Clinical relevance of the HLA system in blood transfusion

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1 REVIEW Vox Sanguinis (2011) 101, ª 2011 The Author(s) Vox Sanguinis ª 2011 International Society of Blood Transfusion DOI: /j x Clinical relevance of the HLA system in blood transfusion C. J. Brown 1 & C. V. Navarrete 2 1 Histocompatibility & Immunogenetics Laboratory, NHS Blood and Transplant Colindale Centre, Colindale Avenue, London NW9 5BG, UK 2 Department of Immunology & Molecular Pathology, Division of Infection and Immunity, University College, London, UK Received: 4 October 2010, revised 14 January 2011, accepted 15 January 2011, published online 3 May 2011 HLA alloimmunization induced by pregnancy, multiple transfusions or transplantation is responsible for some of the serious complications seen in patients receiving blood and blood products. These complications are primarily the result of antibody and antigen triggering an acute immunological reaction, which in some cases can be fatal e.g. TRALI. Some adverse reactions are triggered by HLA antibodies present in the patient whereas others are initiated by antibodies or HLA reactive cells present in the transfused product. The introduction of universal leucodepletion for the prevention of vcjd transmission has resulted in a significant reduction in these reactions by eliminating the main source of alloimmunization, but residual cellular components or platelets are still able to activate the immune system and induce the development of HLA reactive antibodies or T cells. However, the use of more sensitive and specific techniques to detect HLA antibodies and antigens has not only improved the investigation of transfusion reactions and their subsequent diagnosis, but it has also facilitated the implementation of a number of measures such as the use of HLA antibody negative products to further reduce their development. Key words: antigen, human, immune, leukocyte, reactions, transfusion. Introduction The transfusion or transplantation of cells or tissues expressing cell surface molecules that differ from those present on the recipient results in their recognition and subsequent development of strong humoural and cellular responses. Antigens of the human leucocyte antigen (HLA) system are particularly efficient in eliciting immune responses since they can be recognized directly or indirectly by the cells of the immune system (Fig. 1) [1]. The direct pathway involves the direct recognition of HLA molecules on the donor antigen presenting cells (APC) in blood or in the transplanted tissues and it is particularly effective in the activation of immunologically naive T cells. The indirect pathway follows the same pathway of nominal antigen recognition and in this case involves processing and presentation of donor derived HLA peptides by the host APC. This pathway operates more efficiently in previously Correspondence: Colin J. Brown, Histocompatibility & Immunogenetics Laboratory, NHS Blood and Transplant Colindale Centre, Colindale Avenue, London NW9 5BG, UK Colin.Brown@nhsbt.nhs.uk sensitized individuals carrying memory T-cells that can rapidly respond to the antigenic challenge [2]. Another contributing factor to the immunogenic efficiency of the HLA antigens is their high degree of polymorphism which is differentially expressed across different population groups which in turn increases the probability of transfusing or transplanting incompatible blood products, tissues or organs and hence inducing an immune response [3]. The HLA system plays a critical role in many areas of clinical medicine and in the blood transfusion setting. HLA alloimmunization has been shown to be responsible for some of the serious complications occurring following the transfusion of blood or blood products. In this review we provide an update on the HLA system and on the techniques currently available for the identification of the HLA antigens and antibodies. We also describe some of the severe transfusion reactions mediated by HLA alloimmunization such as febrile non-haemolytic transfusion reactions (FNHTR), immunological platelet refractoriness (IPR), transfusion related acute lung injury (TRALI) and transfusion-associated graft-versus-host disease (TA-GVHD). The immunomodulatory role associated with blood transfusions and the impact of universal leucodepletion on this effect is also discussed. 93

2 94 C. J. Brown & C. V. Navarrete HLA class II genes and molecules Fig. 1 Allorecognition. The laboratory procedures used in the investigation of these reactions and possible measures for their prevention or treatment are also reviewed. The HLA system The HLA system consists of a group of closely linked genes coding for highly polymorphic cell surface molecules whose main role is to present antigenic peptides to the immune system in order to initiate an adaptive immune response. A number of co-stimulatory molecules (e.g. CD80, CD86) and adhesion molecules such ICAM-1(CD54) and LFA-3 (CD58) also contribute to these interactions. Based on some of their molecular and functional characteristics, two main types of HLA genes and molecules have been described, HLA class I and HLA class II [4]. HLA class I genes and molecules These genes code for the heavy (a) chain of the classical (HLA-A, -B and -C), the non-classical HLA-E, -F and -G and the major histocompatibility complex class I chainrelated MICA and MICB molecules. HLA class I molecules (A, B, C) are expressed on the majority of tissues and nucleated blood cells including T and B lymphocytes, granulocytes and platelets and to a lesser extent on endocrine tissue, skeletal muscle and cells of the central nervous system. HLA-E, -F & -G show a more restricted tissue distribution and to date HLA-G products have only been found on extra villous cytotrophoblasts of the placenta and mononuclear phagocytes. MICA and MICB molecules are expressed on normal thymus, cornea, fibroblasts, endothelial, intestinal and tumour epithelial cells [5]. These molecules present primarily, but not exclusively, endogenous antigenic peptides to CD8 + + T cells. HLA-A, -C and HLA-G and -E also interact with a new family of receptors present on cells of the innate immune system, the natural killer (NK) cells and on cdt cells and CD8 + T cells [6]. HLA class II genes include the classical HLA-DRB1,-DQB1, -DQA1,-DPA1, -DPB1 and the non-classical HLA- DMA,-DMB, -DOA and -DOB genes which a have a similar structure to the classical class II genes but show limited polymorphism. HLA class II molecules are constitutively expressed on B lymphocytes, monocytes and dendritic cells but can also be detected on activated T lymphocytes and activated granulocytes [7]. HLA class II expression can be induced on a number of cells such as fibroblasts and endothelial cells as the result of activation and or the effect of certain inflammatory cytokines, such as IFN c, TNF a and IL-10. HLA class II molecules (DR, -DQ and -DP) are involved in the presentation of peptides derived from exogenous pathogens to CD4 + + T cells. Once activated, these CD4 + + cells promote the maturation and differentiation of cellular and humoural effectors. Both classical and non-classical HLA class I and class II molecules can also be found in soluble forms and it has been suggested that HLA class I molecules at least may play a role in the induction of peripheral tolerance [1] or immunomodulatory effect of blood transfusion [8]. The HLA region of the MHC region also contains genes coding for other diverse groups of molecules, including the low-molecular-mass polypeptide genes LMP2 and LMP7, the TAP1 and TAP2 transporter and the Tapasin (Tpn) genes involved in the processing, transport and loading of HLAclass I antigenic peptides [4, 9, 10]. Other genes coding for complement components (C4Bf), tumour necrosis factor (TNF) and heat-shock proteins (HSPs) are located between the class I and class II genes. The class I-like gene HFE associated with the development of hereditary haemochromatosis type 1 (HH) is located 4 Mb telomeric to HLA-F. (Fig. 2) Identification of HLA polymorphism The techniques used for the identification of HLA polymorphism are based on the polymerase chain reaction (PCR) amplification of the specific genes or DNA region to be analysed. These techniques include PCR-SSP (sequencespecific priming), PCR-SSOP (sequence-specific oligonucleotide probing) and DNA sequencing-based typing (SBT) [11]. A modification of the PCR-SSOP technique has been described whereby the relevant PCR products are labelled with biotin and the amplicons are then detected with oligonucleotide specific probes coupled to colour beads. Following the addition of R-Phycoerythrin-conjugated streptavidin (SAPE) to the reaction, the fluorescence is then measured using a flow cytometer based instrument, the Luminex LABtype R [12].

3 Clinical relevance of the HLA system in blood transfusion 95 Fig. 2 Map of the human major histocompatibility complex. Detection of HLA antibodies HLA-specific antibodies are induced by pregnancy, transplantation and blood transfusions but the majority of those found in multi-transfused patients are transient multispecific IgM and IgG and tend to be directed at public epitopes. HLA antibodies have been identified in approximately 20% of multiparous women and in 30 50% of multi-transfused patients. HLA antibodies have also been detected in the sera of non-alloimmunized healthy male donors and they have been shown to be directed against HLA-E molecules and probably induced by cross-reactive bacterial antigens and or peptides derived from ingested food or allergens [13, 14]. The clinical significance of these naturally occurring HLA antibodies in the transfusion or transplantation settings is not yet clear. Complement-dependent cytotoxicity (CDC) has been the technique most commonly used for the detection of HLA antibodies. However one of the main limitations of this technique is its inability to differentiate between HLA and non-hla cytotoxic antibodies. As a result solid phase based assays have been developed including the enzymelinked immunosorbent assay (ELISA). Flow cytometer cell based techniques have also been developed but are used primarily to perform cross matches prior to solid organ transplantation. More recently a new solid phase technique has been developed whereby fluorochrome-dyed polystyrene beads are coated with specific HLA antigens which are then used to detect the antibodies in the serum or plasma. The precise ratio of two different fluorochromes creates 100 distinctly coloured beads, each of them coated with a different antigen. The beads are then incubated with the patient s serum and the reaction is developed using a PE-conjugated anti-human IgG (Fc specific) antibody. The positive or negative reactions are then read using a Luminex analyzer which can distinguish between up 100 different bead sets in a single tube. Antibody detection using Luminex is now widely used and it has been shown to be more sensitive than the CDC or ELISA, although the clinical benefit of this increased sensitivity is not yet clearly defined particularly in the transplant setting [15, 16]. HLA mediated immunological transfusion reactions The role of HLA matching in the outcome of clinical transplantation is well documented and every effort is made to identify the best matched donor for each patient [17, 18]. However in the transfusion setting, the provision of HLA matched products has been limited and this has contributed to the development of immunological reactions observed following the transfusion of blood products [19]. These immunological reactions are mediated by HLA antibodies present in the recipient reacting with HLA antigens in the transfused products, e.g. FNHTR or IPR. Others are mediated by HLA antibodies or HLA reactive cells in the transfused product recognizing HLA antigens in the recipient and include TRALI and transfusion associated graft versus host disease (TA-GVHD) respectively [20]. The introduction of universal leucodepletion (LD) in some countries as a measure to prevent vcjd transmission led to the expectation that some of these reactions would disappear but this has not been fully vindicated. Indeed, the

4 96 C. J. Brown & C. V. Navarrete use of LD products may contribute to reduce alloimmunization rates in non-sensitized, immunologically naive recipients by eliminating cells, e.g. DCs that could directly stimulate the immune system. However in an already sensitized recipient such as multiparous women or multi-transfused patients, memory T cells can be rapidly activated by the recipient s own APC presenting antigenic peptides derived from the incompatible HLA molecules present in the transfused product. Published studies indicate that extreme leucodepletion of HLA class II positive B cells may enhance rather than reduce alloimmunization [21] and that genetic factors and or the presence of Tregs may also contribute to the capacity of individual patients to mount an alloresponse [22, 23]. Febrile non-haemolytic transfusion reactions (FNHTRs) FNHTRs are some of the most common transfusion reactions and are characterized by fever, chills and a rise in the temperature of more than 1 or 2 C occurring during or min following the transfusion. Other symptoms such as rigor, flushing, tachycardia, nausea and vomiting can also be present. The differential diagnosis of FNHTR is haemolytic reactions, bacterial contamination of the transfused products and TRALI. Although FNHTR can be triggered by a variety of factors, HLA, HNA and to a lesser extent high titre HPA antibodies present in the recipient and reacting with white blood cells (WBC) or platelets in the transfused product have been shown to be the main immunological trigger of these reactions [24 26] and antibodies against white cells are found in 70% or more of patients who suffer from FNHTRs [25]. In most cases, it is likely that the antibody antigen complex may directly activate the cells to produce pyrogenic cytokines leading to the febrile reaction. A number of studies have reported a reduced incidence of FNHTR following universal pre-storage leucoreduction UPL. Paglino reported a 47Æ1% decrease in incidence of FNHTR for red cell transfusion (pre-upl 0Æ34%, post-upl 0Æ18%) and 93Æ1% decrease for platelet transfusion (pre- UPL 2Æ18%, post-upl 0Æ15%) [27]. The decrease number of leucocytes not only presents fewer targets for leucocyte reactive antibodies but also reduces the probability of sensitization in the non-sensitized patient. FNHTR can occur following first exposure to platelet in patients who have not been previously immunized with leucocytes indicating that in these cases it is likely that the reaction is not antibody mediated and other soluble mediator may be important. The age of the blood product is of importance here as there is a linear correlation between cytokine levels, leucocytes content and the duration of storage with a greater accumulation of cytokines at platelet storage temperature 22 C compared with red cells at 4 C [28, 29]. The use of fresh leucodepleted products should also prevent the majority of cases due to cytokine accumulation during storage, since removal of leucocyte to below per blood component prevents accumulation of IL-8 and inflammatory cytokines such as IL 1b, IL-6 and TNF in both red cell and platelet components [30]. Soluble CD40L has also been implicated in these adverse reactions [31]. Immunological refractoriness to random platelet transfusions Platelet transfusion therapy plays a major role in the management of patients with haematological and oncological disorders with intermittent or long lasting thrombocytopenia. However, approximately 30 50% of transfusiondependent patients become refractory to platelet transfusion as defined by the failure to gain adequate increments (< l), 1 h or up to 24 h post-transfusion. Platelet refractoriness may be the result of immunological and or non-immunological causes. Non-immunological factors include old or badly stored platelets, sepsis, disseminated intravascular coagulation in the patient and certain drugs such as amphotericin B, ciprofloxacin. In contrast, immunological refractoriness caused by antibody-mediated destruction of transfused platelets is normally due to the presence of HLA class I specific antibodies, although antibodies against human platelet antigens (HPA) and high-titre ABO alloantibodies have also been occasionally implicated. The majority of the reported cases of HPA antibodies independently causing platelet refractoriness is not common as most cases involve highly HLA immunized patients [32 34]. Individual cases of refractoriness due to high titre ABO have been reported in patients receiving HLA matched platelets where the transfusion of ABO mismatched platelets into alloimmunized patients can result in a 20% reduction in the platelet increments post transfusion [35]. Circulating immune complexes involving the ABO system have also been shown to affect the survival of transfused platelets [36]. Other anti-platelet antibodies such as drug induced e.g. vancomycin, have also been implicated but their occurrence is rare. Animal models have shown that the indirect allorecognition of platelet derived HLA peptides promote the development of IgG1 responses [37]. However, alloimmunization does not always result in platelet refractoriness which may be due to the presence of low titre or low frequency antibodies in those patients but as platelet destruction occurs

5 Clinical relevance of the HLA system in blood transfusion 97 via the monocyte macrophage system and the FcR expressed on monocytes preferentially binds IgG3 and IgG1, the presence of these antibodies could be more relevant. Studies from our own laboratory of the clinical diagnoses of the 259 patients receiving HLA matched platelet transfusions showed that the majority of patients (57%) suffered from leukaemia (30% AML, 16% CML and 10% ALL), 17% had MDS, 7% aplastic anaemia and the remaining 26% had other haematological disorders. Investigation for IPR patients should normally commence after documented failure to obtain increments using fresh ABO identical single donor platelets. If these fail the patients are screened for the presence of HLA specific antibodies. If positive, HLA compatible products are indicated and HLA typing is performed in order to identify the most suitable HLA matched platelet unit. Although platelets express HLA-A, B and C antigens, most laboratories only perform HLA-A and -B typing and antibody screening for these patients and therefore the clinical significance of HLA-C antibodies in immunological refractoriness remains to be established. Several different approaches have been used for the management of IPR patients which include: (1) The provision of crossmatch compatible platelets; here the patient s serum is crossmatched with cells or platelets from platelet donors or donations. This approach can be useful if the patient is not highly sensitized but has the disadvantage that it has the potential to immunize the patient against mismatched antigens and is not suited to long term platelet transfusion support. (2) The second approach for management of platelet transfusions for IPR patients involves the provision of compatible platelets solely based on the HLA antibody profile of the patient and not matching for the patient s HLA type. This approach can be useful for short term platelet transfusion support but has the potential of immunizing the patient to the mismatched antigens on the transfused platelets which may compromize the efficacy of future transfusions. (3) The third approach involves the provision of HLA selected platelets based on the HLA type of the patient and their HLA antibody profile using a panel of HLA typed donors. In our institution the matching criteria is based on two grades of matches, A grade where there is no mismatch between donor and recipient, and B grade where patient and donor are mismatched with the number of mismatches denoting the type of B match, e.g. B1 one mismatch, B2 two mismatches etc. Mismatching is carried out on the basis of the known serological crossreactivity that exists between different antigens of the HLA-A and -B loci as described by Duquesnoy in 1990 [38, 39]. The English National Blood Service has a panel of over typed platelet donors which facilitates this management strategy (Fig. 3). A number of strategies for assessing HLA compatibility at the molecular or epitope level have been developed. One such strategy is the HLA Matchmaker ( as proposed by Duquesnoy R [40, 41]. Two versions of HLAMatchmaker have been described, the triplet and eplet versions. In triplet analysis, epitopes are defined by linear sequences of triplets of amino acid residues in alloantibody-accessible positions of HLA molecules. Donor- recipient HLA compatibility is assessed by intralocus and interlocus comparisons of these triplet epitopes [42]. The eplets version is similar but also takes account of epitopes formed by nonlinear amino acids brought together by the folding of the molecule. This perhaps is the future direction of HLA matching where there is antibody mediated immune destruction. In addition to the above, the following alternative approaches have been used but with less success; massive transfusions of ABO identical platelets, intravenous immunoglobulin (IVIG) and plasma exchange [43]. More recently, the transfusion of the acid treated platelets resulted in adequate increments in an alloimmunized thrombocytopenic patient [43]. Of these different approaches, the massive platelet transfusion seems to be the most successful. Transfusion related acute lung injury (TRALI) TRALI is a rare but life threatening complication of blood transfusion and can be clinically indistinguishable from adult respiratory distress syndrome [44]. This reaction normally develops within 2 h following the administration of most commonly plasma-containing blood components. Symptoms generally include fever, hypotension, chills, cyanosis, non-productive cough, dyspnoea and sometimes severe hypoxia. Chest X-ray shows severe bilateral pulmonary oedema or perihilar and lung infiltration, without cardiac enlargement or involvement of the vessels. Differential diagnosis includes transfusion associated circulatory overload (TACO), anaphylactic transfusion reaction and bacterial contamination [45]. The Canadian Consensus Conference on TRALI therefore described clinical criteria defining both TRALI and possible TRALI, depending on whether there were other factors which may have caused acute lung injury [46]. There have also been reports of cases of mild TRALI where the patient symptoms did not meet the Canadian Consensus Conference definition but patient specific HLA antibodies have demonstrated in the donor by screening and crossmatch studies. These cases highlight the need for

6 98 C. J. Brown & C. V. Navarrete Patients likely to receive multiple platelet transfusion Assess transfusion response Poor response to random donor platelets on 2 or more occasions Test for HLA specific antibodies HLA antibody test result Positive Use HLA selected platelets Negative Good response to HLA selected platelets Poor response to HLA selected platelets Factors associated with nonimmune platelet destruction Continue transfusing HLA selected platelets Test for HLA antibodies at regular intervals Provide ABO compatible, A grade matches if possible Test for HPA specific antibodies Poor response Absent Consider trial of HLA selected platelets Good response Present Treat cause decide about further platelet transfusion based on clinical status of the patient e.g. increase dose of platelets or discontinue prophylactic transfusions HPA antibody test result Continue transfusing HLA selected platelets Positive Negative Provide HLA and HPA selected platelets Consider 1. Non immune consumption 2. ABO antibodies Fig. 3 Laboratory investigation of refractoriness to platelet transfusion. continued research in the factors leading to mild and severe cases of TRALI [47]. Over 80% of the confirmed TRALI cases are associated with the presence of HLA class I or class II antibodies in plasma containing products with specificities corresponding to the HLA antigen(s) present in the recipient [48]. Granulocyte-specific antibodies e.g. anti-hna1, HNA- 2 and HNA- 3 have also been identified [49]. These antibodies are most commonly found in the donations of multiparous women [50]. Furthermore, patients who received a transfusions from a donor who tested positive for leucocyte antibodies were more likely to develop TRALI than if the transfusion was from an antibody negative donor, Odds Ratio 15 (95% confidence [CI], 5Æ1 45) [51]. The majority of antibody positive cases are associated with female donors. The antibody specificities found more frequently in reported TRALI cases are HLA-A2, -DR4 and -DR52 [50, 52]. All these antigens occur relatively commonly in

7 Clinical relevance of the HLA system in blood transfusion 99 the UK population but antigen frequency does not appear to be the only factor influencing whether the corresponding antibody is implicated in TRALI. HLA-A2 antigen frequency is around 50% in the UK and HLA-A2 concordant antibody was identified in 14% of SHOT cases with concordant antibody. HLA-A1, however, is also relatively common with an antigen frequency of 30% but not a single case of concordant HLA-A1 antibody was identified in our series. The difference may be explained by the fact that HLA-A2 is common in all population groups whereas the frequency of HLA-A1 varies significantly between different population groups. Also in few cases, antibodies present in patients and reacting with the transfused cells have been found and TRALI reactions due to inter-donor antigen antibody reactions have been documented [53]. The majority of TRALI cases involve the transfusion of plasma containing products such as whole blood, platelets and FFP but reactions due to the transfusion of rbc in OAS (optimal additive solution) have also been implicated indicating that small volumes of antibody containing plasma are still able to mediate TRALI [54]. In clinically diagnosed TRALI cases where no antibodies are detected, the granulocyte activation appears to be mediated by a soluble lipid substance which accumulates during the storage of the products [55]. Indeed animal models have shown that TRALI reactions are initiated by the activation of granulocytes by transfused antibodies and or biologically active lipids and that this activation induces the release of anaphylatoxins, cytokines and chemokines which promote neutrophil chemotaxis and aggregation in the lungs. The resulting reaction causes endothelial damage and increased pulmonary vascular permeability and fluid leakage into the alveoli causing non-cardiogenic pulmonary oedema [56], (Fig. 4). As a result, two possible mechanisms have been postulated; to explain the development of TRALI, an antibody-mediated and or a soluble mediatormediated. Both these mechanisms involve the activation of granulocytes and the triggering of an inflammatory process leading to the sequestration of neutrophils in the lung. Also look-back studies have shown that products from donors implicated in TRALI reactions have been transfused into other patients with no reported serious clinical consequences suggesting that other factors, such as the predisposing clinical condition of the recipient, such as TTP [57] or cardiovascular surgery [58], may influence the development of TRALI. These observations have led to the proposal of the Two hits theory for the development of TRALI one involving antibodies or soluble mediators and the second hit, the clinical condition of the patient [59]. It is likely that a number of factors may determine the final clinical response of a patient and these may include the characteristics of the antibody, nature and distribution of the related antigen, the extent of complement activation (in particular liberation of C5a), and the immune status of the recipient. Interestingly, recently published data obtained in an animal model has shown that that recipient T cells are required to modulate the severity of antibody mediated TRALI [60]. Our protocol for the investigation of TRALI cases requires an initial assessment of each case by an expert panel which includes an intensive care and a transfusion medicine specialist. Figure 5 describes the algorithm used in our institution. A serological crossmatch between donor serum and granulocytes and or lymphocytes from the patient should be performed to confirm diagnosis. However Fig. 4 Immunopathology of TRALI.

8 100 C. J. Brown & C. V. Navarrete Suspected Case of TRALI : Acute dyspnoea with hypoxia, bilateral pul monary infiltrates, during or within 6 hours of transfusion, not due to circulatory overload Review by expert panel : intensive care and transfusion specialists Alternative strategy for investigation, where crossmatch is performed first TRALI Suspected NO No Further investigation YES Investigate female donors in the first instance Neg Perform crossmatch : recipient cell vs donor serum Pos HLA/HNA antibody positive Pos Test for HLAHNA specific antibodies Neg HLA/HNA antibody negative HLA class I and II type patient and donor for relevant antigens HNA type patient and donor for relevant antigens Review-non immune factors YES Patient and Donor share HLA/HNA NO Perform crossmatch : recipient cell vs donor serum Report : TRALI not likely to be caused by HLA/HNA specific antibodies Report : TRALI not HLA/HNA antibody mediated Positive Crossmatch result Negative Report : TRALI likely to be caused by HLA/HNA specific antibodies Report : TRALI not likely to be caused by HLA/HNA specific antibodies Fig. 5 Laboratory investigation of TRALI. this is sometimes logistically difficult and therefore in most cases the implicated donors should be investigated for the presence of HLA and HNA antibodies and if positive then proceed to type the patient. Treatment of TRALI includes intensive respiratory and circulatory support. In almost all cases, oxygen supplementation is necessary, although mechanical ventilation may not always be required. Some reports suggest that the administration of corticosteroids may be beneficial and it is likely that prophylactic antibiotics may also help. However, and in spite of these measures, mortality still remains at 6% [61]. The majority of patients improve both clinically and physiologically within 2 or 3 days with adequate supportive care and once recovered there is no residual damage in these patients [62]. In 2003, based on observations and recommendations from SHOT, the English National Blood Service carried out an option appraisal on steps which could be taken to reduce TRALI risk. As a result of this exercise, a number of initiatives to reduce the frequency of TRALI have been implemented based around reducing the use of female donors for manufacturing products containing significant volume of plasma. They include:

9 Clinical relevance of the HLA system in blood transfusion 101 (1) The production of FFP from donations collected from male donors. (2) The use of male donor only plasma to re-suspend pooled platelets. (3) The preferential recruitment of male apheresis donors such that, from 2008, 80% of all apheresis platelets were collected from male donors. (4) HLA and HNA antibody screening of all new female apheresis donors added to the panel. A number of groups have reported the benefits of using male plasma for transfusion. Eder et al. (2010) reviewed 77 cases of probable TRALI reported to the American Red Cross and showed a decrease in TRALI cases when malepredominant plasma was used for transfusion [63]. A Dutch study of TRALI following the introduction of male only FFP showed a 33% reduction in reported cases [64]. Transfusion-associated graft-versus-host disease (TA-GVHD) Acute graft-versus-host disease (GVHD) is a recognized complication of allogeneic haemopoietic progenitor cell transplantation. It results from the presence of viable lymphocytes in the allograft recognizing the host HLA antigen. The clinical syndrome includes fever, diarrhoea, abnormal liver function tests and a characteristic rash particularly affecting the palms. A similar picture may result from the transfusion of viable lymphocytes into immunosuppressed recipients. This TA-GVHD is typically evident from 8 to 10 days post transfusion and it is almost uniformly fatal, with death occurring within one month in over 90% of cases [65]. TA-GVHD cases seem to be under-reported because of lack of recognition or the absence of definitive diagnostic studies. The incidence is unknown, but TA-GVHD is estimated to occur in up to 1% of patients with haematological malignancies or lymphoproliferative diseases. Rare cases of GVHD following transplantation of liver or heart lung have been shown to be due to the presence of passenger lymphocytes in the transplanted organ [66]. TA-GVHD has also been reported in non-immunocompromized hosts, particularly patients undergoing cardiovascular [67], pregnant women, abdominal surgery, patients with active rheumatoid arthritis [20] and trauma cases [68]. The main requirements for the development of GVHD are: (1) the presence of viable lymphocytes in the transfused product; (2) sharing of HLA haplotypes between the recipient and donor but with other differences that make the donor recognize the recipient as foreign; and (3) inability of the host to reject the immunocompetent donor lymphocytes. In a normal recipient, immune cells will far out-number donor derived T cells which are therefore eliminated by a host versus graft reaction. However if a small number of functional T lymphocytes are transfused which derive from a donor who is homozygous for one of the recipient s HLA haplotypes, the recipient will not recognize these cells as foreign. The donor T cells will, however, recognize the host as foreign, undergo clonal expansion and establish TA-GVHD. Recent experiments in selective depletion of recipients CD4 +, CD8 + and NK cells have suggested that CD4 + cells may be involved in the pathogenesis of TA-GVHD, while CD8 + and NK cells appear to be protective. This may explain why TA-GVHD is not reported in patients suffering from AIDS. Furthermore CD4 + and CD8 + cytotoxic as well as CD4 + T cell clones lacking direct cytotoxicity but with lytic supernatants containing tumour necrosis factor (TNFb) have been isolated from the lesion of a patient with TA- GVHD [69]. In addition, the levels of pro-inflammatory cytokines such as interleukin IL-1, IL-2, TNF and c-interferon are greatly increased in TA-GVHD as the result of tissue injury due to infection, chemo radiotherapy or tumour invasion. These cytokines can in turn up-regulate HLA expression and recruit and activate other donor-derived T cells and macrophages. A positive feedback loop is thus established, leading to a full clinical picture similar to the one seen following HSCT [70]. The diagnosis of TA-GVHD depends largely on finding evidence of donor derived cells, chromosomes or DNA in the blood and or affected tissues of the recipient. The detection of chimerism is an important aid in the diagnosis which may otherwise be delayed in view of the severe but initially non-specific clinical features. Since donor leucocytes remain in the circulation of otherwise healthy recipients for periods ranging from days to even weeks, the demonstration of donor lymphocytes in a recipient on one occasion indicating a state of chimerism, does not in itself define TA-GVHD. Evidence of chimerism, preferably on more than one occasion, within the appropriate clinical context is necessary to substantiate the diagnosis. Detection of chimerism using DNA analysis allows a wider range of markers to be employed including HLA genes or other genetic markers. However, for any selected pairs of donors and recipients, these DNA techniques at a particular locus may fail to be informative, e.g. if the donor is HLA homozygous for a haplotype found in the recipient. It may be necessary to look at multiple polymorphic sites in a recipient to identify the presence of multiple alleles such as the short number tandem repeat (STR) profile [71]. Ideally samples for DNA extraction should be obtained from the recipient (before and after transfusion) and from the implicated donors. Post-transfusion samples may be difficult to obtain due to the pancytopenic state in the recipient. Alternative tissues for DNA extraction include skin (both affected and unaffected areas), hair follicles or nail

10 102 C. J. Brown & C. V. Navarrete clippings. Post-mortem samples from the spleen or bone marrow, if available, can be used as a source of DNA. Follow-up samples can provide valuable information about temporal changes to the DNA profile, particularly in the case where the pre transfusion samples are not available. There is no effective treatment of TA-GVHD and the mortality rate is extremely high. Immunosuppressive therapies have been used with little success including steroid therapy, anti-thymocyte globulin, cyclosporin, and cyclophosphamide and anti-t cell monoclonal antibodies. These treatments are sometimes useful in GVHD after stem cell transplantation but are ineffective in TA-GVHD. In the light of the absence of any effective treatment, the prevention of this condition is essential. The critical number of T cells known to cause GVHD following HSCT is between to kg [72]. The precise number of lymphocytes required to initiate TA- GVHD is not known. The introduction of universal LD in the UK has been associated with a significant reduction in the number of reported cases [73]. However LD is not an adequate substitute as the residual lymphocyte numbers may still be above the threshold dose. This has led to the recommendation that all cellular blood products should be irradiated, at a minimum of 25 Gy, prior to transfusion to all at risk patients described in Table 1. The irradiation of cellular blood components renders the donor lymphocytes non-viable and protects the recipient from potentially developing TA-GVHD. Guidelines have been produced by the AABB in the USA and the BCSH in the UK recommending for which patients gamma irradiated products should be available [74, 75]. Currently, because of the low incidence of TA-GVHD in immunocompetent patients receiving donated blood from unrelated donors, Table 1 Patient risk categories for TA-GvHD Patients at risk of TA-GVHD Congenital immune-deficiency disorders Hodgkin s Disease Neonates with erythroblastosis fetalis Recipients of intrauterine transfusions Recipients of haematopoietic stem cell transplants Recipients of blood components donated by relatives Recipient-donor pairs from genetically homogeneous populations Recipients of HLA-matched cellular products Premature neonates Patients possibly at risk Non-Hodgkin s B-cell lymphomas Solid tumours Potential at-risk group Full term neonates Patients receiving immunosuppressive medication such as purine analogues [20, 70]. gamma irradiation is not applied to all transfused cellular blood components. This decision is based upon the extremely low risk in such recipients, and the costs and logistics of universal irradiation, plus the effect on other measurable parameters in components such as potassium content and shelf life. Transfusion related immunomodulation (TRIM) The possible immunomodulatory role of blood transfusion was first described by Billingham in 1953 [76] and has remained a topic for debate for the last 25 years. During this period, a number of studies demonstrating beneficial clinical effect of blood transfusion induced immunomodulation in kidney transplant patients [77], and in women with recurrent spontaneous abortions (RSA) [78] have been published. A similar number of studies have, at the same time, shown a detrimental effect of blood transfusions in the survival of patients with tumours and in the increase risk of infectious complications in patients undergoing major abdominal, cardiac and orthopaedic surgery. However, and in spite of the number of reports, the mechanism involved in the induction of the immunomodulatory effect is still largely unknown [79]. However mediated, the transfusion effect in renal allografting appears to require viable leucocytes such that patients transfused with leucocyte poor, washed or frozen thawed red cells receive less immunological benefit from their transfusions [80]. The precise immunological interactions leading to induction of this immunomodulatory effect are still not fully understood but the requirement of WBC for the induction of this effect is well documented. The cells present in the transfused product can directly activate the mechanisms postulated in the immunomodulation including; induction of suppressor cells, veto cells, anergy or the secretion of immunoregulatory cytokines or prostaglandins. Storage time which has been shown to influence this effect can also act through the accumulation of soluble mediators in the plasma. Plasma or a factor present in plasma, susceptible to temperature changes, are able to suppress mitogen induced T cell responses in vitro [81]. Experimental data from animal models have shown that blood transfusion can lead to the preferential secretion of Th2 type immunoregulatory cytokines such as IL-4 and IL- 10 which favour humoral immunity [82]. Th2 type responses can also downregulate the secretion of Th1 derived cytokines, such as IL-2, and IFNc involved in promoting cellular immunity. Decreased IL-2 secretion may also contribute to the development of T cell anergy, as was seen in renal recipients who received a single transfusion of whole blood which resulted in a decrease of TNFa, IL-2 and IFNc secretion [83].

11 Clinical relevance of the HLA system in blood transfusion 103 The number of APC in the transfused product may play a crucial role in mediating this effect on the T cells, as low number may result in failure to activate T-cells via co-stimulating molecules. In this context, the failure to activate T cells may be due to the lack of cells providing co-stimulatory molecules such as CD40, B7.1 (CD80), B7.2 (CD86) in the transfused product. Alternatively, the type of APC present in blood can also affect T cell activation, e.g. B cells, myeloid or lymphoid dendritic cells or monocytes. B cells could be tolerogenic whereas DCs monocytes could be more immunogenic. More recently, the possible role of soluble HLA molecules and Fas ligand in the immunomodulatory effect of blood transfusion has been documented [84]. Soluble HLA class I molecules are able to induce apoptosis in alloreactive cytotoxic T lymphocytes [85] and are able to block NK recognition of class I molecules [86]. Summary HLA induced antibodies or cellular effectors are responsible for some of the serious complications of blood transfusion. Measures to reduce HLA alloimmunization such as the preferential use of male donors for plasma containing products have contributed to the decreased incidence of some of these reactions. Further strategies may involve: (1) alternative methods of matching [87]; (2) the development of cellular components lacking expression of these alloantigens; [88] (3) universal use of antibody negative donors [50]; and d) better understanding of the genetic basis of alloimmune responses. These strategies will require significant resources and should perhaps be targeted to support specific groups of patients at risk. References 1 Lechler RI, Garden OA, Turka LA: The complementary roles of deletion and regulation in transplantation tolerance. Nat Rev Immunol 2003; 3: Afzali B, Lechler RI, Hernandez-Fuentes MP: Allorecognition and the alloresponse: clinical implications. Tissue Antigens 2007; 69: Navarrete C: The HLA system in blood transfusion; in Contreras M (ed.): New Aspects of Blood Transfusion. London, Baillière s Clinical Haematology, Traherne JA: Human MHCarchitecture and evolution: implications for disease association studies. Int J Immunogenet 2008; 35: Zou Y, Mirbaha F, Stastny P: Contact inhibition causes strong downregulation of expression of mica in human fibroblasts and decreased NK cell killing. Hum Immunol 2006; 67: Groh V, Steinle S, Bauer S, et al.: Recognition of stress-induced MHC molecules by intestinal epithelial cd T cells. Science 1998; 279: Klein J, Sato A: The HLA system. N Engl J Med 2000; 343: Puppo F, Ghio M, Contini P, et al.: Immunoregulatory role of soluble HLA molecules: a new skin for an old subject? Arch Immunol Ther Exp 1998; 46: Bukulmez H, Fife M, Tsoras M, et al.: Tapasin gene polymorphism in systemic onset juvenile rheumatoid arthritis: a family-based case-control study. Arthritis Res Ther 2005; 7:R285 R Horton R, Wilming L, Rand V, et al.: Gene map of the extended human MHC. Nat Rev Genet 2004; 5: Ouwehand W, Navarrete C: The molecular basis of blood cell alloantigens; in Proven D, Gribben J (eds): Molecular Haematology. Oxford, Blackwell Science, Howell WM, Carter V, Clark B: The HLA system: immunobiology, HLA typing, antibody screening and crossmatching techniques. J Clin Pathol 2010; 63: Morales-Buenrostro LE, Terasaki PI, Marino-Vázquez LA, et al.: Natural human leukocyte antigen antibodies found in non alloimmunized healthy males. Transplantation 2008; 86: Ravindranath MH, Taniguchi M, Chen CW, et al.: HLA-E monoclonal antibodies recognize shared peptide sequences on classical HLA class Ia: relevance to human natural HLA antibodies. Mol Immunol 2010; 47: Levin MD, de Veld J, van der Holt B, et al.: Screening for alloantibodies in the serum of patients receiving platelet transfusions: a comparison of the ELISA, lymphocytotoxicity, and the indirect immunofluorescence method. Transfusion 2003; 43: Tait BD, Hudson F, Cantwell L, et al.: Review article: Luminex technology for HLA antibody detection in organ transplantation. Nephrology (Carlton) 2009; 14: Opelz G, Döhler B: Effect of human leukocyte antigen compatibility on kidney graft survival: comparative analysis of two decades. Transplantation 2007; 84: Petersdorf EW: Optimal HLA matching in hematopoietic cell transplantation. Curr Opin Immunol 2008; 20: Brand A: Immunological aspects of blood transfusions. Blood Rev 2000; 14: Taylor C, Navarrete C, Contreras M: Immunological complications of transfusion; in Maniatis A, Van der Linden P, Hardy JF (eds): Alternatives to Blood Transfusion in Transfusion Medicine. Oxford, Wiley-Blackwell, 2010: Semple JW, Speck ER, Cosgrave D, et al.: Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogeneic platelet immunity. Blood 1999; 93: Bao w, Yu J, Heck S, et al.: Regulatory T-cell status in red cell alloimmunized responder and nonresponder mice. Blood 2009; 113: Higgins JM, Sloan SR: Stochastic modelling of human RBC alloimmunization: evidence for a distinct population of immunologic responders. Blood 2008; 112: Brubaker DB: Clinical significance of white cell antibodies in febrile nonhemolytic transfusion reactions. Transfusion 1990; 30: Tazzari PL, Bontadini A, Zamagni C, et al.: Febrile non-haemolytic transfusion

12 104 C. J. Brown & C. V. Navarrete reaction caused by antibodies against human platelet antigen 5a. Transfus Med 2005; 15: Fadeyi EA, Adams S, Sheldon S, et al.: A preliminary comparison of the prevalence of transfusion reactions in recipients of platelet components from donors with and without human leucocyte antigen antibodies. Vox Sang 2008; 94: Paglino JC, Pomper GJ, Fisch GS, et al.: Reduction of febrile but not allergic reactions to RBCs and platelets after conversion to universal pre-storage leukoreduction. Transfusion 2004; 44: Heddle NM: Pathophysiology of febrile non-hemolytic transfusion reactions. Curr Opin Hematol 1999; 6: Heddle NM: Febrile non-hemolytic transfusion reactions to platelets. Curr Opin Hematol 1995; 6: Chudziak D, Sireis W, Pfeiffer HU, et al.: Accumulation of soluble inflammatory mediators between blood donation and pre-storage leucocyte depletion. Vox Sang 2009; 96: Blumberg N, Gettings KF, Turner C, et al.: An association of soluble CD40 ligand (CD154) with adverse reactions to platelet transfusions. Transfusion 2006; 46: Kiefel V, König C, Kroll H, et al.: Platelet alloantibodies in transfused patients. Transfusion 2001; 41: Sanz C, Freire C, Alcorta I, et al.: Platelet-specific antibodies in HLA-immunized patients receiving chronic platelet support. Transfusion 2001; 41: Brand A: Alloimmune platelet refractoriness: incidence declines, unsolved problems persist. Transfusion 2001; 41: Carr R, Hutton JL, Jenkins JA, et al.: Transfusion of ABO-mismatched platelets leads to early platelet refractoriness. Br J Haematol 1995; 75: McVey M, Cserti-Gazdewich CM: Platelet transfusion refractoriness responding preferentially to single donor apheresis platelets compatible for both ABO and HLA. Transfus Med 2010; 20: Bang WA, Speck ER, Blanchette VS, et al.: Unique processing pathways within recipient antigen-presenting cells determine IgG immunity against donor platelet MHC antigens. Blood 2000; 95: Duquesnoy RJ, White LT, Fierst JW, et al.: Multiscreen serum analysis of highly sensitized renal dialysis patient for antibodies toward public and private class I HLA determinants. Transplantation 1990; 50: Park MS, Barbetti AA, Geer L, et al.: HLA class I epitopes selected by serology; in Terasaki PI (ed): Clinical Transplants. Los Angeles, UCLA Tissue Typing Laboratory, Duquesnoy RJ: HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. I. Description of the algorithm. Hum Immunol 2002; 63: Duquesnoy RJ, Marrari M: HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. II. Verification of the algorithm and determination of the relative immunogenicity of amino acid triplet-defined epitopes. Hum Immunol 2002; 63: Duquesnoy RJ, Mulder A, Askar M, et al.: HLAMatchmaker-based analysis of human monoclonal antibody reactivity demonstrates the importance of an additional contact site for specific recognition of triplet-defined epitopes. Hum Immunol 2005; 66: Vassallo RR Jr: New paradigms in the management of alloimmune refractoriness to platelet transfusions. Curr Opin Hematol 2007; 14: Bux J: Transfusion-related acute lung injury (TRALI): a serious adverse event of blood transfusion. Vox Sang 2005; 89: Skeate RC, Eastlund T: Distinguishing between transfusion related acute lung injury and transfusion associated circulatory overload. Curr Opin Hematol 2007; 14: Kleinman S, Caulfield T, Chan P, et al.: Toward an understanding of transfusion related acute lung injury: statement of a consensus panel. Transfusion 2004; 44: Davis A, Mandal R, Johnson M, et al.: A touch of TRALI. Transfusion 2008; 48: Kopko PM, Popovsky MA, MacKenzie MR, et al.: HLA class II antibodies in transfusion-related acute lung injury. Transfusion 2001; 41: Win N, Massey E, Lucas G, et al.: Ninety-six suspected transfusion related acute lung injury cases: investigation findings and clinical outcome. Hematology 2007; 12: Chapman CE, Stainsby D, Jones H, et al.: Serious hazards of transfusion steering group: ten years of haemovigilance reports of the transfusion-related acute lung injury in the UK, and the impact of preferential use of male donor plasma. Transfusion 2009; 49: Middelburg RA, van Stein D, Briët E, et al.: The role of donor antibodies in the pathogenesis of transfusion-related acute lung injury: a systematic review. Transfusion 2008; 48: Bux J, Becker F, Seeger W, et al.: Transfusion-related acute lung injury due to HLA-A2-specific antibodies in recipient and NB1-specific antibodies in donor blood. Br J Haematol 1996; 93: Lucas G, Rogers S, Evans R, et al.: Transfusion-related acute lung injury associated with interdonor incompatibility for the neutrophil-specific antigen HNA-1a. Vox Sang 2000; 79: Win N, Chapman CE, Bowles KM, et al.: How much residual plasma may cause TRALI? Transfus Med 2008; 18: Silliman CC, Bjornsen AJ, Wyman TH, et al.: Plasma and lipids from stored platelets cause acute lung injury in an animal model. Transfusion 2003; 43: Kelher MR, Masuno T, Moore E, et al.: Plasma from stored packed red blood cells and MHC class I antibodies causes acute lung injury in a 2-event in vivo rat model. Blood 2009; 113: Cruz J, Skipworth E, Blue D, et al.: Transfusion-related acute lung injury: a thrombotic thrombocytopenic purpura treatment-associated case report and concise review. J Clin Apher 2008; 23: Silliman CC, Boshkov LK, Mehdizadehkashi Z, et al.: Transfusion-related acute lung injury: epidemiology and a prospective analysis of etiologic factors. Blood 2003; 101: Silliman CC, Ambruso DR, Boshkov LK: Transfusion related acute lung injury. Blood 2005; 105: Fung YL, Kim M, Tabuchi A, et al.: Recipient T lymphocytes modulate the