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Immunomodulation by blood transfusion: an evolving scientific and clinical challenge
THE SCIENCE OF MEDICAL CARE
Immunomodulation by Blood Transfusion: Scientific and Clinical Challenge
An Evolving
Neil Blumberg, MD, Joanna M. Heal, MRCP, Rochester, New York
Ar
llogeneic blood transfusions generally cause upegulation of humoral immunity and. downregulation of macrophage and T-cell immunity. Increased alloantibody formation to HLA-A,B (MHC class I) antigens and decreased cutaneous delayed type hypersensitivity, T-cell proliferation and natural killer (NK) cell function are observed in transfused patients and experimental animals. These changes are hypothesized to be the mechanism underlying experimental and epidemiologic observations that allogeneic transfusions are strongly associated with prolonged survival of organ allografts, increased rates of cancer recurrence, and increased prevalence of postoperative bacterial and viral infections. There is substantial disagreement as to the interpretation of these data. Nonetheless, the quality and quantity of the animal and epidemiologic data are similar to that in the early days of investigations linking smoking to cardiovascular disease and cancer. Interventional trials using autologous transfusions or leukocyte-depleted allogeneic transfusions have yielded convincing results in animal experiments, but mixed conclusions in clinical trials. Recent investigations into the mechanism of allogeneic transfusion-induced immunomodulation suggest that
From the Transfusion (NB) and Hematolowv (JMH) Units, Department of Pathology and Laboratory Medicine (NB) and Department of Medicine (JMH). University of Rochester; and the American Red Cross Blood Services IJMH), New York-Penn Region, Rochester, New York. Requests for reprints should be addressed to: Nell Blumberg, MD, Universitv of Rochester Medical Center, Box 608. 601 Elmwood Avenue, Rochester, NY 14642. Manuscript received December 13, 1995 and accepted In revised form May 9, 1996.
#c 1996 by Excerpta All rights reserved.
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other currently identified risks of mortality uted to allogeneic transfusions.
HISTORICAL
attrib-
PERSPECTIVE
Transfusion of blood from allogeneic donors has had a long and checkered history in the practice of medicine. Most recently, transfusions have been essential supportive measures in the development of high-technology medicine during the 1950sto 1980s. Innovations in open heart surgery, organ transplantation, treatment of leukemia and lymphoma, joint replacement surgery, and care of trauma patients were made possible by transfusion. In contrast, before the ABO blood group system was discovered by Landsteiner a century ago, transfusions were considered ineffective and dangerous (see Figure 1) , and were legally forbidden in some times and places. The discoveries of the ABO and Rh blood groups and preservation techniques for storing allogeneic blood led to dramatic reductions in morbidity and mortality due to hemorrhage by application of transfusion therapy. During the 1940 ‘s through 1960‘s it became apparent that hepatitis occurred very frelquently in transfusion recipients, and infectious disease transmission became the major concern for transfusion risks. With the advent of hepatitis B testing of donor blood in the 1960s this concern faded considerably, only to peak again in the 1980s when AIDS transmission by transfusion became an all-consuming focus of transfusion practice. At the very time when HIV-l and hepatitis C testing of donor blood allowed these concerns to fade somewhat, a new issue arose: transfusion-induced impairment of host cellular immune function, what we and others have termed the “transfusion immunomodulation theory”. For many decades the only common immunologic effect of allogeneic transfusion was thought to be the well known tendency for transfusions to stimulate humoral immunity, as manifested by IgG alloantibodies against red cells, platelets, and white cells. Indeed, prior to the development of tests for such alloantibodies, alloimmunizatiort to ABO and Rh antigens on red cells is what made transfusion so dangerous. The concept that transfusions have an effect on the patient’s cellular immune system originated in the 197Os, when t,he benefit of allogeneic t,ransfusions on renal transplant survival was proven, substantially due to the work of Paul Terasaki and GerOOOZ-9343/96/$15.00 PII SOOOZ-9343(96)00124-6
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Figure 1. The degree over the centuries, with C/G = hepatitis B/C/G; ficiency virus; HbsAg =
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of anxiety and concern about the safety of blood transfusion is depicted some allowance for poetic license. NAN = non-A, non-B hepatitis; Hep B/ CJ = Creutzfeldt-Jakob disease; HlVab = antibody to human immunodeHepatitis B surface antigen.
hard Opelz. Contrary to what had been previously likely to be correct. However, those interested in a believed, allogeneic transfusions enhanced the suc- detailed alternative view of the controversy can find cess of renal allografts rather than impairing their it in reviews in Transfusion by E.C. Vamvakas. function due to alloantibody formation in the recipIMMUNOLOGIC MECHANISMS ients. However, the hypothesis that transfusion-induced immunomodulatory effects might be of broad clinical importance was not formulated until the Downregulation of Macrophage and T-Cell 1980s. At that time the association between trans- Function by Allogeneic Transfusions fusion and increased cancer recurrence, postoperaFrom a scientific standpoint, the notion that allotive infections, viral infection severity, and reduced geneic transfusion could downregulate donor speseverity of inflammatory disease were reported in cific host immunity is relatively old news. Almost. a numerous epidemiologic and animal studies.’ The half century ago, Medawar and his colleagces dem1980s also saw more reports of downregulation of onstrated that administration of allogeneic tissue to macrophage and T-cell function following blood fetal animals often led to tolerance of foreign tissue transfusions, which might theoretically eqlain these grafts, a finding that paved the way for clinical organ epidemiologic associations. In the 199Os, random- transplantation. Actual tolerance in animals and huized trials to assesst,he impact of autologous or leu- mans beyond fetal life has been extremely difficult kocyte-depleted allogeneic transfusions as strategies to achieve. Clinically, intensive and prolonged use of to minimize the hypothesized immunomodulatory ef- immunosuppressive drugs is essential to success in fects of allogeneic transfusions were reported. Fur- the vast majority of organ allografts. The most that ther attempts to dissect the immunologic mecha- can typically be achieved with current methods is nisms underlying these effects were :made. The relative hyporesponsiveness to allografts through a clinical studies were based upon animal studies and mixture of transfusion and pharmacologic immunoextrapolations from the renal transplant literature suppressives such as cyclosporine and corticostethat suggested that allogeneic leukocytes were the roids. major mediators of transfusion immunomodulation, However, a number of investigators, including Daand that syngeneic blood transfusions were less im- vid Sachs, Anthony Monaco, Suzanne Ildstad, and others have demonstrated in animal models that munomodulatory than allogeneic transfusions. The review that follows reflects our own views of more profound tolerance to organ allografts may be achievable by using donor bone marrow transfusions the controversy concerning the immunomodulatory rather than transfusions of peripheral blood. Particeffects of transfusion. There are some who strongly ularly intriguing, and suggestive that viable whole doubt that transfusion has any clinically important effects on host immunologic defenses. We address cells and chimerism may not be essential in all situthe main points raised by those on the opposing side ations, Barry Kahan, Alan Krensky, Carol Clayberger of this controversy, and why we consider them un- and their colleagues have demonstrated that purified 300
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histocompatibility antigens or fragments thereof can be used to downregulate antigen presenting cell and T-cell function in animal models. In organ transplant research, it has been accepted for decades that cellular immune function is critical to organ allograft rejection. Thus the beneficial reduction in renal allograft rejection seen after allogeneic transfusions has been generally thought to be due to downregulation of antigen presenting cell and T-cell function in hemodialysis patients awaiting allografts. Allogeneic donor white cells have been implicated in the animal and clinical literature as the major mediators of this effect. More recently, Lagaaij, Van Twuyver and their colleagues have provided evidence that partial HLA matching between the transfusion donor and recipient may be particularly effective at inducing donor specific tolerance. Major controversy exists as to whether the associations between allogeneic transfusion immunomodulation and increased postoperative infections and tumor recurrence are causal, as has been accepted for the effect on allograft rejection. There is now, however, a general consensus that transfusion downregulates antigen presenting cell and T-cell function. A variety of antigen specific and nonspecific measures of immunity are decreased in transfused animals and patients, including NR cell function, macrophage migration to sites of injury, lymphocyte proliferation and cutaneous delayed hypersensitivity (Table I). These changes have been particularly prominent in the surgical setti.ng, almost certainly because anesthesia and surgery contribute to immunomodulation. In particular the work of Hans Nielsen, Lone Jensen, Paul Waymack and Wesley Alexander, and their colleagues has convincingly demonstrated downregulation of cellular immune function after allogeneic transfusions. Nielsen and colleagues have demonstrated that some aspects of this immunomodulation can be reversed by pharmacologic blockade with ranitidine. Possible explanations for downregulation of antigen presenting cell and T-cell function include peripheral anergy. T-cells encountering antigen in the absence of appropriate costimulatory signals usually become unresponsive (anergic) to that antigen. The costimulatory signals that might be absent include inflammatory/stimulatory cytokines (eg, IL2) or regulatory molecules present on the surface of “professional” antigen presenting cells (eg, B7 on dendritic cells and macrophages). Such cells are more commonly found in skin and mucosa than in circulating peripheral blood. Thus presentation of transfused foreign antigen by peripheral blood cells may be more likely to lead to anergy than when such antigens are encountered at mucosal boundaries. Another possible mechanism for antigen specific hy-
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TABLE I Effects of Allogeneic Transfusions on Recipient Immunologic Functions 1. Reduced responses in mixed lymphocyte culture 2. Decreased Thl and increased Th2 cytokine production In vitro 3. Decreased proliferative response to mitogens or soluble antigens in vitro; impaired delayed type hypersensitivity skin responses 4. Increased CD8 T-cell number or suppressor function in vitro 5. Decreased natural killer cell number and activity in vitro 6. Decreased CD4 helper T-cells number 7. Decreased monocyte/macrophage function in vitro and rn vivo 8. Enhanced production of anti-idiotypic antibodies suppresswe of mixed lymphocyte response in vitro 9. Decreased cell mediated cytotoxicity (LAK) against certain target cells in vitro
poresponsiveness is clonal deletion, in wh.ich actual “holes” in the T-cell repertoire are created. This mechanism is thought to be more important during ontogeny for elimination of auto-reactive clones of T-cells in the immature thymus. Perhaps more directly related to tumor or bacterial immunity, impairments of monocyte /macrophage and natural killer cell function have been observed after allogeneic transfusions in experimental animals and patients. Thus even though humoral immunity is to some extent up regulated after allogeneic antigen exposure, the downregulation of antigen processing functions may lead to less clinically effective humoral immune function. Anergy would be expected to occur in settings in which IL2 secretion is deficient. Indeed, impaired IL2 production has been reported in allogeneically transfused patients and animals by a number of investigators. We have been investigating altered cytokine regulation as a potential mechanism for transfusion immunomodulation. We wondered whether the reduction in IL2 secretion might be accompanied by increased secretion of IL10 and IL4. These two cytokines can oppose IL2 function and are associated with downregulation of antigen presenting cell and T-cell function (Figure 2). This mechanism has been proposed as central to the tolerance of organ allografts.” In preliminary studies the peripheral blood cells of patients undergoing surgery and anesthesia alone exhibit blunted IL2 secretion and dramatic upregulation of ILlO/IL4 production in vitro during the postoperative period. This increase in ILlO/IL4 secretion is further potentiated by allogeneic but not autologous transfusions. Thus in the surgical setting, the immunomodulatory effects of transfusion must exceed or be additive to the effects of operative trauma and anesthesia in order to be detectable.
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of allogeneic transfusions in renal allotransplantation,’ Crohn ‘s disease and women with repetitive spontaneous abortions. In these settings there is evidence that Thl type auto- (Crohn’s) or alloimmune responses are associated with poorer clinical outcomes or active disease. In the clinical setting, allogeneic transfusions favor graft survival, reduce recurrence of inflammatory bowel disease and enhance the probability of successful term pregnancy. We speculate that allogeneic transfusions might ameliorate the pathophysiology in these settings by stimulating a Th2 type response, which might in turn down regulate the pathophysiologic Thl responses. T helper type 1 response
EPIDEMIOLOGIC
T helper type 2 response
Figure 2. A simplified, schematic view of how alloantigens are processed by macrophages and presented to T-cells is shown with emphasis on the distinction between responses that primarily involve Thl or Th2 type cytokines. Allogeneic transfusions appear to evoke primarily Th2 cytokine secretion patterns with reciprocal downregulation of Thl cytokine secretion. DTH = delayed type hypersensitiwty; TCR = T-cell antigen receptor; MHC = major histocompatibility complex; NK = natural killer: IL = Interleukin.
STUDIES
The Association Between Blood Transfusion and Increased Rates of Cancer Recurrence and Postoperative Infection
Our current hypothesis is that the stimulation of allogeneic transfusions drives the immune system toward what has been termed a Th2 (eg, ILlO, IL4) type response with concomitant downregulation of Thl (eg, IL2, ILl2) dependent immune functions. Broadly speaking, Th2 responses favor humoral immunity (ie, immunoglobulin secretion), and Thl responses favor ant,igen presenting cell and T-cell function (T-cell, NK cell and macrophage activation). The cytokines secreted in Th2 type response can downregulate antigen processing and presentation functions that are necessary for many aspects of antibody formation. If this speculation is correct it could explain the apparent paradox that allogeneic transfusions can stimulate antibody synthesis against some blood cell antigens, but simultaneously impair antigen presenting cell and T-cell immunity beneficial for defenses against bacteria, viruses, and tumor cells. Working in animals, Lowry and colleagues have shown that allogeneic transfusions and cyclosporine favor a Th2 type response in successful allografts, and Meryman and colleagues have demonstrated that transfusion of stored, as opposed to fresh allogeneic blood particularly fosters a Th2 type response. In the most unambiguous experiments to date, Babcock and Alexander have recently demonstrated that allogeneic transfusions in mice promote a Th2 type response even in the absence of surgery, drugs or transplanted organs. The concept that allogeneic transfusions generate a Th2 type immune response and consequently impair Thl responses also provides a possible theoretical basis for understanding the beneficial effects
Cancer. That allogeneic transfusions enhance survival of renal allografts, presumably by down regulating antigen presenting cell and T-cell function, led a number of investigators to wonder whether this “immunosuppression” might be generalized, rather than allograft-specific. Could the same effects that were beneficial to patients receiving renal transplants be deleterious to patients undergoing surgical treatment of cancer? In the early 1980sthe surgical research group in Newcastle, England demonstrated an enhancing effect on tumor growth in animals due to allogeneic transfusion. In 1982, Tartter and Burrows reported a dramatic epidemiologic association between increased colorectal cancer recurrence and perioperative allogeneic transfusions. A number of confirmatory studies appeared, involving a wide variety of human solid tumors. These were followed by an almost equal number of st,udies in which this association did not achieve statistical signilicance, or where multivariate analysis could not confirm an independent prognostic role for transfusion. Clearly there was no way the definitive human experiment could be performed-ie, randomizing patients to receive or not receive transfusions. In addition to this obstacle, there are some unique problems in assessing the data linking transfusion with changed clinical outcome. For example, in comparing studies from multiple countries, the manner of preparing blood components may be different. Red cell concentrates in many European nations are relatively leukocyte-depleted compared with North American practice because the buffy coat is removed to prepare platelets. As white cells are a. proposed major effector of transfusion immunomodulation this makes comparing studies from different centers difficult, particularly since blood component preparation methods may have changed during the period of time under study. In some studies, not only is the
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Figure 3. The relative risk of cancer recurrence or death in patients receiving allogeneic transfusions versus the risk in those recerving no transfusions is shown for a varrety of human cancers, based upon epidemiologic studres. 95% confidence intervals of the estimate are shown. A relative risk of 1.0 indicates no additional risk of recurrence or death beyond that seen in non-transfused patients. The relative risk is statistically significantly increased for transfused patients wrth each tumor with the exception of breast cancer. For comparison, the relative risk for lung cancer in lpack-aday cigarette smokers is about 10 compared with nonsmokers, and the relative risk for oral cancer in pipe and cigar smokers is 3.3 compared with nonsmokers. Data are plotted from the meta-analysis results of E.C. Vamvakas (Perioperative blood transfusion and cancer recurrence. Transfusion. 1995;35:760-768).
method of blood component preparation not speci- doubt that transfusions contribute causally to recurfied, but the distinction between different comporence of at least some types of cancer under some nents (eg, red cell concentrates, platelet pools, or circumstances. But practically speaking, whole single donor platelets) is not clearly made. This blood is no longer used clinically. Currently, allogemakes it difficult to know whether the number of neic transfusions are relatively white cell and plasma immunologically disparate donor exposures or total depleted compared with those given in the 1960s to volume of allogeneic blood transfused is the critical 1980s when most of the patients in these retrospecdeterminant of the effect. tive cohorts were treated. Based upon the renal transplant liter,ature, our Many epidemiologic studies have examined the group hypothesized and observed that the greater relative roles of transfusion, clinical or histopathowhite cell and plasma content of whole blood trans- logic tumor stage, anemia, blood loss or duration of fusions is associated with more profound immunosurgery in predicting cancer recurrence, and translogic effects and higher recurrence rates than red fusion has remained an independent prognostic faccell concentrates alone. This finding has generally tor in a substantial number of studies. In epidemiobeen supported by a number of subsequent studies. logic studies there is the well known publication bias There are now approximately 100 epidemiologic effect, in which positive findings are more frequently studies that have examined the relationship between reported than negative findings. We do not think this transfusion and earlier cancer recurrence. About latter factor is operative in the transfusion immutwo-thirds demonstrate a statistically significant del- nomodulation field, because almost as many “statiseterious transfusion effect on clinical outcome. tically negative” studies have been reporteld as posMany of these studies have been subjected to meta- itive studies. analyses recently and the results of one such analyPerhaps the strongest scientific evidence that the sis are shown in Figure 3. The relative risk of re- “transfusion immunomodulation theory” may be relcurrence and/or death in allogeneically transfused evant to human cancer is that the majority of animal patients as compared with cancer patients who are experimental studies demonstrate a deleterious efnot transfused is about 1.5 to 1.8 fold greater in co- fect of allogeneic transfusions on cancer growth or lorectal cancer, barely > 1 for breast cancer, and dra- metastasis. In particular, the work of Morris Blajchmatically higher for head and neck and gastric can- man and colleagues demonstrates convincingly in cers (> threefold and > twofold greater risks of t.wo animal models that white cell depletion of allogeneic transfusions abrogates this metastasis prorecurrence or death). These quantitatively impressive epidemiologic as- moting effect, and that the mechanism is almost cersociations, the differential effects of whole blood tainly immunologic in nature.? Postoperative Infection: Paul Tartter’s work and red cells, and the consistent finding that transfused patients in virtually all studies do worse than on cancer recurrence led him to investigate whether allogeneic transfusions and their immunologic eftheir non-transfused counterparts leave us with little September
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Figure 4. The increase in hospital charges with increasing doses of allogeneic transfusions in hip replacement surgery ISshown, The group with “zero allogeneic transfusions” includes patients receiving no transfusions or up to five units of autologous blood. The dose response IS statistically significant. There is no dose response between units of of autologous blood transfused and hospital costs (data not shown). Figure adapted from Blumberg et al (A cost analysis of autologous and allogeneic transfusions in hip replacement surgery. Am J Surg 1996;171:324-330).
fects might not also increase the risk of postoperative infection. Tartter’s early studies in patients with colorectal cancer and Crohn’s disease demonstrated, and subsequent research in a wide variety of surgical settings has confirmed, that the risk of acute infectious complications is 5% or less in non-transfused patients. With a few exceptions, the 40 or so epidemiologic studies to date demonstrate that a dose related increase in infections is seen in allogeneically transfused surgical patients. The infections seen are predominately bacterial, with a wide variety of organisms. The mean overall increased risk of postoperative infection in allogeneic transfusion recipients is about fourfold (20% prevalence). In many of these studies, transfusion is the quantitatively most important and statistically significant prognostic factor predicting postoperative infection. Work in our center and elsewhere has confirmed that in contrast to allogeneic transfusions, autologous transfusions are associated with infection rates identical to those seen in non-transfused patients. This suggests but does not prove that the allogeneic transfusion effect is causal and immunologically mediated. Markus Heiss and coworkers demonstrated in a randomized study both clinical and immunologic benefit for autologous transfusions in colorectal cancer patients4 Lone Jensen and colleagues demonstrated in a randomized study that recipients of leukocyte depleted allogeneic transfusions experienced a tenfold reduction in postoperative morbidity as compared with recipients of unmodified allogeneic transfusions.” Animal studies, particularly those by 304
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Wesley Alexander and colleagues suggest that the allogeneic transfusion effect on host, defenses against infection is immunologic and may be mediated by transfused leukocytes. The mechanism underlying these observations is presumably downregulation of host immune defenses. While defects in humoral immunity are traditionally associated with bacterial infection, these infections occur within several days of transfusion and surgery, perhaps an insufficient time for antibody responses to be effectively mounted. We speculate that defects in antigen acquisition, processing and presentation by monocytes, macrophages and dendritic cells underlie this apparent defect in antibacterial defenses despite upregulation of some aspects of humoral immunity. For example, Waymack, Alexander and colleagues have demonstrated impaired macrophage migration to sites of infection in an animal model of peritonitis. Recent investigations from our group and Lone Jensen and colleagues have quantitated the additional costs associated with the postoperative morbidity hypothesized to be due to transfusion immunomodulation. We compared costs in patients receiving autologous versus allogeneic blood in joint replacement surgery. Jensen compared recipients of unmodified whole blood and leukocyte depleted whole blood in colorectal surgery. In lboth studies, one conducted in Rochester, New York .andthe other in Aarhus, Denmark, estimates of the incremental postoperative costs due to transfusions were in the same range of $1,000 t,o $2,000 per unit of allogeneic 101
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blood administered. In each study, the recipients of autologous transfusions or leukocyte depleted allogeneic transfusions had morbidity rates and hospital costs indistinguishable from those of patients receiving no transfusions at all. Some of our hospital charge data are shown in Figure 4. Nonetheless, some investigators continue to suggest that the clinical observations supporting the “transfusion immunomodulation theory” are most likely due not to transfusion itself, but to the clinical variables that lead to transfusion. We disagree. The hypothesis that the association of transfusion with altered clinical outcomes is a surrogac;y effect remains weak at present for many reasons: (1) the association between transfusion and increased cancer recurrence and post.operative infection is seen in virtually all studies, although it is not statistically significant in a substantial minority of cancer studies; (2) no confounding variable that might be directly related to transfusion -including anemia, blood loss, duration of surgery, etc.--has been shown to act in a surrogate fashion; (3) potential confounding variables that have been examined are almost always less statistically significant and quantitatively important than transfusion itself; (4) there are extensive data supporting the probable mechanism underlying these changed clinical outcomes-that is, downregulation of macrophage and T-cell function; (5) all studies demonstrate that transfusions are given in a nonsystematic fashion so that transfusion acting as a surrogate variable for some as yet unknown, important clinical variable is not very likely; and (6) animal model data convincingly demonstrate that transfusion alters cellular immune function and markedly alters outcomes in settings such as allograft rejection, tumor growth, and bacterial infection. Finally, several epidemiologic studies suggest that allogeneic transfusions are associated with a two- to fourfold increase in lymphomas (and perhaps other tumors) over the 5 to 10 years after transfusion.
PROSPECTIVE AND INTERVENTIONAL STUDIES Can the Postulated Effects of Transfusion lmmunomodulation be Ameliorated by Use of Autologous Transfusions or Leukocyte Depletion of Allogeneic Transfusions? One must be cautious about drawing definitive conclusions about clinical phenomena from retrospective epidemiologic studies and animal models. Animal experiments cannot precisely model the clinical setting and epidemiologic associations, however credible, are subject to both bias and confounding. Thus it was of great interest when Paul Tatter’s prospective study of the allogeneic transfusion effect on
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colorectal cancer recurrence demonstrated once again a dramatic association between transfusion and poor outcome.” If Tartter’s results are explained by confounding, transfusion must be confounded with a variable that is currently unknown, because the clinical conditions that lead to transfusion, such as anemia and blood loss, clearly did not explain the association. Allogeneic transfusion remains absolutely essential for many types of medical and surgical therapy. Autologous transfusion is not feasible for most patients. Thus attention has focused on a technical innovation, leukocyte depletion, as a potent.ial answer to the scientific and clinical questions surrounding the transfusion immunomodulation theory. If a modification of allogeneic transfusions could be shown in a randomized trial to improve clinical outcomes, it would have the additional benefit of proving once and for all the most general tenets of the transfusion immunomodulation theory. As noted previously, studies of post.operative infection and colorectal cancer recurrence in patients randomized to receive autologous or allogeneic transfusions’, and of postoperative infection in patients randomized to receive leukocyte depleted or unmodified whole blood” have provided convincing evidence that these strategies can provide clinical benefits, at least to patients undergoing colorectal surgery in Munich or Aarhus. However, multicenter randomized tr:ials of autologous blood transfusion from Rotterdam’ and leukocyte depleted blood from Leiden* did not detect significant benefits to either strateg!y in colorectal cancer patients. The reasons for the strikingly different results in the two pairs of studies are not obvious, and it does not appear that fatal methodologic flaws ex.ist in any of them. The effects of transfusion are almost certainly superimposed on profound effects od surgery and anesthesia, which in turn likely vary with surgical and anesthetic technique, and other drugs administered.’ For example, laparoscopic surgery is probably less suppressive of cellular immune responses than laparotomy. Techniques and drugs used vary dramatically from center to center. We hypothesize that because the two smaller, single-center studies demonstrated benefits, and the two larger multicenter studies found no such difference, this difference in study design may have been crucial. If this hypothesis is correct, then the variations in clinical practices inherent in studies employing 15 to 20 centers, each treating a few dozen patients, may have created sufficient “background noise“ to obscure any potential benefit from altered transfusion practice in the multicenter studies. In a single center, with a handful of surgeons and rlelatively
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Estimates of Allogeneic
Transfusion lmmunomodulation Related Deaths in the USA Due to Cancer Recurrence and Postoperative Infection Cancer* Infection+ Tot& Deaths Death Rate per Deaths Death Rate per Deaths Death Rate per per Year lo6 Transfusions per Year lo6 Transfusions per Year lo6 Transfusions 20,000 33,000 1,500 250 21,500 33,250 10,000 16,667 750 125 10,750 16,892 2,000 3,300 150 25 2,150 3,325 200 330 15 2.5 215 332.5
Estimated Proportion of the Effect that is Causal
100% 50%
10% 1%
* Assumptions (Cancer Recurrence and Transfusion): (1) A 10% difference in death rates between transfused and non-transfused patients. per patlent = 3. (3) 200,000 transfused cancer patients per year out of approximately l,OOO,OOO total cancer patients. + Assumptions (Postoperative Infection and Transfusion): (1) A 15% difference in infection rates between transfused and non-transfused Mean transfusions per patient = 3. (3) 6,000,OOO transfusions to surgical patients per year. (4) Mortality due to postoperatwe infection infections. ’ For Comparison: Roger Dodd estimates that the number csf deaths due to HIV-1 infection from transfusion IS currently approximately transfused or about 30 per year total. The corresponding rwmbers for hepatitis C are a fatality rate of 0.75 to 6.63 per million units or per year total. These data include only red blood cell transfusions.
(2) Mean transfusions surgical patients. (2) averages 1 in 200 2.38 per million units about 9 to 80 deaths
consistent use of other drugs and surgical tech- data from our own studies and those of John Ward niques, the benefit predicted from animal models and colleagues at the Centers for Disease Control, may have been more readily detected because the E.C. Vamvakas and Harold Kaplan at New York Unibackground effects of surgery and anesthesia were versity, Elaine Sloand and Harvey Klein al; NIH, and more uniform. laboratory data from Michael Busch at the Irwin The variability in the results of these four ran- Blood Center suggest that transfused HIV-1 infected domized trials has led to confusion and uncer- patients have more rapidly progressive di.seaseand tainty. Some have argued that the failure of two of greater numbers of complications than those receivthe trials to produce the expected effect means ing no transfusions or lower doses of transfusion. that transfusion is probably immunologically be- Based upon these observations, an NIH-fu.nded mulnign. We consider this conclusion untenable given ticenter study is underway to determine if leukocyte the large body of epidemiologic, animal and clinidepletion of transfusions to HIV-1 infected patients cal interventional data that suggests the contrary provides clinical benefits. in a wide variety of disease settings. Furthermore, THE POTENTIAL PUBLIC HEALTH the control arm in three of the four interventional st,udies involved transfusions of buffy coat and IMPACT OF TRANSFUSION plasma depleted red cells. This undoubtedly mini- IMMUNOMODULATION mized the ability to demonstrate a benefit in the Recently, extensive attention has been paid to the experimental arm employing leukodepletion or au- potential mortality associated with better known and tologous transfusion, given the substantial data on accepted risks of allogeneic transfusion. Roger Dodd the role of white cells and stored plasma in both at the American Red Cross Holland Labalratory has clinical and animal studies. estimated that mortality caused by all known infecOt,her studies of leukocyte depletion in surgical tious and alloimmunization transfusion risks, other settings are underway or planned, but we believe than bacterial contamination of platelets, amounts to that studies in medical patients (eg, with gastroin- perhaps 29 to 103 deaths per million units transtestinal bleeding) would also be of great use. Most fused. No attempt has yet been made to assessthe public health implications of the transfusion immuallogeneic transfusions given to medical or pediatric patients are already being leukocyte depleted to pre- nomodulation theory. It has been argued that even vent febrile transfusion reactions, platelet transfu- if the theory is correct, the effects are unlikely to be sion refractoriness or cytomegalovirus transmission large or of much public health significalnce. Howin patients with diseasessuch as thalassemia, sickle ever, postoperative infections are not benign and cell anemia, leukemia and lymphoma. Data from Kal- have a measurable mortality associated with them. evi Oksanen and colleagues and our own group sug- Likewise, earlier death due to tumor recurrence may gest that at least in leukemia and lymphoma, leuko- not be a negligible problem. We performed a theoretical analysis of the potential mortality due to cyte depleted transfusions are associated with decreases in morbidity, mortality, and costs but postoperative infection and cancer recurrence as a these results are not derived from randomized trials consequence of allogeneic transfusion immunomodulation. These results are shown in Table II. and must be viewed as preliminary. Epidemiologic 306
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The assumptions in the table are all based upon estimates reported in the literature. Reported rates of increased postoperative infections associated with allogeneic transfusion(s) range from a few percent to as high as 20% to 30%. The average increase is about 15%. As can be seen from Table II, even if only a small portion of the association between postoperative infection and transfusion is causal, mortality from transfusion immunomodulation induced postoperative infections might still be of equal significance to that from any other single cause. Furthermore, based upon work by Lone Jensen and colleagues, it appears that leukodepletion of allogeneic blood can in some instances completely abrogate the postoperative bacterial infection promoting effects of allogeneic transfusions. Employing the assumptions in the table, and Jensen’s and our data that each allogeneic transfusion is associated with $1,000 to $2,000 in postoperative costs, it is theoretically possible that expending $240 million to leukocyte deplete the 6 million units of blood used in surgery each year could save between $6 and $12 billion dollars in costs, while reducing the number of postoperative infections and deaths by an order of magnitude. We believe these are reasonable speculations, as our personal estimate is that >90% of the association between allogeneic transfusion and postoperative infection is causal. If the transfusion immunomodulation theory is correct and increased rates of earlier cancer recurrence approach 10 to 20% in allogeneically transfused patients, this could dramatically increase the importance of immunomodulation as a public health issue. In transfused colorectal cancer patients the mean increased mortality is about 10%and the mean increase in recurrence rate is 21% as compared with untransfused patients. As is displayed in Table II, the rate of death due to earlier cancer recurrence using these estimates is 3 to 4 orders of magnitude greater than any other transfusion associated mortality risk. Even if transfusion immunomodulation causally accounts for only 1%to 2% of the association observed between allogeneic transfusion and cancer deaths, immunomodulation-induced tumor recurrence still would be one of most important causes of transfusion mortality. If as little as 10% of the association bet,ween allogeneic transfusion and increased postoperative infection and cancer recurrence is causal, the number of deaths annually clue to these causes would be 2,150. We believe both these estimates to be well below the likely actual risk. By comparison with an estimated several thousand transfusion immunomodulation deaths per year, the number of remaining deaths due to HIV is estimated at under 50/year. September
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Therefore even if only 1% of the association observed between transfusion and earlier death due to cancer or infection is causal, transfusion deaths due to immunomodulation would outnumber HIV by a factor of 4 or more. One major uncertainty in assessing the public health impact of the transfusion immunomodulation theory is that while it is clear that transfusion in the surgical setting is associated with increased morbidity, little is known of the potential effects of allogeneic transfusions in non-surgical patients. There is evidence that transfusion immunomodulation may increase morbidity in patients receiving myeloablative chemotherapy for leukemia and lymphoma, but almost nothing is known about potential effects in the treatment of chronic anemia or bleeding. Such studies are urgently needed. These issues are of sufficient potential public health importance that we suggest that further investigation of the transfusion immunomodulation theory is the single most pressing issue in blood transfusion today from both scientific and clinical standpoints. We believe the potential for improving clinical outcome by use of techniques that minimize allogeneic blood use or its immunologic effects, such autologous transfusion and ot.her blood sparing approaches in surgery, leukodepletion of allogeneic blood, and growth factors such as erythropoietin warrant intensive investigation. In addition, insight into the effects of transfusion on immune function may serve as a model for investigations into the potential effects of ‘the many drugs and biologic agents whose effects on the immune system have not yet been adequately addressed.
ACKNOWLEDGMENT The format of this editorial review did not permit comprehensive citation of all relevant work and we wish to thank both the mentioned and unattributed Investigators whose work has contributed to our understanding of transfuston immunology. We thank Drs. John Leddy and R. John Looney for helpful discussions.
REFERENCES 1. Blumberg cer recurrence
N, Heal JM. Effects of transfusion and infectlon. Arch Pathol
Lab
on immune function, Med. 1994;118:371-
can-
379. 2. Lowry RP, Takeuchi T. The Thl, Th.2 paradigm and transplantation tolerance. Austin, TX: R.G. Landes 1994. 3. Blajchman MA, Bardossy L, Carmen R, Sastry A, Singal DP. Allogeneic blood transfusion-induced enhancement of tumor growth: two animal models showing amelioration
by leukodepletion
and passive
transfer
using
spleen
cells.
Blood.
1993;81:1880-1882. 4. Heiss MM, Mempel W, Jauch K-W, Delanoff C, Mayer G, Mempel M. Eissner H-J, Schildberg F-W. Beneficial effect of autologous blood transfusion on Infectious complications after colorectal cancer surgery. Lancet. 1993;342:13281333. 5. Jensen Mortensen
1996
LS, Andersen AJ, Christiansen PM, Hokland J, Moller-Nielsen C, Hanberg-Sorensen
The
American
Journal
of Medicine”
P, Juhl CO, Madsen F, Hokland
Volume
101
G, M.
307
Postoperative infectron in patrents undergoing 516.
and natural killer cell funchon elective colorectal surgery.
6. Tartter PI. The association of perioperative blood cancer recurrence. Ann Surg. 1992;216:633-638, 7. Busch ORC, Hop WCJ, Hoynck van Papendrecht J. Blood
transfusions
and
prognosis
in colorectal
following blood transfusion Br J Surg. 1992;79:513-
8. Houbiers JGA, Brand PJM, Bijnen AB, Pahlplatz Klementschitsch P, Van
transfusion
controlled coat-depleted
with colorectal
MAW, Marquet cancer.
RL, Jeekel
N Engl
J Med.
1993;328:1372-1376.
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A, Van de Watering LMG, Hermans J, Verwey P, Schattenkerk ME, Wobbes T, De Vrres JE, de Maas AHM, Van de Velde CJH. Randomrsed
comparing blood in
transfusion surgery
for
of leucocyte-depleted colorectal cancer.
or buffyLancet.
1994;344:573-578. 9. Blumberg in progress?
101
N, Heal JM. Transfusion
and the immune
Transfusion. 1995;35:879-883.
system:
a paradigm
shaft
Immunomodulation by Blood Transfusion: Scientific and Clinical Challenge
An Evolving
Neil Blumberg, MD, Joanna M. Heal, MRCP, Rochester, New York
Ar
llogeneic blood transfusions generally cause upegulation of humoral immunity and. downregulation of macrophage and T-cell immunity. Increased alloantibody formation to HLA-A,B (MHC class I) antigens and decreased cutaneous delayed type hypersensitivity, T-cell proliferation and natural killer (NK) cell function are observed in transfused patients and experimental animals. These changes are hypothesized to be the mechanism underlying experimental and epidemiologic observations that allogeneic transfusions are strongly associated with prolonged survival of organ allografts, increased rates of cancer recurrence, and increased prevalence of postoperative bacterial and viral infections. There is substantial disagreement as to the interpretation of these data. Nonetheless, the quality and quantity of the animal and epidemiologic data are similar to that in the early days of investigations linking smoking to cardiovascular disease and cancer. Interventional trials using autologous transfusions or leukocyte-depleted allogeneic transfusions have yielded convincing results in animal experiments, but mixed conclusions in clinical trials. Recent investigations into the mechanism of allogeneic transfusion-induced immunomodulation suggest that
From the Transfusion (NB) and Hematolowv (JMH) Units, Department of Pathology and Laboratory Medicine (NB) and Department of Medicine (JMH). University of Rochester; and the American Red Cross Blood Services IJMH), New York-Penn Region, Rochester, New York. Requests for reprints should be addressed to: Nell Blumberg, MD, Universitv of Rochester Medical Center, Box 608. 601 Elmwood Avenue, Rochester, NY 14642. Manuscript received December 13, 1995 and accepted In revised form May 9, 1996.
#c 1996 by Excerpta All rights reserved.
Medica,
Inc.
other currently identified risks of mortality uted to allogeneic transfusions.
HISTORICAL
attrib-
PERSPECTIVE
Transfusion of blood from allogeneic donors has had a long and checkered history in the practice of medicine. Most recently, transfusions have been essential supportive measures in the development of high-technology medicine during the 1950sto 1980s. Innovations in open heart surgery, organ transplantation, treatment of leukemia and lymphoma, joint replacement surgery, and care of trauma patients were made possible by transfusion. In contrast, before the ABO blood group system was discovered by Landsteiner a century ago, transfusions were considered ineffective and dangerous (see Figure 1) , and were legally forbidden in some times and places. The discoveries of the ABO and Rh blood groups and preservation techniques for storing allogeneic blood led to dramatic reductions in morbidity and mortality due to hemorrhage by application of transfusion therapy. During the 1940 ‘s through 1960‘s it became apparent that hepatitis occurred very frelquently in transfusion recipients, and infectious disease transmission became the major concern for transfusion risks. With the advent of hepatitis B testing of donor blood in the 1960s this concern faded considerably, only to peak again in the 1980s when AIDS transmission by transfusion became an all-consuming focus of transfusion practice. At the very time when HIV-l and hepatitis C testing of donor blood allowed these concerns to fade somewhat, a new issue arose: transfusion-induced impairment of host cellular immune function, what we and others have termed the “transfusion immunomodulation theory”. For many decades the only common immunologic effect of allogeneic transfusion was thought to be the well known tendency for transfusions to stimulate humoral immunity, as manifested by IgG alloantibodies against red cells, platelets, and white cells. Indeed, prior to the development of tests for such alloantibodies, alloimmunizatiort to ABO and Rh antigens on red cells is what made transfusion so dangerous. The concept that transfusions have an effect on the patient’s cellular immune system originated in the 197Os, when t,he benefit of allogeneic t,ransfusions on renal transplant survival was proven, substantially due to the work of Paul Terasaki and GerOOOZ-9343/96/$15.00 PII SOOOZ-9343(96)00124-6
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Figure 1. The degree over the centuries, with C/G = hepatitis B/C/G; ficiency virus; HbsAg =
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of anxiety and concern about the safety of blood transfusion is depicted some allowance for poetic license. NAN = non-A, non-B hepatitis; Hep B/ CJ = Creutzfeldt-Jakob disease; HlVab = antibody to human immunodeHepatitis B surface antigen.
hard Opelz. Contrary to what had been previously likely to be correct. However, those interested in a believed, allogeneic transfusions enhanced the suc- detailed alternative view of the controversy can find cess of renal allografts rather than impairing their it in reviews in Transfusion by E.C. Vamvakas. function due to alloantibody formation in the recipIMMUNOLOGIC MECHANISMS ients. However, the hypothesis that transfusion-induced immunomodulatory effects might be of broad clinical importance was not formulated until the Downregulation of Macrophage and T-Cell 1980s. At that time the association between trans- Function by Allogeneic Transfusions fusion and increased cancer recurrence, postoperaFrom a scientific standpoint, the notion that allotive infections, viral infection severity, and reduced geneic transfusion could downregulate donor speseverity of inflammatory disease were reported in cific host immunity is relatively old news. Almost. a numerous epidemiologic and animal studies.’ The half century ago, Medawar and his colleagces dem1980s also saw more reports of downregulation of onstrated that administration of allogeneic tissue to macrophage and T-cell function following blood fetal animals often led to tolerance of foreign tissue transfusions, which might theoretically eqlain these grafts, a finding that paved the way for clinical organ epidemiologic associations. In the 199Os, random- transplantation. Actual tolerance in animals and huized trials to assesst,he impact of autologous or leu- mans beyond fetal life has been extremely difficult kocyte-depleted allogeneic transfusions as strategies to achieve. Clinically, intensive and prolonged use of to minimize the hypothesized immunomodulatory ef- immunosuppressive drugs is essential to success in fects of allogeneic transfusions were reported. Fur- the vast majority of organ allografts. The most that ther attempts to dissect the immunologic mecha- can typically be achieved with current methods is nisms underlying these effects were :made. The relative hyporesponsiveness to allografts through a clinical studies were based upon animal studies and mixture of transfusion and pharmacologic immunoextrapolations from the renal transplant literature suppressives such as cyclosporine and corticostethat suggested that allogeneic leukocytes were the roids. major mediators of transfusion immunomodulation, However, a number of investigators, including Daand that syngeneic blood transfusions were less im- vid Sachs, Anthony Monaco, Suzanne Ildstad, and others have demonstrated in animal models that munomodulatory than allogeneic transfusions. The review that follows reflects our own views of more profound tolerance to organ allografts may be achievable by using donor bone marrow transfusions the controversy concerning the immunomodulatory rather than transfusions of peripheral blood. Particeffects of transfusion. There are some who strongly ularly intriguing, and suggestive that viable whole doubt that transfusion has any clinically important effects on host immunologic defenses. We address cells and chimerism may not be essential in all situthe main points raised by those on the opposing side ations, Barry Kahan, Alan Krensky, Carol Clayberger of this controversy, and why we consider them un- and their colleagues have demonstrated that purified 300
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histocompatibility antigens or fragments thereof can be used to downregulate antigen presenting cell and T-cell function in animal models. In organ transplant research, it has been accepted for decades that cellular immune function is critical to organ allograft rejection. Thus the beneficial reduction in renal allograft rejection seen after allogeneic transfusions has been generally thought to be due to downregulation of antigen presenting cell and T-cell function in hemodialysis patients awaiting allografts. Allogeneic donor white cells have been implicated in the animal and clinical literature as the major mediators of this effect. More recently, Lagaaij, Van Twuyver and their colleagues have provided evidence that partial HLA matching between the transfusion donor and recipient may be particularly effective at inducing donor specific tolerance. Major controversy exists as to whether the associations between allogeneic transfusion immunomodulation and increased postoperative infections and tumor recurrence are causal, as has been accepted for the effect on allograft rejection. There is now, however, a general consensus that transfusion downregulates antigen presenting cell and T-cell function. A variety of antigen specific and nonspecific measures of immunity are decreased in transfused animals and patients, including NR cell function, macrophage migration to sites of injury, lymphocyte proliferation and cutaneous delayed hypersensitivity (Table I). These changes have been particularly prominent in the surgical setti.ng, almost certainly because anesthesia and surgery contribute to immunomodulation. In particular the work of Hans Nielsen, Lone Jensen, Paul Waymack and Wesley Alexander, and their colleagues has convincingly demonstrated downregulation of cellular immune function after allogeneic transfusions. Nielsen and colleagues have demonstrated that some aspects of this immunomodulation can be reversed by pharmacologic blockade with ranitidine. Possible explanations for downregulation of antigen presenting cell and T-cell function include peripheral anergy. T-cells encountering antigen in the absence of appropriate costimulatory signals usually become unresponsive (anergic) to that antigen. The costimulatory signals that might be absent include inflammatory/stimulatory cytokines (eg, IL2) or regulatory molecules present on the surface of “professional” antigen presenting cells (eg, B7 on dendritic cells and macrophages). Such cells are more commonly found in skin and mucosa than in circulating peripheral blood. Thus presentation of transfused foreign antigen by peripheral blood cells may be more likely to lead to anergy than when such antigens are encountered at mucosal boundaries. Another possible mechanism for antigen specific hy-
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TABLE I Effects of Allogeneic Transfusions on Recipient Immunologic Functions 1. Reduced responses in mixed lymphocyte culture 2. Decreased Thl and increased Th2 cytokine production In vitro 3. Decreased proliferative response to mitogens or soluble antigens in vitro; impaired delayed type hypersensitivity skin responses 4. Increased CD8 T-cell number or suppressor function in vitro 5. Decreased natural killer cell number and activity in vitro 6. Decreased CD4 helper T-cells number 7. Decreased monocyte/macrophage function in vitro and rn vivo 8. Enhanced production of anti-idiotypic antibodies suppresswe of mixed lymphocyte response in vitro 9. Decreased cell mediated cytotoxicity (LAK) against certain target cells in vitro
poresponsiveness is clonal deletion, in wh.ich actual “holes” in the T-cell repertoire are created. This mechanism is thought to be more important during ontogeny for elimination of auto-reactive clones of T-cells in the immature thymus. Perhaps more directly related to tumor or bacterial immunity, impairments of monocyte /macrophage and natural killer cell function have been observed after allogeneic transfusions in experimental animals and patients. Thus even though humoral immunity is to some extent up regulated after allogeneic antigen exposure, the downregulation of antigen processing functions may lead to less clinically effective humoral immune function. Anergy would be expected to occur in settings in which IL2 secretion is deficient. Indeed, impaired IL2 production has been reported in allogeneically transfused patients and animals by a number of investigators. We have been investigating altered cytokine regulation as a potential mechanism for transfusion immunomodulation. We wondered whether the reduction in IL2 secretion might be accompanied by increased secretion of IL10 and IL4. These two cytokines can oppose IL2 function and are associated with downregulation of antigen presenting cell and T-cell function (Figure 2). This mechanism has been proposed as central to the tolerance of organ allografts.” In preliminary studies the peripheral blood cells of patients undergoing surgery and anesthesia alone exhibit blunted IL2 secretion and dramatic upregulation of ILlO/IL4 production in vitro during the postoperative period. This increase in ILlO/IL4 secretion is further potentiated by allogeneic but not autologous transfusions. Thus in the surgical setting, the immunomodulatory effects of transfusion must exceed or be additive to the effects of operative trauma and anesthesia in order to be detectable.
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of allogeneic transfusions in renal allotransplantation,’ Crohn ‘s disease and women with repetitive spontaneous abortions. In these settings there is evidence that Thl type auto- (Crohn’s) or alloimmune responses are associated with poorer clinical outcomes or active disease. In the clinical setting, allogeneic transfusions favor graft survival, reduce recurrence of inflammatory bowel disease and enhance the probability of successful term pregnancy. We speculate that allogeneic transfusions might ameliorate the pathophysiology in these settings by stimulating a Th2 type response, which might in turn down regulate the pathophysiologic Thl responses. T helper type 1 response
EPIDEMIOLOGIC
T helper type 2 response
Figure 2. A simplified, schematic view of how alloantigens are processed by macrophages and presented to T-cells is shown with emphasis on the distinction between responses that primarily involve Thl or Th2 type cytokines. Allogeneic transfusions appear to evoke primarily Th2 cytokine secretion patterns with reciprocal downregulation of Thl cytokine secretion. DTH = delayed type hypersensitiwty; TCR = T-cell antigen receptor; MHC = major histocompatibility complex; NK = natural killer: IL = Interleukin.
STUDIES
The Association Between Blood Transfusion and Increased Rates of Cancer Recurrence and Postoperative Infection
Our current hypothesis is that the stimulation of allogeneic transfusions drives the immune system toward what has been termed a Th2 (eg, ILlO, IL4) type response with concomitant downregulation of Thl (eg, IL2, ILl2) dependent immune functions. Broadly speaking, Th2 responses favor humoral immunity (ie, immunoglobulin secretion), and Thl responses favor ant,igen presenting cell and T-cell function (T-cell, NK cell and macrophage activation). The cytokines secreted in Th2 type response can downregulate antigen processing and presentation functions that are necessary for many aspects of antibody formation. If this speculation is correct it could explain the apparent paradox that allogeneic transfusions can stimulate antibody synthesis against some blood cell antigens, but simultaneously impair antigen presenting cell and T-cell immunity beneficial for defenses against bacteria, viruses, and tumor cells. Working in animals, Lowry and colleagues have shown that allogeneic transfusions and cyclosporine favor a Th2 type response in successful allografts, and Meryman and colleagues have demonstrated that transfusion of stored, as opposed to fresh allogeneic blood particularly fosters a Th2 type response. In the most unambiguous experiments to date, Babcock and Alexander have recently demonstrated that allogeneic transfusions in mice promote a Th2 type response even in the absence of surgery, drugs or transplanted organs. The concept that allogeneic transfusions generate a Th2 type immune response and consequently impair Thl responses also provides a possible theoretical basis for understanding the beneficial effects
Cancer. That allogeneic transfusions enhance survival of renal allografts, presumably by down regulating antigen presenting cell and T-cell function, led a number of investigators to wonder whether this “immunosuppression” might be generalized, rather than allograft-specific. Could the same effects that were beneficial to patients receiving renal transplants be deleterious to patients undergoing surgical treatment of cancer? In the early 1980sthe surgical research group in Newcastle, England demonstrated an enhancing effect on tumor growth in animals due to allogeneic transfusion. In 1982, Tartter and Burrows reported a dramatic epidemiologic association between increased colorectal cancer recurrence and perioperative allogeneic transfusions. A number of confirmatory studies appeared, involving a wide variety of human solid tumors. These were followed by an almost equal number of st,udies in which this association did not achieve statistical signilicance, or where multivariate analysis could not confirm an independent prognostic role for transfusion. Clearly there was no way the definitive human experiment could be performed-ie, randomizing patients to receive or not receive transfusions. In addition to this obstacle, there are some unique problems in assessing the data linking transfusion with changed clinical outcome. For example, in comparing studies from multiple countries, the manner of preparing blood components may be different. Red cell concentrates in many European nations are relatively leukocyte-depleted compared with North American practice because the buffy coat is removed to prepare platelets. As white cells are a. proposed major effector of transfusion immunomodulation this makes comparing studies from different centers difficult, particularly since blood component preparation methods may have changed during the period of time under study. In some studies, not only is the
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Figure 3. The relative risk of cancer recurrence or death in patients receiving allogeneic transfusions versus the risk in those recerving no transfusions is shown for a varrety of human cancers, based upon epidemiologic studres. 95% confidence intervals of the estimate are shown. A relative risk of 1.0 indicates no additional risk of recurrence or death beyond that seen in non-transfused patients. The relative risk is statistically significantly increased for transfused patients wrth each tumor with the exception of breast cancer. For comparison, the relative risk for lung cancer in lpack-aday cigarette smokers is about 10 compared with nonsmokers, and the relative risk for oral cancer in pipe and cigar smokers is 3.3 compared with nonsmokers. Data are plotted from the meta-analysis results of E.C. Vamvakas (Perioperative blood transfusion and cancer recurrence. Transfusion. 1995;35:760-768).
method of blood component preparation not speci- doubt that transfusions contribute causally to recurfied, but the distinction between different comporence of at least some types of cancer under some nents (eg, red cell concentrates, platelet pools, or circumstances. But practically speaking, whole single donor platelets) is not clearly made. This blood is no longer used clinically. Currently, allogemakes it difficult to know whether the number of neic transfusions are relatively white cell and plasma immunologically disparate donor exposures or total depleted compared with those given in the 1960s to volume of allogeneic blood transfused is the critical 1980s when most of the patients in these retrospecdeterminant of the effect. tive cohorts were treated. Based upon the renal transplant liter,ature, our Many epidemiologic studies have examined the group hypothesized and observed that the greater relative roles of transfusion, clinical or histopathowhite cell and plasma content of whole blood trans- logic tumor stage, anemia, blood loss or duration of fusions is associated with more profound immunosurgery in predicting cancer recurrence, and translogic effects and higher recurrence rates than red fusion has remained an independent prognostic faccell concentrates alone. This finding has generally tor in a substantial number of studies. In epidemiobeen supported by a number of subsequent studies. logic studies there is the well known publication bias There are now approximately 100 epidemiologic effect, in which positive findings are more frequently studies that have examined the relationship between reported than negative findings. We do not think this transfusion and earlier cancer recurrence. About latter factor is operative in the transfusion immutwo-thirds demonstrate a statistically significant del- nomodulation field, because almost as many “statiseterious transfusion effect on clinical outcome. tically negative” studies have been reporteld as posMany of these studies have been subjected to meta- itive studies. analyses recently and the results of one such analyPerhaps the strongest scientific evidence that the sis are shown in Figure 3. The relative risk of re- “transfusion immunomodulation theory” may be relcurrence and/or death in allogeneically transfused evant to human cancer is that the majority of animal patients as compared with cancer patients who are experimental studies demonstrate a deleterious efnot transfused is about 1.5 to 1.8 fold greater in co- fect of allogeneic transfusions on cancer growth or lorectal cancer, barely > 1 for breast cancer, and dra- metastasis. In particular, the work of Morris Blajchmatically higher for head and neck and gastric can- man and colleagues demonstrates convincingly in cers (> threefold and > twofold greater risks of t.wo animal models that white cell depletion of allogeneic transfusions abrogates this metastasis prorecurrence or death). These quantitatively impressive epidemiologic as- moting effect, and that the mechanism is almost cersociations, the differential effects of whole blood tainly immunologic in nature.? Postoperative Infection: Paul Tartter’s work and red cells, and the consistent finding that transfused patients in virtually all studies do worse than on cancer recurrence led him to investigate whether allogeneic transfusions and their immunologic eftheir non-transfused counterparts leave us with little September
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Figure 4. The increase in hospital charges with increasing doses of allogeneic transfusions in hip replacement surgery ISshown, The group with “zero allogeneic transfusions” includes patients receiving no transfusions or up to five units of autologous blood. The dose response IS statistically significant. There is no dose response between units of of autologous blood transfused and hospital costs (data not shown). Figure adapted from Blumberg et al (A cost analysis of autologous and allogeneic transfusions in hip replacement surgery. Am J Surg 1996;171:324-330).
fects might not also increase the risk of postoperative infection. Tartter’s early studies in patients with colorectal cancer and Crohn’s disease demonstrated, and subsequent research in a wide variety of surgical settings has confirmed, that the risk of acute infectious complications is 5% or less in non-transfused patients. With a few exceptions, the 40 or so epidemiologic studies to date demonstrate that a dose related increase in infections is seen in allogeneically transfused surgical patients. The infections seen are predominately bacterial, with a wide variety of organisms. The mean overall increased risk of postoperative infection in allogeneic transfusion recipients is about fourfold (20% prevalence). In many of these studies, transfusion is the quantitatively most important and statistically significant prognostic factor predicting postoperative infection. Work in our center and elsewhere has confirmed that in contrast to allogeneic transfusions, autologous transfusions are associated with infection rates identical to those seen in non-transfused patients. This suggests but does not prove that the allogeneic transfusion effect is causal and immunologically mediated. Markus Heiss and coworkers demonstrated in a randomized study both clinical and immunologic benefit for autologous transfusions in colorectal cancer patients4 Lone Jensen and colleagues demonstrated in a randomized study that recipients of leukocyte depleted allogeneic transfusions experienced a tenfold reduction in postoperative morbidity as compared with recipients of unmodified allogeneic transfusions.” Animal studies, particularly those by 304
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Wesley Alexander and colleagues suggest that the allogeneic transfusion effect on host, defenses against infection is immunologic and may be mediated by transfused leukocytes. The mechanism underlying these observations is presumably downregulation of host immune defenses. While defects in humoral immunity are traditionally associated with bacterial infection, these infections occur within several days of transfusion and surgery, perhaps an insufficient time for antibody responses to be effectively mounted. We speculate that defects in antigen acquisition, processing and presentation by monocytes, macrophages and dendritic cells underlie this apparent defect in antibacterial defenses despite upregulation of some aspects of humoral immunity. For example, Waymack, Alexander and colleagues have demonstrated impaired macrophage migration to sites of infection in an animal model of peritonitis. Recent investigations from our group and Lone Jensen and colleagues have quantitated the additional costs associated with the postoperative morbidity hypothesized to be due to transfusion immunomodulation. We compared costs in patients receiving autologous versus allogeneic blood in joint replacement surgery. Jensen compared recipients of unmodified whole blood and leukocyte depleted whole blood in colorectal surgery. In lboth studies, one conducted in Rochester, New York .andthe other in Aarhus, Denmark, estimates of the incremental postoperative costs due to transfusions were in the same range of $1,000 t,o $2,000 per unit of allogeneic 101
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blood administered. In each study, the recipients of autologous transfusions or leukocyte depleted allogeneic transfusions had morbidity rates and hospital costs indistinguishable from those of patients receiving no transfusions at all. Some of our hospital charge data are shown in Figure 4. Nonetheless, some investigators continue to suggest that the clinical observations supporting the “transfusion immunomodulation theory” are most likely due not to transfusion itself, but to the clinical variables that lead to transfusion. We disagree. The hypothesis that the association of transfusion with altered clinical outcomes is a surrogac;y effect remains weak at present for many reasons: (1) the association between transfusion and increased cancer recurrence and post.operative infection is seen in virtually all studies, although it is not statistically significant in a substantial minority of cancer studies; (2) no confounding variable that might be directly related to transfusion -including anemia, blood loss, duration of surgery, etc.--has been shown to act in a surrogate fashion; (3) potential confounding variables that have been examined are almost always less statistically significant and quantitatively important than transfusion itself; (4) there are extensive data supporting the probable mechanism underlying these changed clinical outcomes-that is, downregulation of macrophage and T-cell function; (5) all studies demonstrate that transfusions are given in a nonsystematic fashion so that transfusion acting as a surrogate variable for some as yet unknown, important clinical variable is not very likely; and (6) animal model data convincingly demonstrate that transfusion alters cellular immune function and markedly alters outcomes in settings such as allograft rejection, tumor growth, and bacterial infection. Finally, several epidemiologic studies suggest that allogeneic transfusions are associated with a two- to fourfold increase in lymphomas (and perhaps other tumors) over the 5 to 10 years after transfusion.
PROSPECTIVE AND INTERVENTIONAL STUDIES Can the Postulated Effects of Transfusion lmmunomodulation be Ameliorated by Use of Autologous Transfusions or Leukocyte Depletion of Allogeneic Transfusions? One must be cautious about drawing definitive conclusions about clinical phenomena from retrospective epidemiologic studies and animal models. Animal experiments cannot precisely model the clinical setting and epidemiologic associations, however credible, are subject to both bias and confounding. Thus it was of great interest when Paul Tatter’s prospective study of the allogeneic transfusion effect on
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colorectal cancer recurrence demonstrated once again a dramatic association between transfusion and poor outcome.” If Tartter’s results are explained by confounding, transfusion must be confounded with a variable that is currently unknown, because the clinical conditions that lead to transfusion, such as anemia and blood loss, clearly did not explain the association. Allogeneic transfusion remains absolutely essential for many types of medical and surgical therapy. Autologous transfusion is not feasible for most patients. Thus attention has focused on a technical innovation, leukocyte depletion, as a potent.ial answer to the scientific and clinical questions surrounding the transfusion immunomodulation theory. If a modification of allogeneic transfusions could be shown in a randomized trial to improve clinical outcomes, it would have the additional benefit of proving once and for all the most general tenets of the transfusion immunomodulation theory. As noted previously, studies of post.operative infection and colorectal cancer recurrence in patients randomized to receive autologous or allogeneic transfusions’, and of postoperative infection in patients randomized to receive leukocyte depleted or unmodified whole blood” have provided convincing evidence that these strategies can provide clinical benefits, at least to patients undergoing colorectal surgery in Munich or Aarhus. However, multicenter randomized tr:ials of autologous blood transfusion from Rotterdam’ and leukocyte depleted blood from Leiden* did not detect significant benefits to either strateg!y in colorectal cancer patients. The reasons for the strikingly different results in the two pairs of studies are not obvious, and it does not appear that fatal methodologic flaws ex.ist in any of them. The effects of transfusion are almost certainly superimposed on profound effects od surgery and anesthesia, which in turn likely vary with surgical and anesthetic technique, and other drugs administered.’ For example, laparoscopic surgery is probably less suppressive of cellular immune responses than laparotomy. Techniques and drugs used vary dramatically from center to center. We hypothesize that because the two smaller, single-center studies demonstrated benefits, and the two larger multicenter studies found no such difference, this difference in study design may have been crucial. If this hypothesis is correct, then the variations in clinical practices inherent in studies employing 15 to 20 centers, each treating a few dozen patients, may have created sufficient “background noise“ to obscure any potential benefit from altered transfusion practice in the multicenter studies. In a single center, with a handful of surgeons and rlelatively
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Estimates of Allogeneic
Transfusion lmmunomodulation Related Deaths in the USA Due to Cancer Recurrence and Postoperative Infection Cancer* Infection+ Tot& Deaths Death Rate per Deaths Death Rate per Deaths Death Rate per per Year lo6 Transfusions per Year lo6 Transfusions per Year lo6 Transfusions 20,000 33,000 1,500 250 21,500 33,250 10,000 16,667 750 125 10,750 16,892 2,000 3,300 150 25 2,150 3,325 200 330 15 2.5 215 332.5
Estimated Proportion of the Effect that is Causal
100% 50%
10% 1%
* Assumptions (Cancer Recurrence and Transfusion): (1) A 10% difference in death rates between transfused and non-transfused patients. per patlent = 3. (3) 200,000 transfused cancer patients per year out of approximately l,OOO,OOO total cancer patients. + Assumptions (Postoperative Infection and Transfusion): (1) A 15% difference in infection rates between transfused and non-transfused Mean transfusions per patient = 3. (3) 6,000,OOO transfusions to surgical patients per year. (4) Mortality due to postoperatwe infection infections. ’ For Comparison: Roger Dodd estimates that the number csf deaths due to HIV-1 infection from transfusion IS currently approximately transfused or about 30 per year total. The corresponding rwmbers for hepatitis C are a fatality rate of 0.75 to 6.63 per million units or per year total. These data include only red blood cell transfusions.
(2) Mean transfusions surgical patients. (2) averages 1 in 200 2.38 per million units about 9 to 80 deaths
consistent use of other drugs and surgical tech- data from our own studies and those of John Ward niques, the benefit predicted from animal models and colleagues at the Centers for Disease Control, may have been more readily detected because the E.C. Vamvakas and Harold Kaplan at New York Unibackground effects of surgery and anesthesia were versity, Elaine Sloand and Harvey Klein al; NIH, and more uniform. laboratory data from Michael Busch at the Irwin The variability in the results of these four ran- Blood Center suggest that transfused HIV-1 infected domized trials has led to confusion and uncer- patients have more rapidly progressive di.seaseand tainty. Some have argued that the failure of two of greater numbers of complications than those receivthe trials to produce the expected effect means ing no transfusions or lower doses of transfusion. that transfusion is probably immunologically be- Based upon these observations, an NIH-fu.nded mulnign. We consider this conclusion untenable given ticenter study is underway to determine if leukocyte the large body of epidemiologic, animal and clinidepletion of transfusions to HIV-1 infected patients cal interventional data that suggests the contrary provides clinical benefits. in a wide variety of disease settings. Furthermore, THE POTENTIAL PUBLIC HEALTH the control arm in three of the four interventional st,udies involved transfusions of buffy coat and IMPACT OF TRANSFUSION plasma depleted red cells. This undoubtedly mini- IMMUNOMODULATION mized the ability to demonstrate a benefit in the Recently, extensive attention has been paid to the experimental arm employing leukodepletion or au- potential mortality associated with better known and tologous transfusion, given the substantial data on accepted risks of allogeneic transfusion. Roger Dodd the role of white cells and stored plasma in both at the American Red Cross Holland Labalratory has clinical and animal studies. estimated that mortality caused by all known infecOt,her studies of leukocyte depletion in surgical tious and alloimmunization transfusion risks, other settings are underway or planned, but we believe than bacterial contamination of platelets, amounts to that studies in medical patients (eg, with gastroin- perhaps 29 to 103 deaths per million units transtestinal bleeding) would also be of great use. Most fused. No attempt has yet been made to assessthe public health implications of the transfusion immuallogeneic transfusions given to medical or pediatric patients are already being leukocyte depleted to pre- nomodulation theory. It has been argued that even vent febrile transfusion reactions, platelet transfu- if the theory is correct, the effects are unlikely to be sion refractoriness or cytomegalovirus transmission large or of much public health significalnce. Howin patients with diseasessuch as thalassemia, sickle ever, postoperative infections are not benign and cell anemia, leukemia and lymphoma. Data from Kal- have a measurable mortality associated with them. evi Oksanen and colleagues and our own group sug- Likewise, earlier death due to tumor recurrence may gest that at least in leukemia and lymphoma, leuko- not be a negligible problem. We performed a theoretical analysis of the potential mortality due to cyte depleted transfusions are associated with decreases in morbidity, mortality, and costs but postoperative infection and cancer recurrence as a these results are not derived from randomized trials consequence of allogeneic transfusion immunomodulation. These results are shown in Table II. and must be viewed as preliminary. Epidemiologic 306
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IMMODULATION
The assumptions in the table are all based upon estimates reported in the literature. Reported rates of increased postoperative infections associated with allogeneic transfusion(s) range from a few percent to as high as 20% to 30%. The average increase is about 15%. As can be seen from Table II, even if only a small portion of the association between postoperative infection and transfusion is causal, mortality from transfusion immunomodulation induced postoperative infections might still be of equal significance to that from any other single cause. Furthermore, based upon work by Lone Jensen and colleagues, it appears that leukodepletion of allogeneic blood can in some instances completely abrogate the postoperative bacterial infection promoting effects of allogeneic transfusions. Employing the assumptions in the table, and Jensen’s and our data that each allogeneic transfusion is associated with $1,000 to $2,000 in postoperative costs, it is theoretically possible that expending $240 million to leukocyte deplete the 6 million units of blood used in surgery each year could save between $6 and $12 billion dollars in costs, while reducing the number of postoperative infections and deaths by an order of magnitude. We believe these are reasonable speculations, as our personal estimate is that >90% of the association between allogeneic transfusion and postoperative infection is causal. If the transfusion immunomodulation theory is correct and increased rates of earlier cancer recurrence approach 10 to 20% in allogeneically transfused patients, this could dramatically increase the importance of immunomodulation as a public health issue. In transfused colorectal cancer patients the mean increased mortality is about 10%and the mean increase in recurrence rate is 21% as compared with untransfused patients. As is displayed in Table II, the rate of death due to earlier cancer recurrence using these estimates is 3 to 4 orders of magnitude greater than any other transfusion associated mortality risk. Even if transfusion immunomodulation causally accounts for only 1%to 2% of the association observed between allogeneic transfusion and cancer deaths, immunomodulation-induced tumor recurrence still would be one of most important causes of transfusion mortality. If as little as 10% of the association bet,ween allogeneic transfusion and increased postoperative infection and cancer recurrence is causal, the number of deaths annually clue to these causes would be 2,150. We believe both these estimates to be well below the likely actual risk. By comparison with an estimated several thousand transfusion immunomodulation deaths per year, the number of remaining deaths due to HIV is estimated at under 50/year. September
BY BLOOD TRANSFUSION/BLUMBERG
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Therefore even if only 1% of the association observed between transfusion and earlier death due to cancer or infection is causal, transfusion deaths due to immunomodulation would outnumber HIV by a factor of 4 or more. One major uncertainty in assessing the public health impact of the transfusion immunomodulation theory is that while it is clear that transfusion in the surgical setting is associated with increased morbidity, little is known of the potential effects of allogeneic transfusions in non-surgical patients. There is evidence that transfusion immunomodulation may increase morbidity in patients receiving myeloablative chemotherapy for leukemia and lymphoma, but almost nothing is known about potential effects in the treatment of chronic anemia or bleeding. Such studies are urgently needed. These issues are of sufficient potential public health importance that we suggest that further investigation of the transfusion immunomodulation theory is the single most pressing issue in blood transfusion today from both scientific and clinical standpoints. We believe the potential for improving clinical outcome by use of techniques that minimize allogeneic blood use or its immunologic effects, such autologous transfusion and ot.her blood sparing approaches in surgery, leukodepletion of allogeneic blood, and growth factors such as erythropoietin warrant intensive investigation. In addition, insight into the effects of transfusion on immune function may serve as a model for investigations into the potential effects of ‘the many drugs and biologic agents whose effects on the immune system have not yet been adequately addressed.
ACKNOWLEDGMENT The format of this editorial review did not permit comprehensive citation of all relevant work and we wish to thank both the mentioned and unattributed Investigators whose work has contributed to our understanding of transfuston immunology. We thank Drs. John Leddy and R. John Looney for helpful discussions.
REFERENCES 1. Blumberg cer recurrence
N, Heal JM. Effects of transfusion and infectlon. Arch Pathol
Lab
on immune function, Med. 1994;118:371-
can-
379. 2. Lowry RP, Takeuchi T. The Thl, Th.2 paradigm and transplantation tolerance. Austin, TX: R.G. Landes 1994. 3. Blajchman MA, Bardossy L, Carmen R, Sastry A, Singal DP. Allogeneic blood transfusion-induced enhancement of tumor growth: two animal models showing amelioration
by leukodepletion
and passive
transfer
using
spleen
cells.
Blood.
1993;81:1880-1882. 4. Heiss MM, Mempel W, Jauch K-W, Delanoff C, Mayer G, Mempel M. Eissner H-J, Schildberg F-W. Beneficial effect of autologous blood transfusion on Infectious complications after colorectal cancer surgery. Lancet. 1993;342:13281333. 5. Jensen Mortensen
1996
LS, Andersen AJ, Christiansen PM, Hokland J, Moller-Nielsen C, Hanberg-Sorensen
The
American
Journal
of Medicine”
P, Juhl CO, Madsen F, Hokland
Volume
101
G, M.
307
Postoperative infectron in patrents undergoing 516.
and natural killer cell funchon elective colorectal surgery.
6. Tartter PI. The association of perioperative blood cancer recurrence. Ann Surg. 1992;216:633-638, 7. Busch ORC, Hop WCJ, Hoynck van Papendrecht J. Blood
transfusions
and
prognosis
in colorectal
following blood transfusion Br J Surg. 1992;79:513-
8. Houbiers JGA, Brand PJM, Bijnen AB, Pahlplatz Klementschitsch P, Van
transfusion
controlled coat-depleted
with colorectal
MAW, Marquet cancer.
RL, Jeekel
N Engl
J Med.
1993;328:1372-1376.
308
September
1996
The
American
Journal
of Medicine@
Volume
trial
A, Van de Watering LMG, Hermans J, Verwey P, Schattenkerk ME, Wobbes T, De Vrres JE, de Maas AHM, Van de Velde CJH. Randomrsed
comparing blood in
transfusion surgery
for
of leucocyte-depleted colorectal cancer.
or buffyLancet.
1994;344:573-578. 9. Blumberg in progress?
101
N, Heal JM. Transfusion
and the immune
Transfusion. 1995;35:879-883.
system:
a paradigm
shaft