Transfusion-Related Immunomodulation

Chapter 52

Transfusion-Related Immunomodulation Neil Blumberg

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Joanna M. Heal

INTRODUCTION In recent years, a more conservative approach to blood transfusion has begun to become the rule rather than the exception in clinical medicine. To a significant degree, this is the result of clinical outcomes research demonstrating that patients receiving traditional allogeneic transfusions (nonleukoreduced, nonautologous red cells) have dramatically higher rates of morbidity and mortality than do similar patients not receiving transfusions. When patients receiving a traditional therapy are shown to do much worse than those not receiving the therapy, an understandable controversy and growing reluctance exist to use that therapy. To an unknown but probably substantial degree, these inferior clinical outcomes are mediated by a complex phenomenon that has been termed transfusion-related immunomodulation or TRIM. Paradoxically, TRIM is now known to involve both downregulated cellular immunity and upregulated, inappropriate inflammatory responses. Transfusion has long been known to affect the immune system. Humoral immunization to red cell alloantigens after transfusion was recognized as a cause of hemolytic disease of the newborn and hemolytic transfusion reactions in the early 20th century. Over the last 20 years, it has become apparent that transfusion also produces clinically significant immunomodulatory effects on cellular immunity.1,2 Modulation of cellular immunity by allogeneic transfusion may be of importance equal to or greater than that of humoral allosensitization in certain clinical settings, specifically oncology, surgery, and critical care.1,2 Unfortunately, recent reviews of transfusion immunology often fail to address this subject because of its contentious nature, and, perhaps, a reluctance to challenge long-standing clinical practices that have been in use for most of a century.1–7 The insight that transfusions modulate cellular immunity originated with observations in the late 1960s and early 1970s that blood transfusions reduced the rejection rate for cadaveric renal allografts.8 A decade later, the controversial observation was made that transfusion at the time of cancer surgery is associated with increased tumor recurrence.9,10 Tartter and colleagues11 also reported that the incidence of postoperative infections increased in a dose-dependent fashion after perioperative transfusions. Viral infection and autoimmune disorders may be modulated by transfusion,1 and a modest but reproducible benefit of transfusions is found in women with repetitive spontaneous abortion during pregnancy.12 Finally, animal models13,14 and some, but not all, randomized clinical trials15–18 demonstrate that leukoreduction and autologous transfusion19 can reduce the immunomodulatory effects of

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transfusion and minimize resulting morbidity and mortality. Many detailed reviews of TRIM have been published, and the reader is referred to these for summaries of the historical literature.1,3–7

TRANSFUSION AND HUMORAL ALLOSENSITIZATION It has long been known that transfusion or pregnancy can cause formation of clinically significant antibodies to cellular and plasma components of allogeneic blood. Most bloodtransfusion research in the first part of the 20th century was focused on techniques for detecting antibody formation and avoiding transfusion of incompatible red cells. Until the late 1960s and early 1970s, the accepted paradigm was that transfusion stimulated the recipient’s immune system to make humoral immune responses, and that this was virtually the only immunologic complication of transfusions. Two decades later, it became clear that some antigens, when given intravenously in high dose, can promote a type 2 (primarily humoral) immune response while downregulating type 1 (primarily cellular) responses.20–22 Approximately 1% to 5% of individuals receiving ABO and Rh(D) identical transfusions or who become pregnant with an Rh-compatible fetus will mount a humoral immune response to red cell alloantigens. About 50% of patients transfused with cellular blood components or who become pregnant will mount a humoral immune response to human leukocyte antigen (HLA)-A,B antigens present on white blood cells (WBCs). Interestingly, in experimental models when antigens are administered at higher doses, in the absence of costimulatory inflammatory “danger” signals, immune tolerance rather than immunization can occur.23 By 1990, leukoreduction was found to abrogate posttransfusion humoral responses to white cell antigens (primarily antibodies to HLA class I). Filtering out most (at least 99.9%) of the donor white cells before transfusion is effective at preventing 90% or more of HLA antibody sensitizations in patients undergoing myeloablative chemotherapy and transfusion.24 Because HLA antibody formation is the primary mechanism of alloimmune platelet transfusion refractoriness, as platelets carry class I antigens, the incidence of unresponsiveness to platelet transfusions due to allosensitization decreased by an order of magnitude, from approximately 50% to 5% or less, after the introduction of filtered, white cell–reduced transfusions.25 It is possible that leukocyte removal by filtration also decreases the allosensitization rate to red cell alloantigens.26 One plausible mechanism is that white cell removal decreases the type 2 immune

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stimulus that accompanies transfusion of allogeneic white cells. Purified red cells and platelets are known to be less immunogenic than allogeneic white cells. In the absence of allogeneic WBCs, purified red cell components in particular may be less likely to provide inflammatory mediators and costimulatory molecules that activate the recipient’s immune system.

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More than half a century ago, Billingham, Brent, and Medawar27 demonstrated that administration of allogeneic antigen to fetal animals resulted in immunologic tolerance. The science, if not the clinical art, of solid organ transplantation had begun. The initial evidence that blood transfusions affect recipient immunity beyond humoral alloimmunity came from clinical observations in patients receiving cadaveric renal allografts.8,28 During the early history of renal transplantation, acute graft rejection during surgery was observed in alloimmunized recipients. This phenomenon was associated with prior transfusions, which, in the pre-erythropoietin era, were the only treatment for the anemia of renal failure. Some investigators were modestly successful at reducing alloimmunization in these patients through leukoreduction before transfusion. At that time, the sole available method of achieving leukoreduction was the use of washed or previously cryopreserved red cells, which were leukocyte reduced by about 80% to 90%, as well as plasma and platelet reduced. However, other investigators made a startling finding, which was received with substantial skepticism. They observed that transfused patients actually experienced overall improved renal allograft survival compared with nontransfused patients. This effect was later shown to be dependent on the transfusion dose and was not observed in those patients receiving leukocyte and plasma reduced blood transfusions (Fig. 52–1).8,28

One year graft survival rate

80 67

62

60

51 41

40 20

Whole blood

Red cells

Washed Frozen/ red cells thawed red cells

Blood component transfused Figure 52–1 One-year kidney allograft survival in recipients of 1–5 units of allogeneic blood of the type listed is shown as a percentage of total patients with transplants. Transfusions with reduced content of allogeneic white cells, platelets, and stored supernatant plasma are associated with inferior 1-year graft-survival rates. (Data from the UCLA registry. Horimi T, Terasaki PI, Chia D, Sasaki N. Factors influencing the paradoxical effect of transfusions on kidney transplants. Transplantation 1983;35:320–323.)

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TRANSFUSION AND AUTOIMMUNE DISEASES Transfusion appears to affect favorably some autoimmune diseases thought to be mediated by cellular immunity. These include Crohn’s disease (regional enteritis)35 and rheumatoid arthritis.36 In Crohn’s disease, patients with larger sections of inflamed bowel and more-severe disease are more likely to be transfused during surgery for removal of diseased small bowel. Despite these higher risk factors for poor outcomes, transfused patients are actually less likely, or no more likely to have recurrences of their Crohn’s disease than are nontransfused patients who have less-extensive and aggressive disease.37 One potential explanation for these findings is a downregulation of the type 1 inflammatory process by allogeneic transfusion. Similarly, rheumatoid arthritis is thought to be mediated in part by type 1 immunity and inflammation. In pilot studies, disease activity has been favorably influenced by transfusion of allogeneic white cells38 or whole blood.36

TRANSFUSION AND REPETITIVE SPONTANEOUS ABORTION

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0

Animal models supported the critical role of allogeneic white cells in mediating this “tolerogenic” result, whereas other studies29 suggested that a similar effect could be mediated by platelet transfusions. This is of particular interest in light of recent findings that platelets and platelet-derived CD40L can stimulate secretion of prostaglandin E2 (PGE2) and other downregulators of cellular immunity.30,31 Blood transfusions also appear to decrease rejection rates after heart32 and liver33 transplants. Donorspecific transfusions are remarkably effective at preventing rejection in animal models but are infrequently used for patients now that immunosuppressive drugs are highly effective at abrogating allograft rejection. A recent report found that the number of acute rejection episodes was inversely related to the number of blood transfusions in cardiac transplant recipients, most of whom received nonleukoreduced transfusions.34

One of the earliest observations regarding TRIM was that paternal or other unrelated blood transfusions improve the likelihood that women with repetitive spontaneous abortions will carry a pregnancy to term.12 The clinical benefit is modest, and varied transfusion preparations yield different degrees of efficacy, but the effect is reproducible. This observation, although not of major clinical significance, is particularly intriguing, as successful pregnancy, similar to allogeneic TRIM, results in type 2 immune deviation, with increased expression of cytokines such as interleukin (IL)4, IL-5, and IL-10.39,40 Pregnancy is characterized by slightly increased risks from intracellular infectious organisms and tumors, presumably because of the mildly impaired type 1 immune defenses needed for optimal immune surveillance. One potential mechanism for the beneficial effects of transfusion in this setting is that allogeneic transfusions may downregulate pathologic type 1 immune responses that contribute to spontaneous abortion.40 Another parallel between the immunology of transfusion and pregnancy is the high frequency of alloimmunization and

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The allograft tolerizing effects of blood transfusion, by analogy with the drugs used to prevent rejection, was termed an immunosuppressive effect.41 Could this effect, beneficial in organ transplant recipients and patients with autoimmune disease or repetitive spontaneous abortions, lead to unfavorable outcomes in clinical situations in which intact recipient cellular immunity is needed? Would patients undergoing surgery or those with cancer experience negative outcomes from “transfusion immunosuppression?” In the late 1970s, a group from Newcastle, England, observed that animals with tumors had more rapid tumor growth if transfused with allogeneic as opposed to syngeneic blood.42 The clinical analogy would be the use of allogeneic (homologous) versus autologous transfusions. In 1982, Burrows and Tartter9 reported that patients in a colorectal cancer trial had earlier and more frequent recurrences of their cancers if they were transfused at the initial surgical resection. Similar observations were made for most other surgically treatable tumors, although about one third of the studies did not achieve statistical significance despite uniformly poorer outcomes in the transfused patients.43,44 One possible explanation for the association between allogeneic transfusion and earlier tumor recurrence is simply that the need for transfusion is confounded by size or aggressiveness of the tumor. Patients who require transfusions may do so because they tend to have larger, more aggressive, and difficult-to-resect tumors. This confounding certainly explains some of the association. However, a number of studies observed that even in patients closely matched for prognostic factors, transfused patients had more frequent or earlier cancer recurrences.45,46 In addition, transfusion practice is quite variable in almost all clinical settings, due to a lack of accepted, uniform criteria for commencing transfusion therapy. Although transfusion therapy is not given randomly, neither is it given in consistent fashion driven by evidence-based transfusion triggers. Thus it would have been surprising if the two- to threefold difference seen in tumor recurrence is completely due to confounding. In epidemiologic investigations, two- to threefold differences in outcomes usually prove to be causal relations, at least to some extent.47,48 When these observations appeared in the 1980s, the hypothesis that the immune system was involved in cancer outcome was falling out of favor. Failures of immune-based therapies in patients, such as interferon and bacille CalmetteGuérin (BCG) vaccination, which had efficacy in animal models, were largely responsible.49,50 Also, immune responses to human tumors were rarely demonstrable. The possibility that “successful” tumors can evade and/or repress host immune responses had not yet been proposed or proven.51,52 Thus the initial observations of a relation between perioperative transfusion and cancer recurrence were met with intense

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1.0

0.9 Proportion surviving

TRANSFUSION AND CANCER RECURRENCE

skepticism, much as was the case for the initial studies of the allograft-enhancing effects of blood transfusions. Nonetheless, several investigative findings suggest that the association between blood transfusion and cancer recurrence is causal. First, as in the renal transplant data, the type of transfusion affects the likelihood of recurrence. Whole blood transfusions are associated with greater cancer recurrence rates than are equivalent numbers of red cell transfusions (which are partially depleted of plasma, white cells, and platelets) (Figs. 52–2 and 52–3).53–55 Second, two animal models demonstrated that removal of allogeneic leukocytes reduced the number of lung metastases seen after transfusions.13 Third, autologous transfusions reduced the recurrence rate in patients undergoing colorectal cancer resection, as compared with patients receiving allogeneic transfusions, in one randomized trial56 but not in another.57 The single study that examined leukocyte reduction of transfusions as a means of reducing adverse immunomodulatory effects showed no benefit,58 and that was supported by results of another recent report.59 Regrettably, few trials of leukocyte reduction, washing, autologous transfusions, or bloodless medicine and surgery techniques have been published in oncologic surgery. These are extraordinarily challenging and expensive trials to perform because they require adherence to complex protocols, large numbers of patients, and, in particular, long-term follow-up. The one existing trial of washed, leukocyte-reduced transfusions in patients with cancer demonstrated a possible benefit in younger adults with acute leukemia.60 However, this was a small pilot study, and clear-cut benefit was seen only in subgroup analysis, increasing the chances of selection bias and confounding. Fortunately, a trend exists toward decreasing use of transfusions in cancer surgery,17 largely due to the concerns about

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humoral immune responses, precisely as would be expected in clinical settings in which type 2 immune deviation is present. Relevant to this hypothesis, red cell transfusions that are leukocyte reduced may be less likely to result in alloimmunization, not only to WBC antigens, but to red cell alloantigens as well.26 Leukoreduced transfusions may provoke less type 2 immune deviation than do unmodified transfusions.

0.8

0.7

0.6

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2000

4000 Days

No transfusions (n = 130)

Whole blood ≤3 units (n = 58)

Figure 52–2 Kaplan-Meier plot of the proportion of patients remaining alive after initial surgical treatment for colorectal, cervical, or prostate cancer who received either no transfusions or ≤3 units of blood, at least one of which was whole blood. The two curves are statistically significantly different (P < 0.001), with the nontransfused patients having estimated mortality at 5 years of <15%, as compared with almost 40% in those receiving whole blood transfusions.53,54

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Post-operative infection rate (%) or lung cancer incidence/18,000 population

0.9 Proportion surviving

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1.0

0.8

0.7

40

30

20

10

0 None

0.6

Low dose

Medium dose

High dose

Exposure dose Smoking

0.5 0

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4000 Days

No transfusions (n = 130)

≤ 3 units of red blood cells (n = 44)

Figure 52–3 Kaplan-Meier plot of the proportion of patients remaining alive after initial surgical treatment for colorectal, cervical, or prostate cancer who received either no transfusions or ≤3 units of red cell concentrates. The two curves are not statistically significantly different.53,54

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transfusion-transmitted human immunodeficiency virus (HIV) and hepatitis C. It is unknown whether this change in practice has contributed to decreases in recurrence rates in patients with surgically resectable tumors. With the implementation of universal leukoreduction of transfusions throughout much of the developed world, the possibility exists for before and after studies of cancer recurrence, but none has yet been published. When a 5- to 10-year follow-up period is available for patients treated after the introduction of universal leukoreduction, it may be possible to determine whether leukoreduced transfusions are less immunomodulatory in cancer surgery patients than unmodified transfusions. Our speculation is that both leukocyte reduced and washed transfusions may be needed to maximally benefit transfused cancer patients.

TRANSFUSION, POSTOPERATIVE INFECTION, AND MULTIORGAN FAILURE Transfusions are associated with a dramatically increased risk of postoperative bacterial infection, as first reported by Paul Tartter11,61 20 years ago. Preoperative, intraoperative, or postoperative transfusion leads to a severalfold increase in the proportion of patients who develop an infection. This association has been confirmed in many other studies62 and cannot be explained by confounding variables related to transfusion such as anemia, blood loss during surgery, duration of surgery, or hypotension. The quantitative strength of the association is striking. Patients who are transfused with more than 10 units of allogeneic blood experience a 5- to 10-fold increase in their likelihood of developing a postoperative infection.63 This effect is observed in diverse surgical settings, and among studied causal relations is second in magnitude only to the association between smoking and lung cancer (Fig. 52–4). Thus the association between trans-

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Allogeneic transfusions

Figure 52–4 The relative risk of postoperative infection according to number of allogeneic transfusions68,69 is compared with the relative risk of lung cancer according to number of cigarettes smoked.154 The 5- to 10-fold increase in postoperative infections with increasing number of allogeneic transfusions, seen in many studies, which is in part abrogated by leukoreduced or autologous transfusions, provides prima facie evidence for a cause-and-effect relation according to Hill’s principles of causation (including dose relation, size of the effect, underlying plausible mechanism, consistency across studies, and benefits of reducing the exposure).47,48

fusions and postoperative infections is extremely unlikely to be explained by the bias and confounding that are limitations of most observational studies.47,48 It has been suggested that the effects of multiple confounding factors and a surrogacy role for transfusions might explain this association.64 This hypothesis is quite improbable because of the extraordinarily variable and unsystematic way in which transfusion decisions are made.65 A dose-dependent 5- to 10fold increase in postoperative infections after allogeneic transfusion solely accounted for by confounding is extremely remote and would be unprecedented in clinical epidemiology.47,48 One limitation of published studies is that infection is often defined solely as culture-proven infections. This greatly underestimates the incidence of infectious morbidity, given the insensitivity of most culture techniques. This is particularly the case in surgical patients, who invariably receive prophylactic antibiotic therapy. It is preferable also to estimate infections by multiple surrogate measures including days of antibiotic treatment, length of hospital stay, and signs, laboratory tests, and symptoms of infection. Less comprehensive approaches are likely to underestimate infectious morbidity significantly. As discussed previously in this chapter, one additional body of evidence that contradicts the “transfusion as a surrogate marker for confounding variables” hypothesis is that perioperative transfusions are associated with better rather than worse clinical outcomes in patients receiving renal allografts28 or in those with inflammatory bowel disease.35 Were transfusion to be acting purely as a surrogate measure of clinically unfavorable comorbidities, these clinically favorable associations would not exist. To the contrary, as is discussed further, these altered outcomes support a common immunologic mechanism for both improved and poorer clinical outcomes in different clinical settings. Although some analyses that included many unrelated confounders did not find a significant association between transfusion and infection,66 these types of

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Percent with infections

50 40

40 30

25

20 10

7 4

5 0

0 1

2 Units transfused Autologous

3

Allogeneic

Figure 52–5 Patients undergoing posterior or anterior, cervical or lumbar spinal fusion, or primary hip replacement surgery, without malignancy, autoimmune diseases, or diabetes were grouped according to whether they had received only autologous blood or only allogeneic (homologous) blood. The proportion with culture-proven or clinically evident infections, grouped according to number of units of blood transfused, is shown. The differences between autologous and homologous recipients are statistically significant at doses of 2 or 3 units, but not for 1 unit of blood. This dose-response relation also was evident when days of antibiotics and length of hospital stay were measured. Infections were predominantly away from the wound site: two thirds were cellulitis, urinary tract, or pulmonary. Data are pooled from two studies.68,69 (Reprinted from Anderson KE, Ness PM [eds]. Scientific Basis of Transfusion Medicine. Elsevier, 2000, p 437, with permission.)

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the incidence of indwelling vascular-access catheter sepsis in a variety of nonsurgical settings by perhaps 30% to 40%.74 The association between transfusion and postoperative infection is also reflected in observations of a dosedependent increase in multiorgan failure in transfused surgical patients.75–77 Multiorgan failure is often initiated by infection in seriously ill patients. This association is not explained by confounding factors and has been detected in randomized clinical trials as well. Multiorgan failure and death after cardiac surgery appears to be reduced in patients receiving leukoreduced transfusions.18 Platelet transfusions also have been associated with a fivefold increase in postoperative deaths after cardiac surgery, after adjustment for other risk factors.78 Such a large effect is unlikely to be explained primarily by confounding.47,48 The association between platelet transfusion and mortality may be partially abrogated in patients receiving postoperative aspirin.79 Whether the association between platelet transfusion and mortality is mediated by a predisposition to infection due to immunomodulatory effects of transfusions,18,80 a prothrombotic effect of storage activated platelets being transfused,81–83 exacerbation of inflammatory responses from transfused platelet CD40L and sCD40L,30 other bioactive molecules or cells, or other mechanisms, will require further investigation. Wound healing may also be impaired after allogeneic transfusions.84–86 Both randomized trials and before-andafter studies suggest that leukoreduction of transfusions may reduce the risk of postoperative multiorgan failure18 and improve wound healing.87 It is unknown whether storage time of transfused blood influences these complications, but this seems possible.88–90 Recent evidence suggests that postoperative morbidity and death due to immunologic mechanisms may also be increased by transfusion of ABOmismatched blood components containing incompatible antigen or antibody.91–93

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analyses are neither statistically nor scientifically sound.67 Only confounders directly and causally linked to both transfusion and infection are appropriately included in statistical models. Introduction of multiple, causally unrelated, and irrelevant variables can lead to erroneous acceptance of the null hypothesis. A number of animal models confirm that the association of allogeneic transfusion with postoperative infection is most likely cause and effect. Alexander and colleagues14 demonstrated in mice that allogeneic white cells were more potent in mediating this effect than were red cells or plasma. Our group and many others observed that the association between transfusion and postoperative infection is not seen in patients receiving autologous blood, supporting an immunologic mechanism for the effect (Fig. 52–5).68,69 The best evidence for a causal relation between allogeneic transfusion and an increased risk of postoperative infection comes from randomized clinical trials of leukoreduced allogeneic transfusions versus unmodified blood transfusions, as well as studies of autologous versus allogeneic transfusions. These studies, discussed in detail further on in this chapter, demonstrate beyond a reasonable doubt that the incidence of postoperative infection, and probably that of multiorgan failure, in transfused surgical patients can be reduced by use of leukoreduced blood transfusions15–18,58,70–73 or the use of autologous techniques.19 In general, a 10% absolute reduction in postoperative infection incidence is possible by using leukoreduced transfusions compared with unmodified red cells. This means that for every 10 patients treated with leukoreduced transfusions, one less postoperative infection will occur as compared with patients treated with nonleukoreduced transfusions. Recent evidence from our own center suggests that leukoreduction of blood transfusions may also reduce

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TRANSFUSION AND ANTIVIRAL IMMUNITY Given the association between transfusion and postoperative bacterial infection, it was hypothesized that allogeneic transfusions might also impair host resistance to viral infection. Epidemiologic evidence suggests that transfusion accelerates the appearance of clinical manifestations of HIV-194 and cytomegalovirus (CMV) infection.95 However, in end-stage HIV-1 infection, a randomized trial of leukoreduced transfusions failed to identify any immunomodulatory effects that promote viral replication or shorten survival.96 With regard to this last trial, it has become evident in TRIM studies that multicenter studies, which typically involve small numbers of patients at large numbers of hospitals, may be less able than single-center studies to detect improved outcomes because of substantial variations in clinical practices that influence infection or survival. Viral infections play a role in lymphoma in both animal models and humans, and prior allogeneic transfusion has been associated with the development of non-Hodgkin’s lymphoma, specifically the B-cell variants.97 Not all studies have found this association. If the association is causal, it is unclear whether viral transmission or TRIM is primarily responsible, but either or both mechanisms could be involved. TRIM drives B-cell proliferation and maturation

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through promotion of type 2 (Th2) immunity and downregulates type 1 (Th1) immune functions that are thought to be relevant in antiviral immunity and tumor surveillance (Fig. 52–6). Thus although the hypothesized potential of transfusion to impair host defenses to viral infection is plausible, in that transfusion downregulates natural killer cell and macrophage/monocyte cytocidal functions and promotes Th2 immune deviation, no definitive conclusions are possible, given the modest data at hand.

MECHANISMS OF TRANSFUSION IMMUNOMODULATION

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Transfusion involves intravenous infusion of large doses of blood cells, proteins, lipids, and other blood constituents diluted with preservative anticoagulant solution, as well as the breakdown products that result from storage lesions. The only natural circumstances in which the immune system is exposed to such high doses of intravenous antigen are self-antigens and pregnancy.40,98 The immune system has evolved to respond to these two situations with unresponsiveness or tolerance and to provide defense against dangerous foreign antigens that are non-self.23 Pathogens are microorganisms, present in small quantities, at mucosal or skin locations, or altered self (i.e., cancer cells in small numbers), largely in organs. It stands to reason that large quantities of intravenous antigen containing both self and non-self determinants leads to unresponsiveness rather than allosensitization. In particular, this appears to be the case when the clinical setting is that of decreased host immune function (e.g., the immaturity of fetal life, surgical stress). The classic investigations of Medawar and colleagues27 proved that exposure to foreign antigens during fetal life can produce tolerance to skin grafts from the tissue donor.

Felton99 reported that when pneumococcal antigen was administered at high doses, animals have reduced capabilities for subsequent humoral immune responses to antigen administered by routes and in doses that otherwise cause sensitization (“immune paralysis”). In 1951, Snell and Kaliss100 demonstrated that infusions of tissue extracts from allogeneic murine donors before tumor implantation led to accelerated tumor growth. Even serum transfusion before implantation led to the death of most of the mice from tumor progression. Saline-transfused control animals uniformly rejected the implanted tumor and survived. Interestingly, of all the tissue extracts transfused, infusions of lyophilized red cells were the only ones that failed to enhance tumor growth. Whereas most studies have focused on the role of transfused allogeneic white cells and their secreted mediators or breakdown products,101 clinical data suggest that stored supernatant plasma may also be immunomodulatory.94 In addition, recent investigations confirm that platelets are a source of immunomodulatory mediators, such as vascular endothelial growth factor (VEGF), soluble CD40L, and transforming growth factor-β1 (TGF-β1).30,31,102–105 In addition to platelet components, platelet-derived mediators such as soluble CD40L are also present at high concentrations in nonleukoreduced red cell concentrates and whole blood. Allogeneic transfusions in animals result in diminished mixed lymphocyte culture reactivity, diminished antigen processing by macrophages, upregulation of both suppressor/regulatory cells and humoral immunosuppressive mediators, impaired cell killing, and production of anti-idiotypic antibodies that are immunomodulatory in vitro.106,107 These findings indicate that impaired or dysregulated cellular immunity is likely a key mechanism underlying TRIM. Many investigations report that the presence or absence of allogeneic WBCs in the transfusion is critical to observing immunomodulatory effects, but plasma, red cells, and even purified, soluble class I

B cell

B cell Antibody synthesis

Cytotoxic T cell activation (DTH response), NK cell activation

Antibody synthesis

IL-2; IL12 γ-interferon

T cell

T cell TC R

R

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IL-4; IL5; IL6; IL10

Cytotoxic T cell (DTH response)

Antigen

MHC

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T helper type 1 response

Antigen

MHC

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T helper type 2 response

Figure 52–6 A 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 Th1(type 1) or Th2 (type 2) cytokines. Similarly, type 1 or type 2 deviated dendritic cells, macrophages, and CD8 cytotoxic lymphocytes have been identified. Allogeneic transfusions appear to promote primarily type 2 cytokine secretion patterns with reciprocal downregulation of type 1 cytokine secretion. Many cellular immune functions are downregulated by immune deviation, including cytotoxic T cell and NK functions.98,155 DTH, delayed-type hypersensitivity; TCR, T-cell antigen receptor; MHC, major histocompatibility complex; NK, natural killer; IL, interleukin. Reprinted from Anderson KE, Ness PM [eds.]. Scientific Basis of Transfusion Medicine. Elsevier, 2000, p 429, with permission.)

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Table 52–1 Allogeneic Transfusion Effects on Immune Function 1. Decreased Th1 and increased Th2 cytokine production in vitro 2. Reduced responses in mixed lymphocyte culture 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 in vivo 8. Enhanced production of anti-idiotypic antibodies suppressive of mixed lymphocyte response in vitro 9. Decreased cell-mediated cytotoxicity (LAK) against target cells in vitro 10. Humoral alloimmunization to cell-associated and soluble antigens Data from Blumberg N, Heal JM. The transfusion immunomodulation theory: The Th1/Th2 paradigm and an analogy with pregnancy as a unifying mechanism. Semin Hematol 1996;33:429–440; Blumberg N, Heal JM. Transfusion and recipient immune function. Arch Pathol Lab Med 1989;113:246–253; Blumberg N, Heal JM. Effects of transfusion on immune function: Cancer recurrence and infection. Arch Pathol Lab Med 1994;118:371–379.

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T cells are critical to maintaining antitumor, anti-allograft, and antimicrobial immunity. Thus, their dysregulation after allogeneic transfusion provides one plausible unifying mechanism to explain a variety of seemingly unrelated clinical outcomes. To complicate the clinical picture, surgery alone, absent transfusion, causes immune deviation toward type 2 cytokine secretion. This effect likely accounts in part for the reduction in delayed-type hypersensitivity responses seen for the first week or two after surgery.117–119 Whether this altered immunity is due to surgical trauma, anesthesia, or other drugs is not entirely understood. These immune effects are additive with the effects of transfusion in the clinical setting, and impairments of cellular immunity can last for days to weeks. From both immunologic and clinical standpoints, it is of interest that immune dysregulation is not seen or is seen to a lesser degree in patients receiving only autologous blood,115,118,120 or leukocyte-depleted transfusions.15,16,121 Finally, evidence indicates that infusion of apoptotic allogeneic WBCs facilitates type 2 immune deviation,122 organ allograft acceptance,123,124 and multiorgan failure122 in experimental animals. Apoptotic WBCs and platelets, as well as red cells, accumulate during blood storage and are selectively removed by leukocyte-reduction filters.125 Thus, infusion of storage-damaged apoptotic allogeneic cells may provide one mechanism by which T cell, macrophage, and dendritic cell immunity is biased toward type 2 cytokine secretion, and explain why leukocyte reduction reduces adverse immunologic and clinical effects.126 Immune deviation, greater in intensity but similar in principle to that seen in pregnancy, is not the only event that occurs with allogeneic transfusion. However, it is the only current hypothesis that could account for a broad range of seemingly contradictory clinical findings that range from reduced spontaneous abortion, allograft rejection, and autoimmune disease, to increased postoperative infection and cancer recurrence (Table 52–2). In summary, TRIM is a pleiotropic phenomenon. It is extremely unlikely that any single mechanism fully accounts for the effects of transfusion on organ transplants, postoperative infection, cancer recurrence, pregnancy, and autoimmune diseases.

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histocompatibility antigens or peptide fragments of class I molecules108,109 can have similar effects. The simultaneous presence of alloantigen and key mediators probably accounts for many of the clinically observed TRIM results, because autologous or syngeneic blood does not generally mediate these effects. TRIM in the allotransplant setting is mediated at least in part by the effects of increased PGE2 and decreased IL-2 production. Inhibition of PGE2 secretion by indomethacin or anti-PGE2 antibody abrogates the transfusion allograft enhancement effect.110 In another animal model, administration of exogenous IL-2 reversed the blood-transfusion tolerance induction for renal allografts.111 Allogeneic transfusions also impair the ability of murine and human mononuclear cells to secrete IL-2 in response to a variety of in vitro stimuli.22,112 Table 52–1 lists some of the immunologic alterations that have been consistently described in transfused animals and patients.98,106,107 Several mechanisms appear to play a role common to many or even most of these clinical settings. One model for transfusion immunomodulation is depicted in Figure 52–6.20–22,98,112–115 Immune deviation involving increased secretion of type 2 (Th2) cytokines (e.g., IL-10, IL-4) and decreased secretion of type 1 (Th1) cytokines (e.g., γ-interferon, IL-2) is one proposed mechanism by which transfusion immunomodulates recipient immune responses to allogeneic organs, tumors, and the fetus. Effective immune responses against tumors and organ allografts are thought to be primarily Th1 in nature,116 as is rejection of the fetus as an allograft.39 Although this scheme no doubt oversimplifies the intricate biology of such processes, clinical and animal studies demonstrate that allogeneic transfusions alter cellular immunity by promoting immune responses with increased production of IL-10, IL-4, and TGF-β. These mediators downregulate natural killer (NK) and T-effector cell functions, as well as phagocytic cell functions and generally function as anti-inflammatory mediators. NK and

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METHODS TO MITIGATE TRANSFUSION IMMUNOMODULATION AND REDUCE MORBIDITY AND MORTALITY Reducing the damaging effects of transfusion immunomodulation is not straightforward, as the underlying mechanisms have not been entirely elucidated. For example, it is not certain whether transfused cells and molecules other than allogeneic WBCs cause significant changes in clinical outcomes. However, this seems probable. Supernatants from

Table 52–2 Th1 Processes Downregulated by Allogeneic Transfusions Allograft rejection Tumor rejection Rejection of the fetus as an allograft Inflammation of Crohn’s disease and rheumatoid arthritis Inflammation of type 1 diabetes (in animals) Antibacterial and antiviral immunity

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stored platelet concentrates contain significant amounts of immunomodulatory mediators such as TGF-β1 and sCD40L, which are potential causes for some observed associations. Platelets express surface CD40L, which has been linked in preliminary work to acute lung injury.83,127 Storage-damaged apoptotic red cells express phosphatidyl serine, which could interact with receptors on endothelial or other immunocompetent cells. Transfusion components stored under variable conditions for various periods may have differing immunomodulatory effects. Clinical data suggest that transfused whole blood has more potent immunomodulatory effects than do red cell concentrates. Whether this is attributable to variations in platelet or WBC content, supernatant plasma, or other variables is not known. Differences in leukocytereduction methods may yield sufficiently varied numbers of residual platelets in the transfused component that this accounts for some of the inconsistency of the results reported in randomized trials. Despite these caveats and uncertainties, three well-established methods are known for mitigating the immunomodulatory complications of transfusion. The first is entirely avoiding transfusion via bloodless medicine and surgery practices.128 The second is avoiding or reducing allogeneic transfusions by use of autologous transfusion, hemodilution, and blood salvage.19 The third is administering only leukoreduced (also platelet-reduced) red cell transfusions by using high-efficiency WBC adherence filters.15–18,58,70–73,129 Two randomized trials of autologous predeposit versus allogeneic transfusions have produced opposite results. Heiss and colleagues56,118 reported reduced infections and cancer recurrences in patients receiving autologous transfusions, whereas Busch and colleagues57 found no benefit. Both these studies are limited by the most significant problem in studies of autologous or leukoreduced transfusions, which is a high protocol-violation rate. A high percentage of patients in these studies received no transfusions or the wrong types of transfusions because of insufficient availability of autologous or leukoreduced blood. When patients with protocol violations were removed from the database, the benefits of reduced cancer recurrence were much clearer in the study of Heiss and colleagues. Another caveat for both of these studies is that the control arms were buffy coat depleted (60% to 80% leukoreduced) allogeneic red cells. This partial leukoreduction of the control transfusions minimizes the opportunity for detecting a benefit because allogeneic WBCs/platelets are important mediators of TRIM. In addition, a potentially important difference may be that the Heiss study56,118 was a single-center study, whereas the Busch study included multiple centers.57 Most single-center trials of leukocyte depletion or autologous transfusion have demonstrated reductions in morbidity or mortality compared with unmodified allogeneic transfusion recipients. Most multicenter trials have failed to find such benefits. The variability introduced by small numbers of patients treated by many diverse clinical protocols may have obscured potential benefits. Such variations from center to center have been shown to account for the variability in postoperative infection rates in Israeli medical centers.130 It is interesting that an initial multicenter study by investigators in Leiden failed to find a benefit to leukoreduced transfusions in colorectal cancer surgery,58 but a subsequent single-center study from the same investigators found beneficial effects of leukoreduced transfusions on morbidity and mortality after cardiac surgery.18 Variation in infection-control and surgical

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techniques, such as time of administration of prophylactic antibiotics, significantly affects infection rates, and these uncontrolled variables may confound studies of transfusion interventions. One large randomized trial found that leukoreduced transfusions benefited neither mortality, morbidity, length of stay, nor hospital costs.131 These data did support the probability that leukocyte reduction was cost neutral. This study had a number of limitations. Many patients were included who might not be seriously affected by TRIM. Outpatients with anemia of chronic disease, medical inpatients with modest blood loss or hypoproliferative anemias, and other subgroups may affect the power of a large study to detect a benefit in those who are more seriously at risk, such as surgical and critical care patients. Second, as in other studies with high protocol-violation rates, more than one in eight patients in the leukoreduced arm of this study actually received nonleukoreduced blood. Third, the proportion of patients with protocol violations was significantly greater in the leukoreduced arm of the study than in the control arm. A likely explanation for this failure of randomization is that when supplies of leukoreduced blood ran short, technical staff switched patients to the nonleukoreduced protocol. As TRIM is dose dependent, this effectively removed from the leukoreduced arm of the study the heavily transfused patients most likely to benefit. This study thus cannot definitively address whether leukoreduction reduces morbidity and mortality in surgical and critical care patients, which are the settings in which the largest clinical and cost benefits may be observed.132 Over the last decade, a number of “meta-analyses” of randomized trials of leukoreduced transfusions to reduce postoperative infections have been published, claiming to demonstrate that leukoreduction is of little or no benefit in reducing postoperative infection.66,133–139 However, these studies have methodologic problems. One error is failing to calculate a summary odds ratio for the effects of leukoreduced transfusions. Without a summary odds ratio, the single number representing the cumulative results of the published clinical trials, no meta-analysis is produced, but only a review containing the authors’ opinions. The rationale for not calculating a summary estimate was the presence of statistically significant heterogeneity in the published original studies. Statistical heterogeneity is not a valid scientific rationale for failing to calculate a summary odds ratio, but rather is an indication for use of a random effects metaanalysis, and for further exploration of the data to investigate the sources of heterogeneity.140 Even more scientifically questionable is that these analyses include many nontransfused patients. Nontransfused patients compose as many as three fourths of the patients in some clinical trials in the published graphs. Nontransfused patients cannot provide answers to the question of whether TRIM has clinically significant effects, nor whether leukoreduction can reduce such effects. Most seriously, some metaanalyses do not represent the actual data from the original studies. For some of the clinical trials, the meta-analyses used data that represent “imputed” outcomes created by the authors of the review.66,133–139 The original authors of the studies analyzed only the patients who actually received transfusions because these are the only relevant patients for assessing whether leukoreduction has any benefits. Retrospectively to create an “intention to treat” analysis including the nontransfused patients, the

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Overall Van Hilten 04 Bilgin 04 Wallis 02 Titlestad 01 Van de W 98 Tartter 98 Jensen 96 Houbiers 94 Jensen 92 0

5

10 15 20 25 30 35 40 45 50 55 Post-operative infection rate Non-leukoreduced transfusions Leukoreduced transfusions

Figure 52–7 The postoperative infection rates observed in nine randomized trials of leukoreduced versus unmodified transfusions in colorectal or cardiac surgery are shown according to randomization arm (intention to treat). Six of the nine studies found statistically significant reductions in postoperative infections with leukoreduced transfusions, and in eight of nine studies, the infection rate was lower in the leukoreduced arm of the study. Nontransfused patients are excluded from these data, but patients with protocol violations (range, 0–11%) remain in the arm to which they were originally randomized. Overall, the infection rate in 1637 patients randomized to receive leukoreduced transfusions was 23%, as compared with 33% in the 1456 patients randomized to receive whole-blood, buffy-coat-poor, or unmodified red cell concentrates (equivalent to a 36% decrease in relative risk with leukoreduced transfusions; P = .005 in a random-effects model metaanalysis performed by Drs. Gary Lyman, Hongkun Wang, and Hongwei Zhao of the University of Rochester; unpublished data).

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and the confounding effects of multi-institutional observational studies. Five of the seven studies reported reductions in mortality, morbidity, and/or costs,144–148 and three of the studies reported patient cohorts with no apparent clinical benefit.142,143,146 None has demonstrated significant increases in morbidity, mortality, or costs with institution of universal leukoreduction, suggesting that, at worst, leukoreduction is cost neutral and causes no harm, whereas at best it can reduce morbidity, mortality, and costs significantly. These benefits of leukoreduced transfusions are not minor public health concerns.74,149 Depending on the percentage of the epidemiologic association that is causal, the number of TRIM–associated deaths could be as many as tens of thousands per year, far exceeding those due to other transfusion complications.132 The opportunity to improve care involves not only morbidity and mortality averted, but also significant cost savings. Investigators in Rochester, New York, and Aarhus, Denmark, have estimated these savings at $1000 to $2000 per leukoreduced or autologous unit transfused instead of an allogeneic unit.144,150,151 By extrapolating from these data, the estimated savings to the United States health system from universal leukocyte reduction of allogeneic transfusions to surgical patients could be as much as $6 to $12 billion per year, or 1% to 2% of the national health budget at that time.152

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meta-analysts divided in half the number of nontransfused patients and infections reported in these nontransfused patients. They then added these infections and nontransfused patients back to the actual reported data on transfused patients, as originally published. It is a violation of the scientific method to include nontransfused patients in a study of leukoreduced transfusion outcomes, whether using actual or fictional data. Estimates from properly performed metaanalyses that restrict the analysis to actual data from about 3000 patients who were transfused in eight randomized trials recently demonstrated beyond reasonable doubt that leukoreduction decreases the odds of postoperative infections in transfused surgical patients by about 40% to 50% compared with unmodified red cells, whole blood, or buffy coat–poor red cells.129,141 This corresponds to an absolute risk reduction of 10%, yielding a number to treat of 10. For every 10 surgical patients receiving leukoreduced, instead of unmodified, transfusions, one patient will be protected from developing a postoperative infection that otherwise would have occurred (Fig. 52–7). This figure probably understates the benefits of leukoreduced transfusions because protocol violation rates of up to 11% occurred in the leukoreduced arm of these studies. A number of implementation trials of universal leukoreduction have been reported.142–148 These have yielded variable results, perhaps not surprisingly, when derived from before-and-after studies of infection incidence, length of stay, and other variables heavily influenced by possible temporal changes in case mix, other clinical interventions,

SUMMARY Transfusions of allogeneic blood to animals or patients are immunomodulatory. Transfusions alter cellular adaptive immunity, innate immunity, and lead to both favorable and unfavorable reduced or increased inflammatory responses, depending on the clinical setting. Transfused patients are more likely to accept renal allografts. Whole-blood recipients have better allograft survival but higher colorectal cancer recurrence rates than patients receiving red cells alone. Allogeneic transfusion recipients are more likely to develop postoperative infections than are recipients of identical amounts of autologous transfusion. Recipients of leukocytereduced transfusions are less likely to develop postoperative infections than are recipients of unmodified red cells. These observations are compatible with the finding that allogeneic transfusions lead to decreased type 1 and increased type 2 cytokine secretion in animal models and medical and surgical patients. Thus, immune deviation provides an initial concept in formulating a unifying theory of transfusion immunology, which can account for such diverse outcomes as the formation of alloantibodies (primarily a type 2 response), allergic reactions (also type 2 in origin), and downregulation of cellular immunity (a type 1 process). The single explanation that best fits these varied clinical and laboratory observations is that allogeneic transfusions mediate clinically significant immunomodulation by modifying existing normal or abnormal immune responses.

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