molecules bound at closely adjacent sites are needed Several of the antigen-binding sites on a single IgM molecule may bind to antigen sites on the cell surface, giving the molecule the appearance of a staple (see Fig 3.4b), in contrast to the normal star form shown in Fig 3.4a IgA The plasma concentration of IgA is 1.4 – 4.0 g / l; about 40% of total body IgA is intravascular; the plasma t1/2 is days IgA is the only Ig present in epithelial secretions Whereas, in plasma, IgA is predominantly monomeric, in secretions it occurs mainly as dimers, which are held together by a J chain In secretions, IgA has a ‘secretory piece’ that prevents the molecule from being digested The presence of IgA in secretions is believed to be due almost entirely to local production rather than to transport from plasma The function of IgA is evidently to neutralize antigens that might otherwise enter the body via mucosal routes There are two subclasses of IgA, a major component, IgA1, and a minor component, IgA2, which has two serologically distinguishable variants, A2m(1) and A2m(2) Both IgAl and IgA2 bind to an Fc receptor for IgA (CD89) on monocytes and macrophages Inhibition of IgA binding to CD89 by staphylococcal superantigen-like proteins leads to increased survival of staphylococcal bacteria in blood (Langley et al 2005) IgD and IgE Both of these Igs act mainly as cell receptors No free antibodies made of IgD have been described, although IgD is synthesized by, and demonstrable on, the surface of B lymphocytes and is probably involved in the activation of these cells by antigen IgE is synthesized by plasma cells and is present in the plasma in very low concentrations It has a very high affinity for Fc receptors (FcεRI) on basophils 62 and mast cells The binding of antigen to IgE antibody on the surface of these cells leads to crosslinking of receptors and produces degranulation with release of vasocative amines, such as histamine, and of cytokines as well as synthesis of inflammatory mediators of arachidonic acid (e.g leukotrienes) These mediators are responsible for producing such atopic phenomena as asthma and hay fever The role of IgE antibodies in reactions to atopens is discussed in Chapter 15 Markers on immunoglobulin molecules Idiotypic markers Idiotypic determinants arise from the unique configuration of the antigen-combining site of an antibody molecule, which gives the molecule its specificity The idiotype of a particular antibody specificity is composed of several individual determinants called idiotopes, in much the same way that the Rh polypeptides expressed by the R1 haplotype encompass several Rh determinants (i.e D, C, G, e, Rh17, Rh18, Rh29, Rh34, Rh44, Rh46, Rh47, etc.) Some of the individual idiotopes arise from the unique amino acid sequences in the hypervariable region that come into contact with the antigen in the antibodybinding site; others arise from framework amino acid sequences Antisera can be raised to both sets of idiotopes Some idiotypes (public idiotypes) are common to all antibodies of a single specificity; each individual can make 106–107 different antibody specificities Other idiotypes (private idiotypes) are restricted to only a few clones producing antibodies of similar specificity but each differing slightly (by one or two amino acids) in their hypervariable region; a typical polyclonal immune response can be composed of 104 different species of antibody molecules of similar specificity, each species being the product of a separate B cell clone Sometimes, antibodies of quite distinct specificities, for example anti-albumin and anti-influenza virus can exhibit common idiotypes; these are called the major crossreactive idiotypes and represent common amino acid sequences in the framework regions of their binding sites Every antibody is capable of inducing the formation of auto- or allo-anti-idiotypes The formation of autoanti-idiotypes is believed to be important in regulating the immune response IMMUNOLOGY OF RED CELLS Isotypic markers These are antigenic determinants on H chains and on certain L chains, which define and characterize the class or subclass of Ig molecules and which are sequences on the C region of γ and µ chains or on the C region of κ and λ chains Isoallotypic markers (formerly called non-markers) are those which are isotypic markers for one subclass but are allotypic markers in another subclass For example, G4m(a) is an allotype in subclass IgG4 but is an isotype in IgG2 100 IgM IgG Per cent of adult level Allotypic markers These are antigens, found on γ, α and κ chains, the inheritance of which is controlled by alleles of the gene coding for a particular Ig chain For example, H chains of IgG subclass can carry either G1m(z) or G1m(f), the difference being determined by a single amino acid substitution Further details are given in Chapter 13 IgG 50 IgA 0 12 Months Fig 3.5 Concentration of IgG, IgM and IgA in the first year of life, as percentages of the normal adult levels (based on the data of West et al 1962) The dotted line indicates IgG derived from the mother by placental transfer Immunoglobulins in fluids other than plasma Immunoglobulins are found in the following fluids Colostrum and saliva In colostrum, the IgA concentration is about 90% and in saliva is about 20% of that in normal serum (Chodirker and Tomasi 1963) By contrast, concentrations of IgG and IgM in colostrum are respectively 1% and 5–10% of the amounts in normal serum, and in saliva only faint traces of IgG and IgM are present (Adinolfi et al 1966) The titre of IgA antibody tends to be higher in colostrum than in serum (Tomasi et al 1965; Adinolfi et al 1966) Colostrum frequently contains potent IgA anti-A and anti-B, but in saliva these antibodies are usually present only in low concentration (see Chapter 4) Ascitic fluid Several alloantibodies have been harvested from ascitic fluid; anti-Yta (Eaton et al 1956); anti-c and -Lea (MM Pickles, quoted by Race and Sanger 1968, p 255); and anti-E (Zeitlin et al 1958) and anti-K (Longster and Major 1975) In the last mentioned case the titre of the antibody was about the same in serum as in ascitic fluid and the antibody was probably IgG; in contrast to that of anti-K, the titres of anti-A and anti-B were slightly lower in ascitic fluid than in serum Amniotic fluid The IgG concentration at or near term is about 0.1–0.2 g / l, i.e 1/50 –1/100 of that in normal adult serum Production of immunoglobulin in the fetus and infant Changes in Ig levels in the first year of life are summarized in Fig 3.5 IgG Although most of the IgG in the serum of newborn infants is derived from the mother by placental transfer, a small amount is of fetal origin as shown by the fact that it has the father’s Gm allotype (Mårtensson and Fudenberg 1965) The time at which the synthesis of substantial amounts of IgG begins after birth was studied by Zak and Good (1959) in two normal infants born to mothers with severe hypogammaglobulinaemia The IgG level, which was negligible at birth, started to increase between and weeks; antibodies were first demonstrable at about months, by which time the IgG level had reached g / l At 10 –18 months, IgG levels are about 60% of adult values (Buckley et al 1968) IgM IgM is not transferred across the placenta The concentration in cord serum is between 5% and 10% of that found in adult serum IgM antibodies of various specificities can be found in cord serum: anti-I in most infants (see Chapter 7); anti-A and anti-B in occasional infants (see Chapter 4); anti-Gm in most infants (see Chapter 13) and anti-A chain in about 10% of infants (Epstein 1965) Within or days of birth the concentration of IgM starts to rise and reaches 50% of 63 CHAPTER the adult level at 2–3 months and 100% at about months; between the ages of months and years values remain at the adult level, although between and years they are at the lower end of the adult range (West et al 1962) IgA This protein cannot be detected in cord serum By the age of months, the amount in the serum has reached about 20% of the adult level (West et al 1962) and by the age of years about 50% (Buckley et al 1968) Immunoglobulin class and subclass of red cell alloantibodies Red cell alloantibodies may be naturally occurring or immune When naturally occurring they are most often IgM but may be partly IgG or, occasionally, predominantly IgG (see also p 80); when immune they are most often IgG, but may be IgM or a mixture of IgM and IgG; they are sometimes partly IgA Cells derived from a single clone make antibodies of the same specificity but any given cell can make only one class of antibody Determination of the subclass of antibodies is difficult There is more than 95% sequence homology between most of the C regions of human IgG subclasses There are therefore few subclass-specific epitopes and it is difficult to raise antibodies against them (Michaelsen and Kornstad 1987) If insufficiently absorbed sera are used, false-positive results are obtained If well-absorbed sera are used, some antibodies fail to react with all subclass sera Investigations of the subclass composition of different blood group antibodies are referred to in the three following chapters Immunoglobulins on B lymphocytes The Ig on the surface of lymphocytes gives them the capacity to react with an antigen of corresponding specificity and to develop into active antibody-secreting cells The two major classes of Ig demonstrable on the surface of immature B lymphocytes are IgM and IgD; the latter appears to have a role in triggering the cell’s response to antigen In subjects immunized to Rh D, anti-D-bearing lymphocytes can be demonstrated by incubating Dpositive red cells with the blood of the immunized 64 subjects and observing ‘rosettes’, i.e single lymphocytes surrounded by many adherent erythrocytes Rosetting could be demonstrated in two out of eight women immunized by pregnancy and in five out of five hyperimmunized volunteers following a booster injection of red cells; in these latter subjects the number of rosettes increased to a maximum on about the 10th day after re-stimulation (Elson and Bradley 1970) The Ig superfamily The name Ig superfamily is given to all those molecules that contain domains within their structures that are related to either the variable or constant domains of immunoglobulins Most of the molecules concerned play an important role in antigen recognition at the cell surface in cell–cell adhesive interactions or in cell– extracellular matrix interactions The Ig superfamily includes the antigen receptors, i.e immunoglobulins and T-cell receptors, Fc receptors, as well as several other lymphocyte cell surface molecules, such as CD4 and CD8 and MHC class I and II molecules It also includes several blood group active molecules on red cells (Lutheran, LW, Oka, see Chapter 6) Methods of separating and identifying immunoglobulins IgG can be affinity purified with particle-bound bacterial proteins A and G Protein A and Protein G although structurally distinct both bind to the Fc portion of antibody molecules at the interface of the CH2 and CH3 domains (Sauer-Eriksson et al 1995) Protein A binds human IgG1, IgG2 and IgG4 but not IgG3 Immunoglobulins may be separated according to their different charges, for example by ion-exchange chromatography or by their different sizes, i.e by passage through a ‘molecular sieve’ as in gel filtration The greater positive charge on IgG compared with other plasma proteins is exploited in one method of preparing anti-D IgG for immunoprophylaxis, IgG being separated from plasma on a diethylaminoethyl (DEAE)-cellulose column (Hoppe et al 1973) This method of separating IgG from plasma has a much higher yield than that of cold-ethanol precipitation Some IgG antibodies can be separated from one another by fractionation on carboxymethyl (CM) cellulose or hydroxyapatite columns, and results with IMMUNOLOGY OF RED CELLS 100 Anti-D 100 100 Anti-c Anti-K Anti-Fya 100 Anti-A 1 100 7 Anti-Jka Fractions 7 Fractions Fig 3.6 Distribution of the total IgG (– – – –) and of particular antibodies (––––) in fractions eluted from carboxymethyl-cellulose (using buffers or increasing molarity), expressed as percentages of the total amount recovered from the columns The four small figures show the distribution of single antibodies in four sera; the larger righthand figure shows the elution of two antibodies from one serum From Frame and Mollison (1969) these methods will be briefly described, as the methods not seem to have been fully exploited so far Anti-D, and some other red cell alloantibodies, are more positively charged than the bulk of IgG in the serum and can thus be partially separated from it Similarly, the charge on different alloantibodies varies Examples of separation of anti-D from the bulk of IgG and of other alloantibodies from one another, on carboxymethyl-cellulose columns, are shown in Fig 3.6 sensitive than intrasubunit bonds to reduction Mild reduction releases J chain from the IgM molecule Treatment of serum with a reducing agent abolishes the agglutinating activity of most IgM antibodies IgM subunits of anti-D, anti-A and anti-B retain their ability to combine with red cell antigens, but subunits of IgM anti-Lea have no serological activity, indicating that their binding affinity is very low (Holburn 1973) If a monoclonal IgM is mildly reduced and then dialysed against saline to remove the reducing agent, the subunits may slowly reassemble, if the suspending medium does not contain non-specific IgM It would therefore be expected that if a purified IgM antibody, or for example an eluate, were treated in the same way, the serological activity of the antibody would be partially restored On the other hand, when an IgM antibody in whole serum is mildly reduced and the serum is then dialysed, restoration of serological activity is not expected, because the subunits of IgM reassemble at random and most of the IgM molecules in the serum are of different antibody specificities The reassembly of IgM subunits to form 19S IgM can be prevented by treatment with alkylating agents, such as iodoacetamide (IAA) Such agents irreversibly block the SH groups which have been liberated by Identification of immunoglobulins using the antiglobulin test Determination of the Ig class of red cell antibodies is most conveniently performed using Ig class-specific antiglobulin reagents (Polley et al 1962; Adinolfi et al 1966) Effect of reduction on different immunoglobulins Effect on IgM Mild reduction of human IgM with sulphydryl compounds cleaves intersubunit (IgMs– IgMs), intersubunit-J chain and intrasubunit (H–H, H–L) disulphide bonds Intersubunit bonds are more 65 CHAPTER reduction of the protein, as well as any free SH groups originally present Effect on IgG After mild reduction, some IgG incomplete antibodies such as anti-D become weakly agglutinating (Chan and Deutsch 1960; Romans et al 1977) The changes in the properties of the IgG molecule are brought about by the breaking of the disulphide bonds in the hinge region of the molecule, permitting the two antigen-binding sites to move further apart and thus bridge the distance between red cells The two halves of the molecule remain held together by strong noncovalent bonds between the CH3 domains (see Fig 3.2) Reduced IgG3 anti-D monoclonals are more potent direct agglutinators than reduced IgG1 anti-D monoclonals (Scott et al 1989) The modification of the properties of the IgG molecule produced by mild reduction are stabilized by treatment with alkylating reagents (e.g IAA) Practical applications Treatment of serum with the reducing agents 2-mercaptoethanol (2-ME), dithiothreitol (DTT) or its isomer dithioerythritol (DTE), is commonly used in blood group serology to distinguish between IgG and IgM antibodies as, if the agglutinating activity is abolished, it is virtually certain that the antibody in question is IgM DTT is preferred to 2-ME for the following reasons: first, it is more efficient in maintaining reduction; second, it is more resistant to oxidation in air; and third, it lacks an offensive odour Serum that has been treated with 2-ME may be tested for agglutination without preliminary dialysis (Reesink et al 1972), although if the mixture of undiluted serum and 2-ME is tested by the indirect antiglobulin test, false-positive reactions occur (Freedman et al 1976) False-positive reactions also occur occasionally if DTT-treated undiluted serum is left with red cells for more than h False-positive reactions are not observed when dilutions of 2-MEtreated or DTT-treated serum are tested Heat lability of different immunoglobulins Heating at 56°C for h has little effect on anti-D (IgG) but produces a just detectable fall in anti-P titre (IgM) Heating at 63°C for h decreases the IgG anti-D titre by about one-third, whereas heating at 63°C for only h almost completely inactivates anti-P1 (Adinolfi 66 1965) IgA antibodies, like IgG antibodies, are unaffected by heating at 56°C for h (Adinolfi et al 1966) Naturally occurring antibodies In blood group serology, the term naturally occurring is used for antibodies (Table 3.5) found in the serum of a subject who has never been transfused or injected with red cells containing the relevant antigen or been pregnant with a fetus carrying the relevant antigen Landsteiner (1945) concluded that natural antibodies had a dual origin, antigen induced and spontaneous As examples of the first kind, anti-A and -B may be cited These antibodies are believed to be heteroagglutinins produced as a response to substances in the environment, which are antigenically similar to red cell alloantigens (Wiener 1951b) In ‘germ-free’ chicks, no heteroagglutinins against group B human red cells developed in the first 60 days of life, whereas in ordinary chicks such agglutinins usually develop within the first 30 days In germ-free chicks, anti-B developed promptly after the administration by mouth of Escherichia coli O86; the antibodies had serological characteristics similar to the ‘naturally occurring’ agglutinins; that is, they reacted slightly more strongly at 0°C than at 37°C In germ-free chicks, very weak agglutinins developed 2–3 months after hatching but this was attributed to traces of non-living antigenic contaminants Although the stimulus to the formation of these naturally occurring agglutinins was traced to the environment, the authors pointed out that there was evidence that the time of onset and extent of the response depended upon genetic factors (Springer et al 1959) A direct demonstration that anti-B titres can be increased in humans by the ingestion or inhalation of suitable bacteria was provided by Springer and Horton (1969) In both infants and adults, the oral administration of killed E coli O86, with blood group B specificity, produced rises in anti-B titre in a proportion of subjects; responses were commoner in subjects with an intestinal disorder, suggesting that antigen was absorbed more readily when the mucosal surface was damaged Four out of 12 adults showed an increase in anti-B titre after receiving a nasal spray of E coli O86 Similar observations have been reported for anti-T and antiTn (Springer and Tegtmeyer 1981) Other antibodies which may be heteroagglutinins pk include anti-H, -PP1 and -Pk, found in virtually all IMMUNOLOGY OF RED CELLS Table 3.5 Naturally occurring alloantibodies to red cell antigens System (or antigen) Specificity of antibodies Subjects in whom antibodies occur ABO/Hh A, B, H A1 All without corresponding antigen 1–8% of A2 22–35% of A2B Occasional A1 and A1B 20% of Le (a–b–), ABH secretors Often accompanies anti-Lea; rare on its own All without corresponding antigen Common in P2 Most i 0.02% of M–N+ 0.002% of M+N– Very rare in S– 1–2% of population 0.1% of D positives Very rare 0.15–0.3% of D negatives 1–2% of population 1% of population Rare Rare 0.02% of population Lewis P/P1 I MNSs Rh Sd Di Ge Kk Lu HI Lea Leb Pk, PP1Pk P1 I M N S VW, Mg E C, Cw, Cx D (cold) Sda Wra K Lua The list is incomplete: specificities that have been found occasionally include antiIna, -Lub, -LW and -Yta amongst others; naturally occurring antibodies to several low-frequency antigens other than Vw, Mg and Wra are common; the figures given for frequencies are only approximate; further details are given in the relevant chapters on blood groups subjects who lack the corresponding antigen, and anti-A1, -HI, -Lea and -P1, found in only some subjects who lack the corresponding antigen In some cases, the formation of anti-K has evidently been due to infection with a microorganism producing a K-like substance There is abundant evidence that some natural antibodies are spontaneous, i.e generated without the intervention of antigens: natural antibodies are present in newborn nude, germ- and antigen-free mice, sometimes in higher proportions than in normal mice (Avreamas 1992) Examples of red cell antibodies that are probably spontaneous in origin are anti-E and various others in the Rh system (see Chapter 5), antibodies to lowincidence antigens, and anti-Lua, -Dia and -Xga (see Chapter 6) and anti-HLA, found in about 1% of normal donors (see Chapter 13) In autoimmune haemolytic anaemia, it is common to find several antibodies to low-incidence antigens, for example to Wra, Swa, Cx, Mia and Vw, to which the patient has clearly not been exposed (Cleghorn 1960) Further evidence that antibodies are produced in the absence of antigenic stimulation is supplied by work referred to on p 74: antibodies such as anti-D, -E and -Kpb were grown from heavy and light chains of serum from normal subjects Naturally occurring antibodies and class of immunoglobulin Most, but by no means all, naturally occurring antibodies are cold agglutinins (IgM) and immune antibodies of the same specificity are either warm IgM or IgG antibodies 67 CHAPTER Anti-A and anti-B are always partly IgM and may be wholly IgM; in group O subjects anti-A and -B are always partly IgG and may be partly IgA Anti-A1 in A2 and A2B subjects seems always to be wholly IgM, but anti-A1 separated from group O serum may be only IgG (see Chapter 4) Examples of anti-HI and -H have been solely IgM (Adinolfi et al 1962; Chattoraj et al 1968) Although most examples of anti-Lea behave on ordinary serological testing as if they were solely IgM, an IgG component can often be demonstrated by appropriate methods Rare examples of anti-Lea and -Leb are solely IgG Naturally occurring antibodies of the MNSs system may be IgM or IgG In the past, many examples of naturally occurring anti-E were considered to be solely IgM because they gave a negative indirect antiglobulin test but, with the introduction of more sensitive methods, an IgG component is usually detectable and some examples may be solely IgG Rather surprisingly, cold-reacting anti-D, demonstrable in the AutoAnalyzer, has been found to be IgG References supporting the foregoing statements and further details will be found in Chapters – Naturally occurring antibodies to low-frequency antigens (e.g anti-Wra) are mainly IgM but a large proportion of them have an IgG component and some are solely IgG (Lubenko and Contreras 1989) Immune responses to red cell antigens Immune responses to foreign antigens are mediated by lymphocytes There are two types of immune response, humoral and cellular, but only humoral responses are relevant to blood transfusion In the humoral response, antigen is recognized by receptors on T cells, which subsequently stimulate B lymphocytes The T-cell receptors are extremely diverse and are capable of recognizing all of the foreign molecules that may be encountered A few antigens can stimulate B cells directly Stimulation of B lymphocytes results in their proliferation and, eventually, in the production of plasma cells Some of the primed B cells not differentiate and remain, in the absence of further antigenic stimulation, as memory cells On re-stimulation, memory cells mount a secondary (anamnestic) response; this response is characterized by the rapid production of antibody that is more potent and specific than that produced in the primary response 68 Antigen recognition Before a humoral response can be mounted, antigen must be brought into contact with antibody-forming cells, i.e B lymphocytes The way in which this is done and the role of HLA antigens in the process are described in Chapter 13 Primary and secondary responses to red cell antigens Primary responses to any particular antigen can, by definition, be studied only in subjects who have not previously encountered the antigen In the case of some antigens, for example A and B, it can be presumed that there has been previous exposure, as antigens identical with or similar to the human ones are found in a wide variety of organisms, and in the case of others, for example Lea, P1, previous exposure to the antigen is possible Although it is difficult or impossible to study primary responses to these antigens, such responses can be studied in many other systems, for example Rh, K, Fy and Jk, in which naturally occurring antibodies are rare As Rh D is the most immunogenic of the latter antigens, it has been used in most of the systematic work that has been done As described in Chapter 5, after a first injection of ml of D-positive red cells to D-negative subjects who have not previously been exposed to the antigen and who have no trace of anti-D in their serum, antibody can first be detected in some subjects after about weeks; in these subjects, the concentration of antibody increases slowly and after –10 weeks reaches peak values not exceeding about 4µg/ml (Samson and Mollison 1975; Contreras and Mollison 1981) In other subjects, antibody can first be detected only after two injections, given at an interval of months or more; less commonly, antibody is first detected only after three or more injections There is evidence that in all subjects in whom antibody is ultimately detected, primary immunization is induced by the first injection of red cells because in such subjects red cells injected on the second occasion invariably have a shortened survival (see Chapter 10) There are some subjects in whom no antibody is formed even after many injections given over a period of 2–3 years, and these subjects are classified as non-responders In these subjects, D-positive red cells have a strictly normal survival even after many injections of D-positive red cells have been given IMMUNOLOGY OF RED CELLS The production of anti-D after a first injection of D-positive red cells cannot be hastened by injecting a much larger amount of red cells, although the number of responders increases, as does the amount of antibody produced in the primary response (see Chapter 5) It is known that in animals, after a first injection of antigen has been given, there is a certain period during which the animal will not respond well to a second injection For example, in horses injected with tetanus toxoid, the period was at least months, although less than a year (Barr and Glenny 1945) In humans, the number of D-negative subjects forming anti-D within months did not appear to be greater when injections were given every weeks than when only a single injection was given (Archer et al 1969) In subjects already immunized to Rh D, following a (further) transfusion of D-positive red cells, the concentration of anti-D in the serum starts to increase about days after transfusion and then rises logarithmically to reach a peak value, which may be as high as about 1000µg/ml (i.e 10% of the total IgG), some 10–20 days later (see Chapters and 11) The response to a first injection of group A or B red cells in a group O subject is similar in that antibody concentration rises rapidly and reaches a peak within about 12 days (see Figs 4.1 and 4.2), indicating that these responses should be regarded as secondary The differences between primary and secondary responses to the D red cell antigen in humans are very similar to those between primary and secondary responses to bacterial toxins in animals (Burnet and Fenner 1949) There have been few systematic studies of immune responses to alloantigens other than D; the production of serologically detectable anti-K, -E and -C within weeks of a first transfusion has been reported in children with burns (Bacon et al 1991) The production of anti-Jka and anti-P1 within weeks of a first transfusion has been reported in a 7-year-old child (Cox et al 1992) Class of immunoglobulin produced in red cell alloimmunization In the primary response it is usual for IgM antibody to be formed initially and for production then to be switched to IgG antibody, but it has proved difficult to confirm that this generalization applies to the production of red cell alloantibodies in humans For example, in investigating responses to the Rh D antigen, using manual tests, anti-D can often first be detected by the agglutination of enzyme-treated red cells at a time when the indirect antiglobulin test is negative As it is known that both IgM and IgG anti-D in low concentrations will agglutinate enzyme-treated cells, and as all serological reactivity is usually lost if attempts are made to fractionate serum containing such antibodies, it is usually impossible to decide the Ig class of the antibody present Although it is not known with certainty whether IgM antibody is the first to be made in subjects responding to the Rh D antigen, it is quite clear that in the majority of subjects IgG antibody soon predominates and is often the only type that can be identified at any time In a minority of subjects IgM antibody is also produced in substantial amounts In hyperimmunized donors anti-D is also quite often partly IgA (see Chapter 5) Responses to several other red cell antigens, for example K, Fya and Jka, appear to be similar to responses to D; that is to say, most antibodies are predominantly IgG, although in some subjects a mixture of IgM and IgG antibodies is found Lutheran antibodies may sometimes be partly IgA (see Chapter 6) Blood group systems in which naturally occurring antibodies are found demand separate consideration In the ABO system perhaps all subjects should be regarded as immunized Moreover, ABO-incompatible pregnancies and injections of various animal products cause both quantitative and qualitative changes in anti-A and anti-B (see Chapter 4) Perhaps the most interesting fact about the production of ABO antibodies is that immune anti-A and anti-B are predominantly IgM in A or B subjects but may be largely IgG (and partly IgA) in group O subjects Alterations in binding constant IgG antibody formed late in the immune response tends to have a higher binding constant than antibody formed early in the response; for example, an increase in the binding constant of anti-D during the secondary Rh D immunization was demonstrated by Holburn et al (1970) Increased heterogeneity of antibody in hyperimmunized subjects There is evidence that as immunization progresses the antibody tends to be more diverse with regard to Ig 69 CHAPTER class and subclass Data with regard to anti-D are given in Chapter Persistence of IgM and IgG antibodies IgM antibodies tend to decline rapidly in concentration after the last stimulus and usually become undetectable after 1–2 years Some IgG antibodies (e.g antiRh D) decline far more slowly and may be readily detectable 30 years after the last stimulus (see Chapter 5); others (e.g anti-Jka) may become undetectable a few months after the last stimulus In a follow-up with a median period of 10 months, of 160 patients in whom 209 antibodies had been detected, some of the findings were as follows: of 39 antibodies belonging to the Lewis, MN or P1 systems (presumably IgM), 28 became undetectable, whereas of 170 belonging mainly to the Rh, K, Fy and Jk systems, which were presumably IgG, only 49 became undetectable Antibodies of the Kidd system became undetectable more frequently than those of the Rh system, in 11 out of 21 compared with 27 out of 98, although this difference may have been partly due to the relative potency of the antibodies concerned; of antibodies (of all specificities) with initially weak reactions, 41 out of 84 became undetectable whereas of those with initially very strong reactions, only out of 18 became undetectable (Ramsey and Larson 1988) In a retrospective study, 5–10 years after first being identified, 14 out of 36 alloantibodies could no longer be detected on at least one occasion Of a further 22 antibodies looked for after more than 10 years, only 10 could still be detected (Ramsey and Smietana 1994) Relation between immunoglobulin class of antibody and serological behaviour IgM antibodies Most IgM antibodies will agglutinate red cells suspended in saline Agglutination by some IgM antibodies, for example anti-A (Polley et al 1963) is not enhanced in a medium of serum whereas that of some other antibodies is Using purified IgM anti-D, it was found that the agglutinin titre was greater by four or five doubling dilutions when using serum rather than saline as a diluent; the effect was observed with serum diluted up to in 32 (Holburn et al 1971a) The enhancing effect of serum is, therefore, important only when comparing sera with titres of more than 32 or when determining the titre of purified antibodies 70 The titre of IgM antibodies is about four times higher with enzyme-treated red cells than with untreated cells (Aho and Christian 1966; Holburn et al 1971a) Evidently, very weak IgM antibodies may be detectable only with enzyme-treated red cells Naturally occurring IgM antibodies of the ABO, Lewis and P systems agglutinate red cells more strongly at 0°C than at higher temperatures For example, the titre of anti-A and anti-B is about eight times higher at 0°C than at 37°C (Kettel, cited by Wiener 1943, p 19) Although anti-A and -B almost invariably agglutinate appropriate red cells at 37°C, other antibodies of the aforenamed systems, for example anti-A1, -HI, -Lea -Leb and -P1, not usually agglutinate red cells above 20 –25°C; occasional examples of these antibodies will agglutinate up to about 30°C and even give trace reactions at 37°C; examples with such a wide thermal range as this will invariably bind complement The behaviour of anti-Lea is slightly different in that although it will usually not agglutinate cells above a temperature of about 20 –25°C, it will usually bind to red cells and fix complement at 37°C and thus give a strongly positive indirect antiglobulin test Anti-D agglutinins produced in an immune response are ‘warm’, i.e they are as active at 37°C as at lower temperatures (Levine et al 1941) A few IgM antibodies are ‘incomplete’, for example occasional examples of anti-Jka (Adinolfi et al 1962; Polley 1964) IgG antibodies In most blood group systems IgG antibodies will not agglutinate untreated red cells suspended in saline; such non-agglutinating antibodies used to be described as ‘incomplete’ The term ‘incomplete’ was introduced by Pappenheimer (1940) for horse antibodies against ovalbumin that produced no visible precipitation but inhibited the reaction of precipitating antisera The term was subsequently introduced into blood group serology by Race (1944) to describe the behaviour of Rh D antibodies that failed to agglutinate D-positive red cells suspended in saline but blocked the subsequent agglutination of the red cells by an agglutinating anti-D serum; such antibodies were described as ‘blocking’ by Wiener (1944) Potent examples of IgG anti-D either in undiluted serum, or in some cases in serum diluted 1:2 in saline, will agglutinate saline-suspended red cells (Hopkins 1969, 1970) In fact, it has subsequently become clear that really potent IgG anti-D, even at a dilution of in 10 or more, will agglutinate saline-suspended cells IMMUNOLOGY OF RED CELLS (unpublished observations, M Contreras) Potent IgG anti-K and anti-M may also agglutinate salinesuspended red cells IgA antibodies IgA fractions containing anti-A and anti-B will agglutinate red cells suspended in saline; the titre is increased about four-fold by using the indirect antiglobulin test with an anti-IgA serum (Adinolfi et al 1966) Sera containing IgA anti-D as well as IgG antiD not as a rule agglutinate red cells suspended in saline although they sensitize red cells to agglutination by anti-IgA A serum containing potent IgA anti-D, such as a purified IgA fraction from the same serum, which did agglutinate saline-suspended red cells, is mentioned in Chapter In tests on five murine IgA monoclonals, three anti-A and two anti-A,B, agglutinating activity was associated only with tetramers or higher polymers (Guest et al 1992) Individual differences in response Individuals vary widely in their response to different antigens The recognition of nearly all antigens depends on their presentation, bound to HLA class II molecules by antigen-presenting cells, i.e dendritic cells Evidently then, specific immune responsiveness is influenced by the products of the HLA-DR alleles (see review by Benacerraf 1981; see also Chapter 13) Control of antibody responses is also influenced by genes outside the HLA system and segregating independently from it, which control the quantitative production of antibody tested for alloantibodies In another series it was found that 44% of alloantibodies could not be detected before autoabsorption (Wallhermfechtel et al 1984) There has been one report indicating that red cell alloimmunization is rare in WAIHA (Salama et al 1992) but the patients’ sera were not absorbed with autologous red cells before being tested and alloantibodies may have been overlooked Subjects with hypogammaglobulinaemia have a greatly diminished capacity to form alloantibodies As described in Chapter 4, their serum may lack anti-A and anti-B In one case a patient of group A whose serum lacked anti-B was given a series of injections of B substance of animal origin but failed to form anti-B (Barandun et al 1956) Among patients with various diseases, receiving regular transfusion over a period of a year or so, only those with chronic lymphocytic leukaemia failed to produce any new red cell alloantibodies; in contrast, patients with acute myeloid leukaemia on intensive chemotherapy produced alloantibodies as frequently as those with aplastic anaemia or gastrointestinal bleeding (Blumberg et al 1983) In another study of patients receiving regular transfusions, the frequency of formation of red cell alloantibodies was significantly lower in patients with chronic lymphocytic leukaemia than in those with other diagnoses (Blumberg et al 1984) Patients on haemodialysis may have a reduced tendency to form red cell alloantibodies; of 405 patients who had received a total of almost 7000 red cell transfusions, only seven developed alloantibodies attributable to the transfusion (Habibi and Lecolier 1983) Alterations in antibody response in disease Subjects with autoimmune disease appear to have an increased risk of forming red cell alloantibodies One early example was a patient with systemic lupus erythematosus, who formed five different immune alloantibodies (Race and Sanger 1950, p 240) There have been several reports of a high incidence of alloantibodies in patients with the warm-antibody type of autoimmune haemolytic anaemia (WAIHA) who have been transfused In three series, the frequency was 32–38% and was as high as 75% in patients who had received more than five transfusions (Branch and Petz 1982; James et al 1988; reviewed by Garratty and Petz 1994) In these three series, the patient’s serum was absorbed with autologous red cells before being Formation of immune red cell alloantibodies in infants The immune response in newborn infants appears to be similar to that of newborn mice; the response is typically limited to the production of low-titre IgM antibodies, with reduced switching to IgG production and the repertoire of available antibody specificities is highly restricted In the mouse, the repertoire is 100 times smaller in the newborn than in the adult In newborn human infants, class II HLA molecules are weakly expressed (Riley 1992) although the response to foreign antigens is normal (Roncarolo et al 1994) It is uncommon for red cell alloantibodies other than anti-A and -B to be produced in the first few months of 71 CHAPTER life Some examples are: (1) anti-c in a 7-week-old child of phenotype CCDee, who had been transfused during surgery weeks previously (S Kevy, unpublished observations, reported by Konugres 1978); (2) anti-Lub in a 2-month-old infant who had been transfused month previously (unpublished observations, M Contreras); (3) IgG anti-E in an infant aged 11 weeks who had received 31 transfusions in the previous weeks, all from donors whose plasma had been screened for alloantibodies (DePalma et al 1991); and (4) anti-K in a premature infant born at 30 weeks and given 28 red cell transfusions in the first months, anti-K was found for the first time 18 days after the last transfusion; the antibody was still present year later (Maniatis et al 1993) In two series of newborn infants transfused with blood from an average of nine donors during the first few months of life no unexpected red cell antibodies were detected The first series consisted of 53 premature infants, about one-half of whom were tested at least months after birth (Floss et al 1986) and the second of 90 full-term infants tested not less than weeks after their last transfusion (Ludvigsen et al 1987) In contrast, in a series of adults, primary immunization to red cell antigens was observed in some 6% of subjects (see below) Role of the spleen In splenectomized subjects, the response to sheep red blood cells (SRBC) is much lower than in control subjects (Rowley 1950; McFadzean and Tsang 1956) However, the spleen is not essential in the formation of antibodies against red cell antigens For example, the patient described by Collins and colleagues (1950), who formed so many blood group antibodies, had had her spleen removed before receiving the series of blood transfusions that immunized her Moreover, in subjects with sickle cell disease, whose spleens are usually infarcted and functionless, alloimmunization is no less frequent than in other patients (see later in text) Association between weakening of red cell antigens and the appearance of allo- or autoantibodies in the serum There are many examples of this phenomenon In pregnant women, the agglutinability of red cells with Lewis antibodies tends to diminish (see 72 Chapter 4), a change that appears to be correlated with an increase in the frequency with which Lewis antibodies are detected in the serum Transient weakening of Rh D antigens has been observed in an infant with autoimmune haemolytic anaemia (AIHA) (see Chapter 5) Temporary weakening of LW may be associated with the appearance of anti-LW in the serum (see Chapter 5) The appearance of Kell antibodies in the serum may be associated with weakening of Kell antigens; in one case there was a second episode in which a Lutheran antibody appeared concomitantly with weakening of Lu antigens (see Chapter 6) In a patient whose red cell phenotype changed from Jk(a+ b –) to Jk(a– b –), loss of Jka was associated with the appearance in the serum of anti-Jk3 (see Chapter 6) In a patient in whose serum anti-Glycophorin C appeared, Glycophorin C was temporarily absent from the red cell membrane; when Glycophorin C reappeared, the antibody disappeared; the cells now reacted with serum taken earlier (Daniels et al 1988) Temporary depression of AnWj has been associated with the presence of auto-anti-AnWj (see Chapter 6) Monoclonal antibodies Each kind of antibody molecule is made by a line of plasma cells derived from a single lymphocyte The progeny of a single cell is called a clone and a monoclonal antibody population consists of the identical molecules produced by such a clone When an antigen is injected into an animal, numerous different lymphocytes, each producing antibodies with the capacity to react to some extent with the antigen, are stimulated Polyclonal antibodies consist of such collections of molecules In 1975, Köhler and Milstein described a method of obtaining monoclonal antibodies, which depended on fusing mouse lymphocytes with mouse myeloma cells The lymphocytes were obtained from the spleen of mice immunized with a particular antigen Unfused splenic lymphocytes die in laboratory culture within 48 h and unfused myeloma cells in the culture die because the composition of growth medium is selected so that it does not support DNA synthesis in these cells As a result, the fused ‘hybridoma’ cells flourish and cell lines secreting specific antibody that grow indefinitely in culture can be obtained by cloning IMMUNOLOGY OF RED CELLS Cloning is achieved by culturing the hybrid cells at low cell concentrations in microplates, recovering cells from the well containing the antibody of interest, and then repeating the process two or three times to obtain a stable clone of cells When stable clones secreting antibody of interest at high concentrations (50 –100 µg/ml) are obtained it is possible to culture the cells in large fermenters to obtain grams of antibody for diagnostic or therapeutic use The ability to make monoclonal antibodies has proved to be of vast importance, on both a theoretical and a practical level fuse human lymphocytes with murine myelomas to form heteromyelomas (see review by Thompson and Hughes-Jones 1990) Examples of monoclonal anti-D have been used in analysing the different D epitopes (see Chapter 5) and in the experimental clearance of D-positive red cells from the circulation of volunteers (see Chapter 10); they are used routinely in blood grouping (see Chapter 8) Other human monoclonal antibodies available as diagnostic reagents include anti-C, -c, -E, -e, -G, -K, -Jka, -Jkb, -H, -Lea, -Leb, -IgG and –C3d Some disadvantages of monoclonal antibodies Mouse monoclonal antibodies Some monoclonal antibodies produced from lymphocytes from mice injected with human red cells have specificities corresponding to those of human alloantibodies, for example anti-A, -B, -A,B, -H Type 1, -H Type 2, -P, -P1, -Pk, -I, -Y (as in Ley), -Lea, -Leb, -M, -N, -T, -Tn, -k, -Lub (First and Second International Workshop on Monoclonal Antibodies 1987, 1990) and -Ge3 (Loirat et al 1992) Others react with structures carrying the antigenic determinants rather than the antigens themselves For example, they may react with glycophorin A, rather than with M or N; or they may react with Rh-related glycoprotein and thus with all human red cells except those that are Rhnull; or they may react with Kell glycoprotein and thus with all except KO cells (see Chapter 6) Murine monoclonal antibodies against A and B and against complement components have largely replaced polyclonal reagents in diagnostic blood typing procedures Human monoclonal antibodies The first human monoclonal antibodies with blood group specificity were produced by transformation of human B lymphocytes with Epstein–Barr virus (EBV) The lymphoblastoid cells that result have the property of growing in tissue culture and of secreting antibody By taking lymphocytes from Rh D-immunized donors, both IgG (Koskimies 1980) and IgM (Boylston et al 1980) anti-D have been obtained EBV-transformed cell lines are unstable, often ceasing to produce antibody after a few months (Melamed et al 1985), although many stable EBV-transformed cell lines have been described (Goosens et al 1987; Kumpel et al 1989a) Another way of obtaining stable cell lines is to As mentioned elsewhere in this chapter, changes in pH alter the binding affinity of antibodies The effect is particularly striking with monoclonal antibodies and may have a profound effect on apparent specificity in tests in a system such as HLA, which involves many similar but non-identical antigens Monoclonal antibodies which give non-specific reactions at normal pH can be made monospecific by lowering pH to 6.0 (Mosmann et al 1980) A problem associated with some high-affinity monoclonal antibodies is crossreactivity It may seem paradoxical that an antibody apparently specific for a single antigenic determinant should crossreact at all Nevertheless, single antibody molecules are not specific for one particular epitope The combining site on the end of the Fab arm is capable of binding closely with a number of different epitopic configurations (Talmage 1959) The higher the affinity of an antibody, the more it tends to crossreact (Steiner and Eisen 1967) Presumably, the more strongly an antibody combines with any particular determinant, the more certain it is to bind to some extent to related antigenic determinants By no means all monoclonal antibodies have a high affinity On the contrary, the affinities of monoclonal antibodies produced by different clones are similar to those of polyclonal antibodies, although all antibodies of a single clone have the same affinity, whereas the antibodies in a polyclonal population have a range of affinities Monoclonal antibodies even of average affinity are more likely than polyclonal antibodies to crossreact This is because all the molecules of a monoclonal antibody population have the same paratope and will therefore all recognize the same crossreacting antigen In contrast, in a population of polyclonal antibodies, the paratopes will differ and 73 CHAPTER only a proportion of the molecules will recognize any particular crossreacting antigen Crossreactivity explains the demonstration that certain examples of monoclonal anti-D react with the cytoplasmic component vimentin (Thorpe 1989, 1990), giving rise to the false conclusion that D is present on tissue cells On the other hand, the reaction of certain monoclonal anti-A with B cells and of certain monoclonal anti-B with A cells is due to the potency of the antibodies concerned which enables them to detect the small amounts of B and A, respectively produced by A and B subjects (see Chapter 4) A different problem is provided by monoclonal antibodies with ‘pseudo-specificity’, for example: (1) an anti-glycophorin B, which, in agglutination tests, behaved as anti-S but in other tests reacted with both s+ and S+ red cells (Green et al 1990a) and (2) an anti-J chain that reacted more strongly with Gm(a+) than with Gm(a–) molecules but could be shown to be reacting with the same determinant on the γ chain in both cases (De Lange 1988) Copying variable regions and constructing new antibodies For the construction of new antibodies, the genes encoding the V regions can be amplified using the PCR and can then be cloned into expression vectors (e.g bacteriophage) Complete antibody V domains with specificity identical to the parent antibody can then be displayed on the surface of the bacteriophage Completely new antibodies can be constructed (McCafferty et al 1990) Another approach has been to link VH and VL gene repertoires together at random to encode a repertoire of single-chain FV (scFV) antibody fragments, which are then displayed on the surface of bacteriophage Phage are selected by binding to the surface of mobilized antigen (red cells) and then used to infect E coli Antibodies produced include anti-B, -D, -HI, -E and -Kpb (Marks et al 1993) and -HPA-1a (Griffin and Ouwehand 1995) Frequency of immune red cell alloantibodies By definition, an immune red cell alloantibody is one that develops in a subject who has been exposed to a red cell alloantigen Evidently then, the frequency of immune red cell antibodies will be zero in subjects who have never been transfused or been pregnant, and will 74 be greatest in subjects who have received many transfusions For two reasons, pregnancies constitute a smaller stimulus than transfusions: first, the number of foreign antigens is limited to those possessed by the father of the fetus (although, of course, some women have infants by more than one man), and, second, in many pregnancies the amount of red cells transferred from fetus to mother is too small to stimulate a primary response The frequency of alloantibodies is expected to be relatively low in blood donors compared with patients in hospital requiring blood transfusion; blood donors have a lower average age and are far less likely to have received blood transfusions in the past The frequency with which particular antibodies are found depends on the sensitivity of testing (usually relatively low in screening healthy donors) and on the ethnic group being tested (e.g anti-D is rare in Chinese people) D is the most immunogenic red cell antigen (excepting A and B), but the frequency of immunization to D has been reduced to a low level, first, by using only Dnegative donors for transfusion to D-negative subjects and, second, by administering anti-D Ig to D-negative women who would otherwise become immunized as a result of a pregnancy with a D-positive infant As a result of these measures, the frequency with which anti-D is encountered has fallen greatly in the last few decades In contrast with the diminishing frequency of antiD, that of anti-K, -Fya, etc has increased, evidently due to the increased number of multitransfused patients and the absence of measures for preventing immunization to antigens other than D Frequency of immune anti-D (including anti-DC and anti-DE) In healthy blood donors In more than 200 000 donors screened between 1971 and 1975 in Seattle, the frequency was 0.22% (Giblett 1977) In 36 000 donors screened in London in 1988, the frequency (0.25%) was almost identical although in 35 000 screened in 1990 the frequency was 0.16% (R Knight, personal communication) In pregnant women Before the introduction of immunoprophylaxis, anti-D was found in approximately in 170 pregnant white women, both in England (Walker 1958) and in North America (Walker 1984) Following the introduction of immunoprophylaxis IMMUNOLOGY OF RED CELLS with anti-D in the late 1960s, the frequency with which anti-D is found has fallen progressively and was, for example, in 963 in 1988 in one North American survey (RH Walker, personal communication 1991) and in 1600 in another (Heddle et al 1993) The frequency in one English region for 1988 had fallen to a lesser extent, being in 497 (GJ Dovey, personal communication), although a slightly greater fall, namely to one in 584, was found in another English region for 1993 –95 (A Rankin, personal communication) Figures for the changing incidence of HDN are given in Chapter 12 clinically significant antibodies other than anti-D were found in 100 out of 17 568 women, but in 58 instances the antibody was present in the first trimester and it was concluded that the frequency of antibodies stimulated by the current pregnancy was 42 out 17 568 or approximately 0.24% (Heddle et al 1993) In pre-transfusion tests on potential recipients Frequencies were: in 1974 –75, 1.12% (Giblett 1977); in 1970 –75, 0.39%; in 1976 –81, 0.45% and in 1982–87, 0.6% (Walker et al 1989) In recipients following solid organ transplants The frequency with which anti-D develops in D-negative recipients of D-positive grafts and the apparent influence of cytotoxic drugs is mentioned in Chapter In random recipients following transfusion The figures in the preceding paragraph are for patients, only some of whom had been transfused or been pregnant previously In a prospective study of 452 patients, all of whom had been transfused in relation to elective surgery, pre-transfusion samples and 10 serial, 2-weekly, post-transfusion samples were taken Additional red cell alloantibodies developed in 38 out of 452 (8.2%) patients between and 18 weeks after transfusion, but 10 of these had been transfused before and one had been pregnant, leaving an apparent frequency of primary immunization of about 27 out of 441 (6.1%); 13 out of the 27 antibodies were anti-E and if these are excluded (because their immune nature is in some doubt) the frequency falls to 14 out of 428 (3.3%) Of all the newly found antibodies, 50% were initially detectable only by a two-stage papain technique or by the manual polybrene test, and not by the indirect antiglobulin test (IAT) (Redman et al 1996) Frequency of immune red cell antibodies other than anti-D Relative frequencies of antibodies other than anti-D In healthy blood donors In the two series referred to above, the frequencies were 0.10% in Seattle in 1975 (Giblett 1977) and in London 0.19% in 1988 and 0.18% in 1990 (unpublished observations, M Contreras) In the London series, about 30% of the antibodies were anti-E and 20% were anti-K Combined figures from 20 different blood grouping laboratories were reported by Grove-Rasmussen (1964); these figures and those from a much smaller series reported by Tovey (1974) are shown in Table 3.6 No information was given as to the relative numbers of parous women and transfusion recipients in the two series As will be seen, Rh antibodies (other than anti-D) constituted about 54% of the total and anti-K and -Fya about 40%, leaving only 5% for all other specificities In the series of Walker and colleagues (1989), if the figures for the three 5-year periods (in each of which approximately 100 000 patients were screened) are pooled, the absolute frequencies for antibodies of various specificities were as follows: Rh In pre-transfusion tests on potential recipients Although there appears to have been a fall between the mid-1950s and mid-1970s in the frequency with which anti-D (and -DC) was found in transfusion recipients, there has been no obvious fall since then Some figures are as follows: in 1956 –57, 0.77%; in 1974 –75, 0.52%, based on testing 60 000 patients in Seattle (Giblett 1977); in 1970–75, 0.55%, in 1976–81, 0.29% and in 1982–87, 0.27%, based on screening about 100 000 patients in each 5-year period in Michigan (Walker et al 1989) In a smaller series (more than 12 000 patients) tested in England in 1990, 0.56% ( J Sangster, personal communication) In pregnant women In approximately 175 000 women (about 85% of whom were D positive), antibodies other than anti-D were found in 0.14%; rather more than one-half of these were within the Rh system and, of these, one-half were anti-E; the next commonest antibody, found in 0.025% of the women, was anti-K (Kornstad 1983) In a more recent survey, 75 .. .Mollison’s Blood Transfusion in Clinical Medicine Mollison’s Blood Transfusion in Clinical Medicine 11 TH EDITION Harvey G Klein MD President of the American Association of Blood Banks;... ISBN -1 3 : 97 8-0 -6 3 2-0 645 4-0 ISBN -1 0 : 0-6 3 2-0 645 4-4 Blood Transfusion Blood groups I Mollison, P L (Patrick Loudon) II Anstee, David J III Mollison, P L (Patrick Loudon) Blood transfusion in clinical. .. dextrose in water Fluid compartment Fluid Table 2 .1 Characteristics of selected resuscitation fluid 11 0 15 4 15 4 15 4 15 4 10 0 13 0? ?16 0 13 0? ?16 0 10 9 98 98 11 97 0 10 0 Cl– (meq/l) 0 3 0 Mg2+ (meq/l) 0 0 0