ABSTRACT

Based on the proceedings of the International Convocation on Immunology held recently at the State University of New York at Buffalo, this up-to-date resource provides a state-of-the-art examination of blood transfusion practice and its future possibilities.
Explains the immunological effects of blood transfusion as well as its immunological and

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THE ERNEST WITEBSKY MEMORIAL LECTURE

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, 22,

658-662 (1992). 17. C. F. Hogman, J. Gong, A. Hambraeus, C. S. Johansson and L. Eriksson, Transfusion, 654-657 (1992). 18. T. L. Cover and R. C. Aber, N. Engl. J. Med., 221, 16-24 (1989). 19. A. P. Gibb, K. M. Martin, G. A. Davidson, B. Walker and W. G. Murphy, Lancet, 240, 1222-1223 (1992).

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PART II TESTING FOR INFECTIOUS AGENTS

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PART ΠΙ: ALLOTYPES

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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 [1]. The antibodies to some antigens have caused haemolytic disease of the newborn and/or 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 antigens or to assign it to a system. 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 of a technique, 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 of 4 high incidence antigens Kna, McCa, Sla 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 al [6] and Rao et al [7] independently showed that these antigens were carried on the CR1 (complement receptor 1, CD35) protein. The related antigen Csa was not located on CR1, so Csa and Csb were left in the Cost collection [3].

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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-molecular complex 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 (E11) 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-Sla 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 1:1 ratio indicating a negative result (Table I). 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 CR1 but had a low copy number of CR1 molecules per cell [9]. 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 significantly higher than for antigen negative cells for Kna, McCa and Yka [8]. MAIEA has confirmed that Kna, McCa, Sla and Yka but not Csa are associated with the CR1 molecule in the red cell membrane and can detect weak expression of CR1 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.

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Reactions of human antibodies with CR1 immobilised by mouse monoclonal antibody E11 Red cell phenotype Murine MAb Human anti- Absorbance Ratio Kn(a+) ] Kn(a-) E11 Kna 0.755 0.195 4:1 McC(a+) 0.538 McC(a-) E11 McC 0.136 4:1 Yk(a+) 0.315 Yk(a-) E11 Yka 0.120 26:1 Sl(a+) 0.342 Sl(a-) E11 Sla 0.074 4.6:1 Cs(a+) 0.139 Cs(a-) E11 Cs 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 antigens on that 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 Kell protein have been used in MAIEA to study the relationships of the Kell system antigens [10]. The decay accelerating factor DAF, CD55, is detected by several monoclonal antibodies. Three antibodies BRIC 230, BRIC 110 and BRIC 216 were known from competitive binding assays to bind to different short consensus repeats (SCR) [11]. So three of the four SCRs of the DAF molecule were positively identified (Table II). Strong positive reactions were observed with all three BRIC antibodies and anti-Cr3, anti-WES8, and anti-WESb showing that MAIEA is a useful techique for studying this system [12]. The results showed that Cr8, WESa, and WESb are not on the first three SCRs and must

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therefore be on the fourth SCR or on the serine/theonine rich region. By sequencing genomic DNA from Cr(a-) people, Telen and colleagues showed that a mutation in the fourth SCR was responsible for Cr3 [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 some inhibition with one of the BRIC antibodies [12]. MAIEA provided biochemical evidence that Esa is indeed a Cromer system antigen [12]. Esa was thought to be a Cromer related antigen because of the failure of 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 and,therefore, immunoblotting experiments could not be done. Strong positive results with BRIC 216 and 110 but a negative result with BRIC 230 suggested that Esa is located on DAF, possibly on the first SCR. Similarly, a negative result with BRIC 230 and Tca suggests that it too is on the first SCR (Table II) [12]. The results of the MAIEA tests for Cromer antigens are summarised in Table II. They agree with those known from DNA studies, Dra on SCR III [15,16,17] and Cr3 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 MAIEA to 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 appear to 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 introduction of Rh immunoprophylaxis, anti-D was the most frequent cause of haemolytic disease of the newborn and neonatal death [1]. Many Rh antigens are good immunogens. Since its initial recognition in the nineteen-forties, the Rh system has become very complex. There are 48 numbered antigens, that is serologically defined determinants, the numbers have reached 50 because two numbers have been declared obsolete [2,3,18,19]. Some antigens are polymorphic and others are of high or low incidence.

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oPnosDsA ib F le m lo o c le a c ti uolnesdoefdCurcoemdefrrosm ys t M em AI EaAntt ig e e st nss Understanding of the biochemical structures and molecular basis of Rh antigens is emerging rapidly. Absence of Rh antigens, as occurs in the RhnuN phenotype, compromises the integrity of red cells and cells from people with an RhnuN 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, Paris 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 there is evidence that the C and E 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 carried the Rh antigens. Two bands are co-precipitated by anti-D: one with an apparent Mr 30,000 called D30 polypeptide by the Bristol group and the other a diffuse band of 50-100 kD called the D50 polypeptide. Similar bands were observed when immune precipitation were done using anti-c, -E or R6A [see 20-22]. The D30 polypeptide was an unusual membrane protein because it was not glycosylated, the gene producing this protein and the other Rh protein were subsequently cloned. Assignment of the genes to chromosome 1p34-p36 confirmed that they are responsible for the Rh polymorphism [see 22]. The role of the Rh glycoproteins, the diffuse band of 50-1 OOkD, is not yet understood: the gene encoding the Rh glycoprotein when cloned was assigned to chromosome 6p21-qter [29].

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Both types of RhnuN cells (amorph and regulator types) lacked the Rh proteins and other proteins, and showed some functional abnormalities. No Rh glycoprotein is immune precipitated by anti-D but it is present in membranes from some RhnuNs as shown by immunoblotting studies with the monoclonal antibody MB-2D10 [30]. The LW protein is missing and some other antibodies only react weakly: anti-U, Duclos, anti-FY5, BRIC 125 (anti-CD47) and the monoclonal antibody 1D8. That so many determinants encoded by genetically independent loci are not fully expressed in RhnuN cells has led to the idea of a protein complex or cluster which involves the Rh proteins, Rh glycoproteins and other proteins [31,32,33]. Since D and CE proteins are integral proteins with about 12 transmembrane domains, it is 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 alterations in 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 34]. 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 III shows some of the Blood Group Unit’s results of testing samples with rare Rh phenotype against polyclonal anti-C. ComC represents commercial anti-C reagents, all others are single donor antibodies. The first three reagents do not contain any anti-D, the next three contain incomplete anti-D so are not suitable for tests with enzyme treated cells. 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 antibodies to low incidence Rh antigens (Table III). Low incidence antigen JAL (RH48) JAL+ cells have a very weak C antigen, it is most easily detected with commercial reagents (Table III). The antibody in Mrs S Allen’s serum was studied for many years in several laboratories interested in low incidence antigens. There were some hints that it might be an Rh antigen, although Mrs Allen’s husband had been an unremarkable cDe. 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

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baby, was found in Switzerland. This antibody was used in Bern to screen donors. Four new JAL+ propositi were found in 90,000 donors, all four were French speaking. The frequency of the antigen was calculated to be 0.004% overall but 0.06% in French-speaking Swiss [36]. The families of the antibody maker and the 4 JAL+ donors were studied. The results proved that JAL was part of the Rh system or encoded by a very closely linked locus [36]. 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]. It is noteworthy that the anti-JAL of Mrs S Allen was stimulated by pregnancy and her husband was cDe JAL+ but J Pas’s anti-JAL, also stimulated by pregnancy, was made in response to a depressed C JAL+ complex. So the expression of JAL does not depend on the C or c with which it is associated. Both these anti-JAL were responsible for haemolytic disease of the newborn [35]. Low incidence antigen FPTT (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 III). FPTT presents a much more difficult problem than JAL. Adsorption/elution tests are needed to identify the antigen. Family studies showed that FPTT is associated with at least 5 different depressed antigenic complexes (Table IV). One complex, that of propositus 1 [37], had depressed C and e antigens, the complex of propositus 2 [37] had depressed C antigen and depressed e antigen

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So FPTT is associated with two different types of D antigen and three different types of Ce antigens (Table IV). These results suggest that a similar amino acid sequence corresponding to the FPTT 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 may have occurred. Several mechanisms could be involved: mutation, recombination or gene conversion have been invoked in other blood group systems to explain rare phenotypes. The large number of Rh antigens and their quantitative and qualitative variants will not be easy to explain. Variation in the Rh genes may explain some variants but we know that Rh expression is affected by suppressors unlinked to RH, homozygosity of one unlinked suppressor causes the regulator type of Rhnu|j. Mutation in one of the genes encoding a non-Rh protein required for formation of the Rh protein complex may affect the presentation of some Rh antigens at the cell surface. Rh groups will continue to be clinically and 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 of PBDX, a candidate gene for XG [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 MIC2 gene product, has been numbered CD99 at the fifth Leucocyte Workshop and this

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designation will be used for the 12E7 antigen. CD99 was first detected by 12E7, 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 [see 42]. Independently, CD99 was identified as E2, a T-cell adhesion molecule, and as a marker antigen for Ewing’s tumours [see 40]. CD99 is expressed on many tissues including red cells. By somatic cell hybridization and biochemical studies, Goodfellow and his colleagues have shown that MIC2, the structural locus encoding the 12E7 antigen, is located on the short arm of the X chromosome and on the short arm of the Y chromosome within the pairing regions [43]. MIC2 has been cloned [44]. XG is X-borne. On red cells, CD99 expression 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 different monoclonal antibodies and variability of expression 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 Mr and in their sialic acid content [49]. Immunostaining 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 band of 28 kD which was found in mouse cell lines in addition to human cell lines, platelets, lymphocytes and red cells but it was not encoded by the MIC2 gene [47]. Immunoblotting assays have shown that Xga was associated with two diffuse bands of 22-25 kD and 26.5-29 kD [49]. These findings supported the evidence that Xga and CD99 were products of different structural loci. However, XG appears to regulate CD99 expression on red cells and Latron and colleagues found that 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 band is seen on X-ray film after 2 minutes in membranes

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from CD99 high expressors but membranes from CD99 low expressors required exposure of 5 minutes before the 32 kD band was apparent [50]. Unfortunately, these tests gave no information about the Xga protein because the position of the Xga band was masked by 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 stained by immunoblotting Xga-immunoprecipitates with 12E7. The luciferin-enhanced luminescent proceedure to detect the avidin-biotin label is very 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 understanding of this relationship. The important blood group genes have been cloned but two big 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 red cells. 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. P.L. Mollison, C.P. Engelfreit and M. Contreras, Blood Transfusion in Clinical Medicine. Blackwell Scientfic Publications, Oxford (1993). 2. M. Lewis (Chairman) et al, Vox Sang., 61_, 158-160 (1991). 3. G.L. Daniels, J.J. Moulds (chairman) et al, Vox Sang., 65, 77-80 (1993). 4. A.C. Petty, J. Immunol. Meth., 161. 91-95 (1993). 5. J. M. Moulds, in Immunobiology of Transfusion Medicine. G. Garratty ed. Marcel Dekker. Inc., New York, (1994) pp. 273-297. 6. J.M. Moulds, M.W. Nickells, J.J. Moulds, M.C. Brown and J.P. Atkinson, J. Exp. Med., 173, 1159-1163 (1991). 7. N. Rao, D.J. Ferguson, S-F. Lee and M.J. Telen, J. Immun., 146, 3502-3507 (1991). 8. A.C. Petty, (abs) Transfusion Medicine 3 Suppl 1, 84 (1993). 9. J.M. Moulds, J.J. Moulds, M. Brown and J.P. Atkinson, Vox Sang. 62, 230-235 (1992).

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A.C. Petty, G.L. Daniels and P. Tippett, Vox Sang, 66, 216-224 (1994). 11. K.E. Coyne, S.E. Hall, E.S. Thompson, M.A. Arce, T. Kinoshita, T. Fujita, D.J. Anstee, W. Rosse and D.M. Lublin, J. Immun. 149. 2906-2913 (1992). 12. A.C. Petty, G.L. Daniels, D.J. Anstee and P. Tippett, Vox Sang., 65, 309-315 (1993). 13. M.J. Telen, N. Rao, E.S. Thompson and D.M. Lublin, (abs) Transfusion, 32, suppl 47S (1992). 14. G. Daniels, Vox Sang., 56, 205-211 (1989). 15. M.J. Telen, in Blood Groups:Ch/Rq. Kn/McC/Yk, Cromer. J.M. Moulds and B. Laird-Fryer, eds. American Association of Blood Banks, Bethesda MD, (1992) pp. 45-63. 16. D.M. Lublin, E.S. Thompson, A.M. Green, C. Levene and M.J. Telen, J. Clin. Invest., 87, 1945-1952 (1991). 17. D.M. Lublin, G. Mallinson, M.E. Reid, J. Poole, E.S. Thompson, B.R. Ferdman, M.J. Telen, D.J. Anstee and M.J.A. Tanner, (abs) Transfusion, 32, suppl 47S (1992). 18. P.D. Issitt, Transf. Med. Rev., 3, 1-12 (1989). 19. C. Lomas, W. Grassman, D. Ford, J. Watt, A. Gooch, J. Jones, M. Beolet, D. Stern, M. Wallace and P. Tippett, Transfusion in press. 20. P. Agre and J-P. Cartron, Blood, 78, 551-563 (1991). 21. J-P. Cartron and P. Agre, Seminars Haemat., 30, 193-208 (1993). 22. D.J. Anstee and M.J.A. Tanner, in Baillieres’s Clinical Haematology. M.J.A. Tanner and D.J. Anstee, eds. Bailliere Tindall, London (1993) pp. 401-422. 23. N.D. Avent, K. Ridgwell, W.J. Mawby, M.J.A. Tanner, D.J. Anstee and B. Kumpel, Biochem. J., 256 1043-1046 (1988). 24. C. Bloy, D. Blanchard, W. Dahr, K. Beyreuther, C. Salmon and J-P. Cartron, Blood, 72, 661-666 (1988). 25. A.M. Saboori, B.L. Smith and P. Agre, Proc. Natl. Acad. Sci. USA, 85, 4042-4045 (1988). 26. N.D. Avent, K. Ridgwell, M.J.A. Tanner and D.J. Anstee, Biochem. J., 271.821-825 (1990). 27. B. Cherif-Zahar, C. Bloy, C. Le Van Kim, D. Blanchard, P. Bailly, P. Hermand, C. Salmon, J-P. Cartron, Y. Colin, Proc. Natl. Acad. Sci. USA, 87, 6243-6247 (1990). 28. I. Mouro, Y. Colin, B. Cherif-Zahar, J-P. Cartron and C. Le Van Kim, Nature Genet., 5, 62-65 (1993). 29. K. Ridgwell, N.K. Spurr, B. Laguda, C. MacGeoch, N.D. Avent and M.J.A. Tanner, Biochem. J., 287, 223-228 (1992). 30. G. Mallinson, D.J. Anstee, N.D. Avent, K. Ridgwell, M.J.A. Tanner, G.L. Daniels, P. Tippett and A.E.G. von dem Borne, Transfusion, 30, 222-225 (1990).

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W. Dahr, in Recent Advance in Blood Group Biohchemistrv, V. Vengelen-Tyler and W.J. Judd, eds. American Association of Blood Banks, Arlington, VA (1986) pp. 23-65. 32. J-P. Cartron, in Monoclonal antibodies against human red blood cell and related antigens. P. Rouger and C. Salmon, eds. Arnette, Paris (1987) pp. 69-97. 33. D.J. Anstee, Vox Sang., 58, 1-20 (1990). 34. P. Tippett, in Blood Group Systems: Rh. V. Vengelen-Tyler and S. Pierce, eds. American Association of Blood Banks, Arlington, VA (1987) pp. 25-53 35. C. Lomas, J. Poole, N. Salaru, M. Redman, K. Kirkley, M. Moulds, J. McCreary, G.S. Nicholson, H. Hustinx and C. Green, Vox Sang., 59, 39-43 (1990). 36. J. Poole, H. Hustinx, H. Gerber, C. Lomas, Y.W. Liew, and P. Tippett, Vox Sang., 59, 44-47 (1990). 37. M. Bizot, C. Lomas, F. Rubio and P. Tippett, Transfusion, 28, 342-345 (1988). 38. N.A. Ellis, T-Z. Ye, S. Patton, J. German, P.N. Goodfellow and P. Weller, Nature Genet., 6, 394-400 (1994). 39. C. Gelin, F. Aubrit, A. Phalipon, B. Raynal, S. Cole, M. Kaczorek and A. Bernard, EMBO J., 8, 3253-3259 (1989). 40. M.N. Dworzak, G. Fritsch, P. Buchinger, C. Fleischer, D. Printz, A. Zellner, A. Schollhammer, G. Steiner, P.F. Ambros and H. Gadner, Blood, 83, 415-425 (1994). 41. R. Levy, J. Dilley, R.l. Fox and R. Warnke, Proc. Natl. Acad. Sci. USA, 76, 6552-6556 (1979). 42. G.S. Banting, B. Pym, S.M. Darling and P.N. Goodfellow, Mol Immunol., 26, 181-188 (1989). 43. P. Goodfellow, G. Banting, D. Sheer, H.H. Ropers, A. Caine, M.A. Ferguson-Smith, S. Povey and R. Voss, Nature, 302. 346-349 (1983). 44. S.M. Darling, G.S. Banting, B. Pym, J. Wolfe and P.N. Goodfellow, Proc. Natl. Acad. Sci. USA, 83, 135-139 (1986). 45. P.N. Goodfellow and P. Tippett, Nature, 289. 404-405 (1981). 46. P. Tippett, M-A. Shaw, C.A. Green and G.L. Daniels, Ann. Hum. Genet., 50, 339-347 (1986). 47. G.S. Banting, B. Pym and P.N. Goodfellow, EMBO J., 4, 1967-1972 (1985). 48. F. Latron, D. Blanchard and J-P. Cartron, Biochem. J., 247, 757-764 (1987). 49. R. Herron and G.A. Smith, Biochem. J., 262. 369-371 (1989). 50. A.C. Petty and P. Tippett Submitted.

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THE EFFECT OF BLOOD TRANSFUSION ON IMMUNE FUNCTION Since homologous blood is never given to normal volunteers, the effect of blood transfusion on immune function in normal man is unknown. In patients who receive homologous blood, changes in immune response are evaluated in the context of the disease for which the blood is given and extrapolated to the effect of blood in the absence of disease. 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 washed/filtered homologous blood can be compared to patients who are receiving routinely prepared homologous blood. The blood is given within the context of a surgical procedure as a consequence of operative blood loss which is due to trauma and trauma itself is associated with changes in immune function. In Vitro Lymphocyte Responsiveness 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 the procedure and in proportion to the amount of blood lost. Certain anesthetic agents, notably ether and cyclopropane, are associated with more profound suppression of immune function than halothane and nitrous oxide, for example (1). Patients with malignancies have low lymphocyte responses and declines with surgery are more precipitous than for patients without malignancies. Operated patients who receive homologus blood have declines in lymphocyte responsiveness compared to untransfused patients undergoing the same procedure. Thorough well-controlled studies have also observed the opposite, causing Munster et al. to comment that continued investigation " into the effect of PHA and ConA on post-traumatic lymphocyte transformation in many laboratories has produced no conclusive and repeatable pattern." (2) Prolonged depression in in vitro lymphocyte responsiveness is noted within hours of surgery and recovers over the next several days. The inhibition is due to both intrinsic and extrinsic factors since lymphocyte responsiveness can be partially restored by testing in plasma from normal blood donors. Homologous blood transfusion adds to the depressed state of the lymphocytes, but may cause stimulation in unoperated patients. The in vivo counterpart of in vitro testing of lymphocytes is delayed cutaneous hypersensitivity to antigens. Delayed Cutaneous Hypersensitivity There exists a correlation between in vivo and in vitro lymphocyte testing and preoperative evaluation of in vivo lymphocyte function is predictive of postoperative infection and subsequent course after surgery. Anergy is associated with low serum albumin and reduced polymophonuclear neutrophil chemotaxis. Patients with gastrointestinal bleeding, recipients of homologous blood, are often anergic (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.

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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 and serum albumin, the authors reanalyzed their data with 64 pairs of patients matched for these variables with the same results. The predictive value of delayed hypersensitivity skin testing for sepsis and mortality has not been accepted by all investigators. Brown et al. (5) agree that anergic patients have significantly higher rates of sepsis and mortality than normal responders, however "careful study of the temporal relationship between skin reactions and clinical events in individual patients suggested that these differences were not of value in clinical practice. Abnormal reactions usually followed obvious complications such as sepsis or secondary hemorrhage rather than predicted them. Anergy to skin testing may be related to a circulating serum factor which appears after trauma and causes lymphocyte suppression. There is no proven association of blood transfusion with serum suppressive activity or with anergy. Infectious complications and hospital stay are both significantly related to immunosuppressive serum and anergy. Lymphocyte Subsets Lymphocytes, B cells, T cells, helper cells and suppresser cells drop significantly five days after surgery and the decline is twice as great in the transfused patients compared to the untransfused (6). Helper cell number declines in transfused patients cause the helper/suppresser ratio to decrease significantly despite a significant decline in suppresser cell number. Changes in cell numbers recover somewhat by ten days so the differences between transfused and untransfused patients are no longer statistically significant although cell numbers in transfused patients are still lower than those in untransfused patients. Lymphocyte responses to ConA and PHA decline significantly in transfused groups, remaining below preoperative levels even one year following surgery. Response to ConA and PHA and MLR's in untransfused patients are significantly higher than in transfused patients at 90 days and 45 - 60 days respectively. Significant declines in immunoglobulin G, A and M cells are noted postoperatively in both transfused and untransfused patients. Other authors have not observed consistent changes in lymphocyte subsets in relation to transfusion. Changes in the numbers of lymphocytes in the various subsets in relation to surgery with and without blood transfusions studied in patients tested before and after surgery 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 surgery is followed by significant decreases in peripheral blood lymphocyte numbers affecting all lymphocyte subsets to some degree. Declines in helper cell numbers are associated with a significant decrease in the helper/suppresser ratio. It is not clear if transfused patients exhibit greater declines in lymphocytes due to the transfusion, due to the operative trauma, or due to pre-existing anemia which caused physicians to transfuse blood.

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Natural Killer Cytotoxicity In a prospective study of colorectal cancer patients, the number of natural killer cells increased significantly in both transfused and untransfused patients who had potentially curative surgery (8). Natural killer cytotoxicity declined significantly in untransfused patients while 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. Removing leukocytes from the blood to be transfused abrogates the changes in natural killer cytotoxicity (9) These studies conflict with the findings of a prospective study of colorectal cancer (9). Patients were randomized to receive whole blood or filtered whole blood, removing 99.98% of the leukocytes and platelets. Natural killer cytotoxicity declined significantly in patients receiving unfiltered whole blood and remained significantly depressed 30 days following surgery. Natural killer cytotoxicity in untransfused patients and in patients receiving filtered blood declined significantly with surgery but fully recovered by 30 days. Since declines in natural killer cytotoxicity can be prevented in cancer patients by simply filtering blood and since natural killer cytotoxicity is of proven prognostic significance, filtered blood may improve the outcome for patients with malignancies. Immune Function Following Transfusion of Dialysis Patients The effect of transfusion on dialysis patients is both immune enhancing and immune 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. Suppressor lymphocytes begin to appear in the serum of transfusion recipients at the same time lymphocytotoxic antibodies are appearing and their appearance is probably also mediated by antibodies -antibodies which play a role in regulating immune function. Suppresser cells suppress lymphocyte responses to antigens on the cells of transfused blood and on the cells of the transplant. 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 patients who receive blood transfusions lymphocyte responses to antigens, mitogens and homologous lymphocytes decline to 50% one week following a single unit of blood in comparison to lymphocyte reactivity measured immediately before transfusion (10). Lymphocyte reactivity declines progressively with additional units of blood given and returns to pretransfusions levels if blood is withheld for six weeks. Suppresser activity is enhanced following transfusion but not at one week when lymphocyte reactivity 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 patients and remains low following transplant, although a correlation between natural killer cytotoxicity and graft survival has not been shown (12).

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These studies indicate that surgery depresses immune function because both anesthetic agents and physical trauma cause circulating levels of all lymphocyte subsets to decline after surgery with general anesthesia causing 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 vivo by loss of response to skin testing. Lymphocyte functional inhibition may be related to disproportionate declines in T cell subsets or related to the appearance of immunosuppressive serum factors which inhibit lymphocytes. Transfusion potentiates whatever mechanism is responsible for lymphocyte inhibition; surgery accompanied by transfusion is followed by more profound decreases in lymphocyte numbers and in lymphocyte functional activity than surgery without transfusion. It is difficult to extrapolate these observations to retrospective clinical studies linking transfusion to increases in risk of infection or recurrence of malignancy. The study by Jensen et al.(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 The hypothesis that transfusion causes immune suppression leading to infections is confounded by the observation that the magnitude of the injury directly correlates with the degree of immune suppression and the necessity for transfusion. Potential confounders must be considered in any study of infections following surgery: confounders in one clinical situation are not significant or non-existent in another. Each field of surgery 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 tissue destruction and bacterial contamination can be calculated statistically using stepwise logistic regression (13). This type of analysis is commonly used in medical studies, ignoring the basic precept that the independent variables must be truly independent. The independent variables are not genuinely independent: the magnitude of the procedure, the duration of surgery, the blood loss and the tissue damage are all related to one another and all 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 has been applied to 23 populations of patients undergoing procedures ranging from bone marrow harvesting to coronary artery bypass graft. In 22 studies transfusion was a statistically significant risk factor for infection and in 17 of the 23 it was the most significant determinant of infectious complications 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 blood transfusion, indicating that 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 predictor of infection after controlling for variables reflecting tissue destruction and contamination.

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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 factors related to the procedure. To control for these factors one must compare patients transfused with red cells from different sources or prepared in a manner which minimize infection risk. Patients transfused with homologous blood have infection rates several fold higher than recipients of equal values of autologous blood undergoing the same operative procedure (20-23). Homologous blood recipients have significantly longer hospital stays attributed to treating infections. The cost of a blood transfusion exceeds the cost of collection, storage and administration because of transfusion's association with length of stay. In this era of 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 to immune suppression and infection risk. A prospective randomized trial comparing the infection rates among colorectal cancer patients receiving filtered and unfiltered blood has been conducted (9). There were 17 infectious complications among the 56 recipients of whole blood and one infectious complication among the 48 recipients of filtered blood. Infections were prevented by the seemingly simplistic addition of a $25/filter to every bag of blood transfused. These clinical studies are very convincing: homologous blood transfusion is associated with increased risk of infection in every clinical situation examined. In multivariate analyses transfusion was a significant predictor of infection after consideration of other variables measured and in the majority of those studies transfusion was the single most significant factor. Patients receiving homologous blood exhibited an incidence of infectious complications that was approximately four times higher than patients receiving autologous blood. The association of transfusion with infection is found among patients undergoing surgery for cardiac, orthopedic and gastrointestinal disorders and for trauma as well as among unoperated patients transfused for bums and gastrointestinal bleeding. The observation that nosocomial infections are increased in these studies argues strongly that the association of transfusion with infection is not simply a reflection of transfusion as a marker of tissue destruction and contamination. Infections that develop in transfused patients away from the site of trauma or in the absence of trauma, cannot be attributed to the quantity of tissue destroyed or to the degree of bacterial contamination. Filtered blood can remove leukocytes and prevent postoperative infections. Since filtering blood can significantly reduce the incidence of infection among transfused patients, all transfused blood will be passing through filters in the very near future. EXPERIMENTAL STUDIES RELATING BLOOD TRANSFUSION TO INCREASED RISK OF INFECTION Patients are extremely heterogeneous and even in prospective randomized trials, factors which influence patients' participation affect the outcome despite double-blinding and randomization. In animal studies using syngeneic strains with identical housing, lighting, access to food and water, control over the extent of injury, use of antibiotics and exposure to other variables the influence of a single variable such as blood transfusion can be measured. Dr. Waymack's laboratory has intensively studied parameters which interact with transfusion in

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affecting survival following septic challenge in animal models. Using a pseudomonas contaminated burn model they found that the effect of transfusion was not dose-related (24). They also demonstrated with this model that transfusion within 24 hours of pseudomonas 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) nor transfusion affected survival of animals given intravenous injections in comparison to untransfused unanesthesized animals given the same intravenous dose of E. coli (26). Both allogeneic transfusion and anesthesia caused significantly increased mortality compared to controls when 10^ E. coli were injected into the peritoneal cavity. The timing of transfusion relative to septic challenge and the severity of the septic challenge interact in determining the significance of allogeneic blood for increasing susceptibility to infectious agents (27). Immunosuppressive thromboxane and prostaglandins E and Fla production by macrophages is increased following allogeneic transfusion (28) and macrophage migration into the peritoneal cavity is reduced in animals previously transfused with allogeneic blood (29). Macrophages from animals transfused with allogeneic blood also exhibit impaired ability to phagocytose and kill bacteria in culture. Leukotrienes are immunostimulatory metabolites of arachidonic acid and their production is inhibited following allogeneic transfusion. Macrophages and macrophage supernatants from transfused rats suppress lymphocyte responses 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 experimental studies reproducibly demonstrate that allogeneic blood transfusion causes inhibition of cellular antibacterial mechanisms which cause increased susceptibility to bacterial pathogens. The models support the hypothesis that transfusion-induced immune suppression leads to enhanced susceptibility to bacterial pathogens in the recipient. CANCER RECURRENCE In 1981 a letter in The Lancet suggested that the immunosuppressive properties of transfusion which are beneficial for dialysis patients may be detrimental for patients with malignancies (32). There are now over one hundred published studies investigating the relationship between homologous blood transfusion and cancer recurrence. Meta-analysis of 20 colorectal studies representing 5,236 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 and extent of surgery. Since preoperative anemia often leads to blood transfusion 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 with anemia. Advanced malignancies necessitate extensive surgery, require more time and cause greater blood loss. Procedure, duration of surgery and blood loss are associated with transfusion and may account for transfusion's association with recurrence. Prognostic factors cannot be adequately controlled 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

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risks of homologous blood transfusion, such a study is unethical. Less controversial would be randomization of patients likely to be transfused into an autologous blood program. A study utilizing multiple institutions in the Netherlands with over 500 colorectal cancer 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 patients and 2.11 for recipients of 1 - 2 units of homologous blood. Both autologous and homologous transfusions were bufify 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. A randomized prospective study of colorectal cancer patients by Weiss et al., (34) from Munich randomized 120 patients to receive either homologous or autologous blood if transfusion were needed. With median follow-up of 21 months (9 - 48), the recurrence rate among 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 identified homologous 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 produces profound changes in the immune systems of experimental animals which are analogous to those observed in man. Experimental studies have observed promotion or inhibition of tumor growth following allogeneic blood transfusions because the effect of transfusion on tumor growth 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 is inhibited by transfusion while pulmonary nodules are unaffected. Timing of transfusion relative to tumor inoculation also determines subsequent tumor growth. Studies of tumor growth in experimental animals lack analogy to the situation in the cancer patient. The tumor has been present for years in patients with malignancies and some immunologic interaction between the host and the tumor has preceded the effects of surgery and blood transfusion. In experimental studies, tumor inoculation generally followed allogeneic transfusion. MISCELLANEOUS PHENOMENA ASSOCIATED WITH BLOOD TRANSFUSION Recurrent Abortion One of the most exciting, intriguing and controversial areas in which transfusion affects the outcome and has a therapeutic role is in the treatment of recurrent abortion. During pregnancy, lymphocyte function, as measured by responses to antigens, mitogens and homologous lymphocytes (MLR), is suppressed. Inhibition of lymphocyte function is due to serum factors, blocking antibodies which develop in response to trophoblast antigens. When spouses share HLA antigens, trophoblast antigens are not recognized by the pregnant woman's immune system, blocking antibodies are not produced, and the fetus is rejected. In 1981 Taylor and Faulk (36) induced suppressive sera in women suffering from recurrent spontaneous abortion and sharing HLA antigens with their spouse by transfusing the women with leukocyte-enriched plasma from

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multiple donors. Three women had normal pregnancies and deliveries at term. Several groups have replicated this work with spouse leukocytes and successful deliveries result in more than 50% 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. Recurrence of disease following surgery is common -nearly half of the patients 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 same way steroids affect the course to the disease. Most of the studies observed that untransfused patients exhibited higher rates of recurrence than transfused patients (37-40). The studies suggest that transfusion may influence the course of diseases which are thought to have an immune or autoimmune basis and clinically respond to steroids. Crohn's disease patients with more severe disease, those with lower hemoglobins and serum albumins, undergoing resection of more bowel, should have higher recurrence rates. Yet, these patients when transfused have recurrence rates comparable to untransfused patients with higher hemoglobins and albumins and less bowel 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, migration and collagen synthesis. In vivo depletion of lymphocytes impairs skin wound healing. Since transfusions 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 blood were sacrificed three and seven days following surgery and the bursting pressure of the anastomosis measured. Bursting pressure was significantly lower following transfusion with syngeneic or allogeneic blood in comparison to saline. Hydroxyproline content of the anastomoses was reduced and anastomotic abscesses were common in the transfused animals. This study clearly implicates blood transfusion in impaired wound healing. D iabetes In man, insulin dependent diabetes mellitus is associated with decreases in both the number and functional activity of suppresser T lymphocytes. In the Bio-Breeding rat, diabetes develops when the animals develop pancreatic insulitis, suggesting a cell-mediated immune pathogenesis. Diabetes is prevented in these animals by treating them with immunosuppressive agents such as anti-lymphocyte serum, steroids, cyclosporin, irradiation, neonatal thymectomy, or blood transfusion (42).

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These studies indicate that homologous blood transfusion affects the outcome of clinical diseases in both beneficial and adverse ways. Experimental situations are not suitable for randomized clinical trials - transfusions cannot be given to prevent the onset of diabetes or wound strength measured in man following receipt of homologous or autologous blood. These experimental observations indicate 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 studies indicate that changes in immune function following transfusion are permanent. The number of clinical phenomena associated with immune suppression and attributable to blood transfusion is unknown. SUMMARY Given the evidence presented here it would be foolish to suggest that transfusion of homologous blood has 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 is present one 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 lower natural killer cytotoxicity than colorectal cancer patients who have never been transfused (44). Transfusion of neonates causes suppression of lymphocyte reactivity which is still demonstrable 25 to 30 years later (45). There is evidence that transfusion at any time prior to elective surgery increases susceptibility to infectious complications (14) and otherwise healthy transfused individuals may be at increased risk of developing malignancies (46). All the longterm consequences of blood transfusion are not negative: Survival of transplants is prolonged by pretransplant transfusion and some women suffering from 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. Studies presented 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 10^/unit. An alternative which is increasingly being utilized is autologous blood programs. Physicians are discovering that patients tolerate hemoglobin levels which were previously unacceptably low and many patients prefer being anemic over the risks of receiving homologous blood. Since transfusion is an identifier of high cost hospitalized patients, alternatives to routine blood use are being studied in hopes of safely reducing the costs of transfusion. REFERENCES 1. Jubert AV, Lee ET, Hersh EM, McBride CM. J Surg Res 15:399-403, 1973. 2. M 19 u4n ( s3t ) e3r4A6-M 35 , 2 W , i1n9c8h1u . rch RA, Keane RM, Shatney CH, Ernst CB, Nuidema GD. Ann Surg

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M 18 a3c ( L 3) e : a2n07L -D 21 , 7 M , e1a9k7i5n . s TL, Taguchi K, Duignan TP, Dhillon KS, Gordon J. Ann Surg 4. Nielsen HJ, Hammer JH, Moesgaard F, Kehlet H. Surgery 105(6):711-719, 1989. 5. B 67 ro 6 w , n19R8 , 2 . Bancewicz J, Hamid J, Tillotson G, Ward C, Irving M. Ann Surg 196(6):672-6. Fernandez LA, MacSween JM, You CK, Gorelick M. Am J Surg 1613:263-270, 1992. 7. H 57 a , m 1 id 98J4 , . Bancewicz J, Brown R, Ward C, Irving MH, Ford WL. Clin Exp Immunol 56:49-8. Tartter PI, Steinberg B, Barron DM, Martinelli G. Arch Surg 122:1264-1268. 1987. 9. J M en o s ll eenr -N LS ie , ls A en ndCe , rsH en anAbJe , rg C -S hr oirse ti nasnesnenF , PHMo , klH an odk la M n . dBP, r J Ju Shul rg CO7 , 9 M :51 ad 3 s -5 en 16G , , 19M 92 o . rtensen J, 10. Fisher E, Lennard V, Siefert P Kluge A, Johannsen R. Human Immunol 3:187-194, 1980. 11. L 10 e1n5n , ar1d9V 83 , . Maassen G, Grosse-Wilde H, Wernet P, Opelz G. Transplant Proc 15(1): 1011-12. F1o9r8d7 . CD, Warnick CT, Sheets S, Quist R, Stevens LE. Transplant Proc 19( 1): 1:456-457, 13. Cox DR. Analysis of binary data, Methuen: London, 1970. 14. Murphy PJ, Connery C, Hicks GL Jr, Blumberg N. J Thoracic Cardiovasc Surgery (in press). 15. A Pa rc tc hheSnu rg Deerlyl in 1g2e3r ( E 1 , 1 ) M : 1i3 ll 2e0r -1 S3D2 , 7 , W1e9r8 tz 8 . MJ, Grypma M, Droppert B and Anderson PA. 16. D 12 e 3 ll : i1n3g2e0r -1 E3P2 , 5 M , 1 il 9 le 8r8 , SD, Wertz MJ, Grypha M, Droppert B, Anderson PA. Arch Surg 17. Dawes LG, Aprahamian C, Condon RE and Malongi MA. Surgery 100:796-803, 1986. 18. Tartter PI. Br J Surg 75:789-792,1988. 19. A Lo gsarAwnagleN le , s , MAuprrpihly1J9G 92 , . Cayten CG, Stahl WM. Presented to the Surgical Infection Society, 20. Truilzi DJ, Vanek K, Ryan DH and Blumberg N. Transfusion (accepted for publication). 21. Murphy P, Heal JM and Blumberg N. Transfusion 31:212-217,1991. 22. Mezrow CK, Berstein I and Tartter PI. Transfusion 32:27-30, 1992. 23. BMuesdch3R2C8 , : 1 H 37 o2p , W 19 C9J3 , . Hoynck van Zpapendrecht MAW, Marquet RL, Jeekel J. N Engl J 24. W 19 a8y7m . ackJP, Warden GD, Miskell P, Gonce S, Alexander JW. World J Surg 11:387-391, 25. WaymackJP, Robb E, Alexander JW. Arch Surg 122:935-939, 1987.

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WaymackJP, Miskell P, Gonce S. Anesth Analg 69:163-198, 1989. 27. W 19 a8y7m . ackJP, Warden GD, Alexander JW, Miskell P, Gonce S. J Surg Res 42:528-535, 28. JWSauyrmgaRceksJP4 , 9 M :3 o2l8d -a 3 w 32 er , L 19 L 9 , 0 L . owry SF, Guzman RF, Okerberg CV, Mason AD, Pruitt BA. 29. WaymackJP and Yurt RW. J Surg Res 48:147-143, 1990. 30. AWnanym Su arcgk JP, 20M4( c6N ): e6a8l1N -6 , 8W5, a 1 rd 9e8n6 . GD, Balakrishnan K, Gonce S, Alexander JW, Miskell P. 31. W BA aJyrm . aAcrkcJhPS , u H rg e rn 1a2n6d : e5z9 -G 62 , , C1a9p9p1e . lli PJ, Burleson DG, Guzman RF, Mason AD Jr, Pruitt 32. Gantt CL. Lancet ii:363, 1981. 33. Chung M, Steionmetz OK, Gordon PH. BrJ Surg 80:427-432, 1993. 34. W 19 e9i2 ss . MM, Jauch KW, Delanoff CL, Memple W, Schildberg FW. Proc ASCO 11:172, 35. H Si anaggh , 1S9K8 . 8. The Blood Bank, Rotterdam: Cip - Gegevens Koninklijke Bibiliotheek, Den 36. Taylor C and Faulk WP. Lancet ii:68-69, 1981. 37. Peters WR, Fry RD, Fleshman JW, Kodner IJ. Dis Col & Rect 32(9):749-753, 1989. 38. Williams JG and Hughes LE. Lancet H31-132, 1989. 39. SGta eu st proW en H te , roBl ra SnudpA pl , 2W6: e 8 te 1 r -m 86 a , n1I9T9 , 1 . Zwinderman KH, Lamers CBHW, Gooszen. Scand J 40. Scott ADN, Ritchie JK, Phillips RKS. BrJ Surg 78:455-4587, 1991. 41. Tadros Tamer, Wobbes T, Hendriks T. Ann Surg 215(3):276-281, 1992. 42. R 74 o : s3s9 in -i 46A , A 1 , 9 F 84 a . ustman D, Woda BA, Like AA, Szymanski I, Mordes JP. J Clin Invest 43. Tartter PI, Heimann TM, Aufses AH Jr. Am J Surg 151: 358, 1986. 44. T ca anrc tt eerrpPaIt . i en Ttrsa . ns V fu osxioSnanhg is to 5r6y : , 80T , c1e9l8l9s . ub sets and natural killer cytotoxicity in colorectal 45. Beck I, Scott JS, Pepper M, Speck EH. Am J Repro Immunol 1:224, 1981. 46. Tartter PI. Transfusion 28:593-596, 1988.

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mimi

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Measurement Of Platelet Potency

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Studies Of Processing Effects on Potency

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PART V: TRANSFUSION STRATEGIES

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