Transfusion Immunology and Medicine
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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
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
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
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.
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.
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
