Platelet transfusion reaction associated with interdonor HLA incompatibility

An HLA-compatible platelet transfusion was followed by chills, fever, and severe respiratory distress in a multitransfused patient with chronic lymphocytic leukemia. During the previous 7 days the patient had received blood products without incident, including 8 units of red blood cells (RBC), 24 units of pooled random donor platelet concentrates, and five HLA-compatible platelet pheresis products. The patient had no demonstrable RBC, HLA lymphocytotoxic, platelet or granulocyte antibodies. The platelet donor, a multiparous female, had no granulocyte or RBC antibodies but had lymphocytotoxic antibodies against HLA-A2 CREG (cross-reacting group A2, A28, A23, A24) which reacted not with lymphocytes of the patient but with lymphocytes of the donor whose RBC were transfused 24 h prior to the platelet transfusion reaction and whose HLA type is A23, A24; B44, B57. No RBC donors had HLA lymphocytotoxic, granulocyte, or platelet antibodies against the platelet donor. The patient received three subsequent platelet transfusions from the same donor after removal of the antibody-laden plasma with no adverse reaction. These data suggest an interdonor reaction caused by the presence of cells from the RBC donor received by the patient 24 h prior to the transfusion of donor lymphocytotoxic antibody to HLA-A2 CREG antigens.     

Platelet transfusion therapy for alloimmunized patients: selective mismatching for HLA B12, an antigen with variable expression on platelets

There can be wide variation in the expression of the HLA B12 antigen and its "splits," B44 and 45, on the platelets and lymphocytes from the same individual. One hundred sixty-two single donor platelet transfusions mismatched only for this antigen group were administered to 54 alloimmunized patients who were refractory to random donor platelets. Satisfactory increments (one-hour post-transfusion corrected- count increment [CCI] greater than 7,500) were seen following 111/162 transfusions (69%). In 31 patients (57%), all transfusions (n = 85) produced CCI greater than 7,500, and 76% of patients received some transfusions that were satisfactory. Of note is that ten patients had excellent increments despite either preformed lymphocytotoxic antibody against the mismatched antigen or positive lymphocytotoxic cross- matches with the donor. In contrast, poor increments were seen in ten recipients under similar circumstances, implying disparities in antigen expression on the platelets and lymphocytes of different donors. There was no obvious pattern of other donor HLA antigens which could be correlated with these differences. The HLA B12 antigen group is relatively common (found in approximately 25% of the population), and these data indicate that selective mismatching for these antigens can be an effective donor-selection strategy to increase the number of donors for alloimmunized recipients.     

HLA antigens on platelet membranes. In vitro and in vivo studies

In order to determine whether HLA-A,B antigens of platelets are integral membrane constituents or rather represent adsorbed plasma proteins, their presence in plasma and their adsorbability onto platelet membranes was studied by in vitro and in vivo experiments. The amount of HLA antigens was quantitated by inhibition of lymphocytotoxicity (LCT) and by enzyme-linked immunosorbent assay (ELISA) using operationally monospecific polyclonal HLA antibodies or murine HLA-specific monoclonal antibodies, respectively. We found that in 11 out of 13 HLA-A2 and in 9 out of 10 HLA-B13 experiments, platelets from antigen-negative donors pretreated with plasma from the same number of antigen-positive donors inhibited LCT to the same extent as platelets from antigen-positive donors. Nevertheless, the in vitro adsorbed HLA antigens onto antigen-negative platelets were, unlike those on antigen-positive platelets or in plasma, not reactive with monoclonal antibodies as quantitated by ELISA. Similarly, infusion of HLA-A2-negative platelets from single donors into 3 HLA-A2-positive, thrombocytopenic patients with bone marrow failure led to a good platelet increment, but did not convert the HLA type of donor platelets, neither at 2 h nor at 18 h posttransfusion. On the basis of these results, we conclude that soluble HLA antigens can be taken up by human platelets from plasma in small amounts. However, the major portion of HLA antigens appears to be integral membrane constituents.     

Flow-cytometric approach to the prompt laboratory diagnosis of TRALI: a case report

OBJECTIVES: Transfusion-related acute lung injury (TRALI) is a rare but serious complication which can occur after transfusion of blood components. In this report we describe our flow-cytometry approach to the laboratory diagnosis of a case of TRALI in a recipient of fresh frozen plasma containing human leukocyte antigen (HLA) class II antibodies. METHODS: The post-transfusion reaction work-up included the direct and indirect Granulocyte Immunofluorescence Test (GIFT) on the recipient's neutrophils collected before and after the reaction and on the serum from the recipient and from all implicated donors; flow-cytometry bead-based screening and identification assay for HLA class I and II antibodies in donor sera and flow cytometry cross-matching on T and B patient's lymphocytes. Finally, we investigated the reactivity of one donor serum, containing HLA class II antibodies, with the patient's neutrophils activated in vitro to induce expression of HLA class II. RESULTS: We found an increased level of IgG bound on patient's granulocytes collected after TRALI, in the absence of detectable granulocyte and HLA class I antibodies in the five implicated donors. One of them showed HLA-DR 1 and -DR 51 antibodies, which determined a positive cross-match with patient's B lymphocytes and in vitro activated granulocytes. Both HLA class II antigens were present in the recipient and absent in the donor. CONCLUSIONS: In some pathological conditions, HLA class II antibodies can react with activated granulocytes expressing HLA-DR antigens, and activate TRALI reaction. HLA class II antibodies screening and flow cytometry cross-matching techniques should be added to the current diagnostic algorithm of TRALI.     

Use of leucocyte-poor blood components and HLA-matched-platelet donors to prevent HLA alloimmunization

Recent studies have shown that the incidence of alloimmunization due to repeated platelet transfusions from random donors may be reduced by the use of leucocyte-poor blood components. These results were confirmed by this study, where 16% of patients with acute leukaemia undergoing initial chemotherapy and receiving leucocyte-poor blood components developed lymphocytotoxic antibodies, compared with 48% of patients in a control group receiving standard (non-leucocyte-depleted) blood components. In a third group, who received leucocyte-poor blood components and HLA-matched platelets, none of the patients developed lymphocytotoxic antibodies. There was a low incidence of platelet-specific antibodies (8%) but no difference between the three groups. Improved methods of removing leucocytes from blood components appear to offer the best approach for minimizing HLA alloimmunization, as the provision of HLA-matched platelet donors for prophylactic platelet support of all patients is not feasible.     

Donor-HLA-incompatible marrow transplantation with an anti-donor cytotoxic antibody in the serum of the patient

A 42-year-old female with acute mixed lineage leukemia received a marrow transplantation from an HLA non-identical sibling. The serum of the patient showed a positive crossmatch for anti-donor lymphocytotoxic antibody and exhibited a complement-mediated cytotoxicity to donor hematopoietic progenitor cells. In an attempt to reduce the risk of graft rejection, a large volume plasma exchange was performed, which was followed by an infusion of irradiated donor lymphocytes to eliminate remaining antibodies from her serum. The level of anti-donor antibody fell below the sensitivity of the anti-human immunoglobulin lymphocytotoxicity test after the infusion of donor lymphocytes. The cytotoxic activity against donor progenitor cells also disappeared from the serum. Cyclosporin had been administered for 2 weeks before marrow infusion, and methylprednisolone and prednisolone for 1 week before the initiation of conditioning chemoradiotherapy. Conditioning comprised cytosine arabinoside 5.6 g/m2, cyclophosphamide 4500 mg/m2 and fractionated total body irradiation with 15 Gy followed by an infusion of 4.0 x 10(8) cells/kg of unmodified marrow cells. Engraftment of donor cells was documented by HLA typing of peripheral lymphocytes. A sustained engraftment may be obtained in a donor-incompatible HLA non-identical marrow transplantation with anti-donor antibody by elimination of the antibody and achieving an intensive immunosuppression in the recipient before marrow infusion.     

Frequency and severity of transfusion-related acute lung injury – German haemovigilance data (2006–2007)

Objective  In an observational cohort study (2006–2007) the Paul-Ehrlich-Institut collected epidemiological data to investigate the frequency and causes of TRALI.
Methods  Diagnosis of TRALI was confirmed according to criteria of the European Haemovigilance Network. Subsequent testing of white blood cell antibodies (WBC-Ab) against HLA or human neutrophil alloantigens was performed.
Results  Of a total of 187 reported TRALI cases, 44 could be confirmed consisting of 35 cases of antibody-mediated TRALI and nine cases of non-immune-mediated TRALI. Eight of 44 affected patients (18%) had a fatal outcome, seven cases with WBC-Ab positive plasma donors and one case with red blood cell donors. WBC antibodies were found in one male and 39 female donors. In 34 female donors, a history of pregnancy was confirmed. WBC-Ab positive donors presented four HLA class I antibodies, 15 HLA class II antibodies, 13 HLA class I and class II antibodies, one HNA-2a, and seven HNA-3a antibodies. WBC antibodies matching with recipient antigens were found exclusively in 28 female donors; 26 FFP donors, one platelet donor and one red blood cell donor. Reporting frequency of immune-mediated TRALI was 1 : 66 000 for fresh frozen plasma, 1 : 2.86 million for red blood cell concentrates and 1 : 420 000 for platelet concentrates. Reporting frequency of TRALI-related fatalities was 1 : 285 000 for FFP.
Summary  Haemovigilance data show the significance of female donors with a history of pregnancy for the development of antibody-mediated TRALI. Manufacturing of FFP from male plasma and female donor screening for WBC-Ab could represent preventive measures.     

Fatal graft-versus-host disease following blood transfusion in Hodgkin's disease documented by HLA typing

Fatal graft-versus-host disease (GVHD) developed in a patient with Hodgkin's disease treated with combined chemotherapy and radiotherapy following the transfusion of 2 U of packed red blood cells. Clinical features of the GVHD included the development of exfoliative dermatitis, progressive hepatic dysfunction, aplastic anemia, and finally progressive fatal pneumonia. GVHD was documented by skin biopsy and chimerism by HLA typing. The HLA phenotype of the patient's skin fibroblasts [A3, Bw44 (w4)/A2, B15 (w4)] was appropriate for parental haplotypes and probably represented her true HLA phenotype. Lymphocytes from the patient (peripheral blood and lymph node biopsy) were of a different HLA phenotype (A3; Bw35, w38, w4, w6; Cw4), which was inappropriate for parental HLA haplotypes but identical to the HLA phenotype of one of the blood donors. The HLA-DR typing of the patient's family and of the blood donor demonstrated that the patient and the donor probably were HLA-DR identical (DRw5/DRw6), although no B lymphocytes could be obtained from the patient for direct DR typing. We are currently irradiating all blood products administered to patients with Hodgkin's disease receiving intensive treatment. Further observations will be necessary to determine whether transfusions to other cancer patients with immunodeficiency states should be restricted to irradiated blood products.     

A case report of transfusion-related acute lung injury during plasma exchange therapy for thrombotic thrombocytopenia purpura

Transfusion-related acute lung injury (TRALI) is a transfusion reaction that is often under recognized and underreported. Implications for diagnosis not only influence treatment considerations but also extend to donor selection, donor deferral and ultimately the safety of the final blood product. We report a case of a previously well 19-year-old female who presented a one week history of flu-like symptoms and mucosal bleeding. Laboratory results confirmed the diagnosis of thrombotic thrombocytopaenia purpura (TTP) and she was commenced on plasma exchange. During her second day of plasma exchange, she developed dyspnoea and rigors. Examination and investigation findings were consistent with a clinical diagnosis of TRALI. Granulocytes immunofluorescent test (GIFT - flow cytometry) was performed and cross reactivity was demonstrated between the patient's granulocytes and plasma from one of the nine donor fresh frozen plasma (FFP) packs. She made a full recovery. TRALIa accounts for 7% of all adverse events reported in the Serious Hazards of Transfusion (SHOT) database and has a mortality rate between 5-25%. Apheresis patients are a particularly vulnerable group of patients where clinical recognition and rapid laboratory confirmation of TRALI is imperative to minimize the risk of further patient exposure to donor granulocyte or human leukocyte antigen (HLA) antibodies. The provision of plasma from male donors may additionally reduce exposure. On a wider scale, rapid donor identification and deferral maintains the safety of the national blood supply.     

HLA-B8 in caucasian patients with systemic lupus erythematosus.

HLA antigen frequencies were determined in 27 Caucasians with systemic lupus erythematosus (SLE). A statistically significant association between HLA-B8 and SLE was found. HLA-B8 occurred in 48% of patients and in 18% of controls (P corr approximately equal to 0.005). The relative risk of SLE for HLA-B8 carriers is 4.23. In addition, SLE patients with later onset of disease more frequently had HLA-A1 and/or HLA-B8. There was no association between any HLA antigen and clinical (renal, central nervous system, and lung involvement) or serological (antinuclear and anti-nDNA antibodies) parameters.     

Presentation of human minor histocompatibility antigens by HLA-B35 and HLA-B38 molecules.

Cytotoxic T lymphocyte (CTL) clones specific for human minor histocompatibility antigens (hmHAs) were produced from a patient who had been grafted with the kidneys from his mother and two HLA-identical sisters. Of eight CTL clones generated, four recognized an hmHA (hmHA-1) expressed on cells from the mother and sister 3 (second donor); two recognized another antigen (hmHA-2) on cells from the father, sister 2 (third donor), and sister 3; and the remaining two clones recognized still another antigen (hmHA-3) on cells from the father and sister 3. Panel studies revealed that CTL recognition of hmHA-1 was restricted by HLA-B35 and that of hmHA-2 and hmHA-3 was restricted by HLA-B38. The HLA-B35 restriction of the hmHA-1-specific CTL clones was substantiated by the fact that they killed HLA-A null/HLA-B null Hmy2CIR targets transfected with HLA-B35 but not HLA-B51, -Bw52, or -Bw53 transfected Hmy2CIR targets. These data demonstrated that the five amino acids substitutions on the alpha 1 domain between HLA-B35 and -Bw53, which are associated with Bw4/Bw6 epitopes, play a critical role in the relationship of hmHA-1 to HLA-B35 molecules. The fact that the hmHA-1-specific CTLs failed to kill Hmy2CIR cells expressing HLA-B35/51 chimeric molecules composed of the alpha 1 domain of HLA-B35 and other domains of HLA-B51 indicated that eight residues on the alpha 2 domain also affect the interaction of hmHA-1 and the HLA-B35 molecules.     

Human neutrophil antibodies in a blood donor population: a lookback study

Background and Objectives Human neutrophil antibodies (HNA) have been associated with severe transfusion‐related acute lung injury (TRALI). We identified HNA antibodies in a blood donor population and performed an observational lookback on patients who received products from these donors to determine whether TRALI was associated with these transfusions. Materials and Methods Human neutrophil antibodies were determined in 1171 blood donors (388 non‐transfused males, 390 human leucocyte antigen (HLA) antibody–negative females and 393 HLA antibody–positive females) for IgG and IgM antibodies using a flow cytometric assay. Selected positive samples had a monoclonal antibody immobilization of granulocyte antigen (MAIGA) and neutrophil genotyping performed to confirm specificity. Lookback was performed on patients receiving blood from donors with positive samples by extracting recipient data from hospital medical records. An expert panel of three pulmonary critical care physicians reviewed the summarized data and assigned a diagnosis of TRALI, possible TRALI, cannot distinguish between TRALI and TACO, TACO and other. Results Eight donors had HNA antibodies of which five contributed to this lookback (3‐HNA‐specific antibodies, 2‐HNA non‐specific antibodies). Seventy‐six blood products were transfused from these donors into individual patients. One patient developed TRALI that was associated with a donor with a non‐specific HNA antibody as well as class‐I and class‐II HLA antibodies. Conclusion The incidence of TRALI in this lookback was low and combined with low frequency of HNA antibodies in the donor population suggests not screening donors for HNA antibodies at this time is acceptable.     

Klebsiella ''modifying factor'': binding studies with HLA-B27+ and B27- lymphocytes.

On the basis that extracts of some klebsiella organisms bind selectively to the lymphocytes of HLA-B27+ individuals and induce the appearance of new antigens, attempts were made to detect the binding of klebsiella products to HLA-B27+ and B27- lymphocytes by a number of different techniques. Firstly, blocking of the binding of two different HLA-B27 specific monoclonal antibodies to HLA-B27+ lymphocytes has been examined following exposure of the lymphocytes to a cell-free culture filtrate from K. pneumoniae K21 and K43. There was no reduction in the cytotoxicity of either antibody, suggesting that neither of the epitopes detected by the anti-HLA-B27 monoclonal antibodies is a binding site for klebsiella products. Secondly, we have studied the binding of partially purified, radiolabelled klebsiella products to healthy HLA-B27+ and B27- lymphocytes. There was no significant difference either in terms of numerical counts bound or by comparing, by SDS-PAGE analysis, the molecules bound to each cell type. At the level of sensitivities of these techniques we can detect no difference in binding of klebsiella products to the lymphocytes of healthy HLA-B27+ and B27- individuals.     

Transfusion-Associated Graft-versus-Host Disease in an Immunocompetent Patient following Cardiac Surgery

Objectives: We determined which of the 22 blood components obtained from unrelated donors and transfused to an apparently immunocompetent patient following open heart surgery caused transfusion-associated graftversus-host disease (TA-GVHD). Methods: Serologic and molecular methods were used to type the donors, the patient's family members, and the patient's postmortem tissues for HLA and a genetic marker on chromosome 17. Results: Two donors were homozygous for the HLA class I antigens A1 B8, for which the patient was heterozygous. Both donors were heterozygous, not homozygous as expected, for the class II alleles. One of them had the same class II alleles as the patient (DRB1*0301, DRB3*0101/DRB1*0404, DRB4*0103). The patient's tissues were chimeric for restriction fragments at 17 p 13 of this donor. Conclusion: One-way HLA match leading to TA-GVHD can be caused by donor blood that is homozygous for class I and heterozygous for class II alleles. Two blood components given to our patient had such one-way HLA match. Class II alleles of the lymphocytes in one component were identical with those of the recipient and caused TA-GVHD. Class II alleles of the lymphocytes in the other component differed from those of the recipient and were eliminated either by the immune system of the patient or the lymphocytes that caused the TA-GVHD (graft versus graft).     

Cold reactive lymphocytotoxic activity in autoimmune thyroid disease

An increased incidence of cold-reactive lymphocytotoxic activity (LCTA) has been demonstrated in the sera of patients with autoimmune thyroid disease. Twenty-six of 79 (33%) patients with Graves' disease and 9 of 21 (43%) patients with Hashimoto's thyroiditis had cold-reactive LCTA detected by microcytotoxicity assay compared to 6 of 42 (14%) normal controls. There was no correlation between LCTA and age, sex, MCHA titre or TGHA titre. A positive correlation with FTI and LCTA in Hashimoto's patients was demonstrated, but no such correlation was demonstrable in Graves' patients. The lymphocytotoxic activity was directed preferentially against B cells. There was no preferential lysis of T-cell subsets as defined by monoclonal antibodies, and the lymphocytotoxins were equally reactive with normal lymphocytes and toxic Graves' lymphocytes. The significance of cold-reactive lymphocytotoxic activity in the pathogenesis of autoimmune thyroid disease remains to be determined.     

Relationship of HLA and platelet-reactive antibodies in alloimmunized patients refractory to platelet therapy

Platelet crossmatching assays have been used to predict the outcome of platelet transfusions in alloimmunized patients by detecting antibodies against platelets. The transfusion failure of HLA-matched platelets predicted by platelet crossmatching may be related to HLA antibodies undetected by lymphocytotoxicity but detected by platelet immunoglobulin-binding assays or platelet-specific antibodies (both antibodies defined here as platelet-reactive antibodies). To differentiate platelet-reactive antibodies from lymphocytotoxic HLA antibodies, we used HLA characterized lymphocytes in parallel with platelets from individuals to form separate frozen panels. Sera from 10 allosensitized patients were studied in the lymphocyte panel by lymphocytotoxicity and in the platelet panel by enzyme-linked immunoassay (ELISA). By comparing pattern and percent wells reacting in each panel, lymphocytotoxic HLA antibodies and antibodies reactive with platelets in ELISA were detected separately. In all 10 allosensitized patients, platelet-associated antibodies were present and 7 had additional lymphocytotoxic HLA antibodies. Using this double parallel panel technique, we found platelet-reactive antibodies important in platelet alloimmunization, unrecognized by lymphocytotoxicity. These data indicate platelet-crossmatching be solely used in the selection of platelets for allosensitized patients.     

Matching of histocompatibility (HL-A) antigens for platelet transfusion.

The average in vivo platelet survival was measured by both per cent recoveries (%R) and half-lives (t 1/2) of 51Cr-labeled platelets in 69 transfusions given to 43 thrombacytopenic patients. The results demonstrated that (1) increase in the number of HL-A incompatibilities in the platelet donor was significantly associated with decreased %R and t 1/2 of the infused platelets; (2) survival of transfused platelets from HL-A-matched single donors was consistently superior to those pooled from several grossly mismatched donors; and (3) survival of platelets infused when patients had circulating lymphocytotoxic antibodies was consistently lower than when patients did not have such antibodies, regardless of whether it was the first, second, or third such infusion.     

Transplant of rhesus-positive bone marrow in a rhesus-negative woman having anti-rhesus D alloantibodies

A woman affected by acute myeloblastic leukemia was grafted with HLA A, B and D compatible rhesus-positive bone marrow from her brother. Before grafting, she had anti-D alloantibodies (1/512 IAT, 2.9 micrograms/ml). To prevent the destruction of donor red blood cells, four plasma exchanges and a conditioning regimen (total-body irradiation 800 rad, cyclophosphamide, methotrexate) were carried out to decrease anti-D from 2.9 to less than 0.02 micrograms/ml on day 0. The anti-D level was 0.8 micrograms/ml on day 12 and was decreased to 0.2 micrograms/ml by eight plasma exchanges until day 35. Anti-D antibodies were undetectable with Lalezari's technique on day 45. Engraftment was obtained on day 25 (3,000 leukocytes/mm3 and 50% erythroblasts in bone marrow). The patient died from aspergillosis and graft-versus-host disease on day 54. This observation shows that an engraftment of rhesus-positive bone marrow in a recipient with anti-D antibody is possible.     

Endothelial Monocyte Antigens Are Coded for by Loci Centromeric of HLA-B

900 pregnancy sera were screened for monocyte antibodies. 23 sera (2.6%) were found to be reactive with monocytes in an allotypic pattern distinct from blood group ABO, HLA-A,B,C, DR and DQ specificities. Because of strong reactions and high reproducibility, 4 sera with pure endothelial monocyte (EM) reactivity and 2 sera also positive with B lymphocytes of certain donors were selected for population and family studies. The frequency of positive reactions in a panel of 26 random donors obtained with the pure EM sera was 13, 8, 8 and 13% with clearly distinct patterns. Analysis of the tentatively designated EM antigens 1.1, 1.2, 2.1, and 2.2 in 7 families revealed definite segregation with HLA. By means of two crossing-over events within the HLA region the two hypothetical EM loci were localized centromeric of HLA-A and one of both loci centromeric of HLA-B.     

Class I and Class II HLA Antigens in a Homogeneous Argentinian Population with Whipple''s Disease: Lack of Association with HLA-B 27

The prevalence of class I and class II HLA antigen was analyzed in 14 patients (12 males, two females) with Whipple's disease, diagnosed an average of 9.7 yr (range 6 months to 25 yr) before the typing. They were compared with 174 healthy control subjects of the same geographic area in Argentina. Class I antigens (locus A, B, C) were studied by lymphocytotoxic test, and class II antigens (locus DR, DQ) were detected by the double immunofluorescence technique. HLA-B27 was positive in one patient (7.7%) and in 4% of the control population. No significant association was found with the antigens tested. We observed no difference in the clinical picture or in the frequency of arthralgias, compared with those reported in the literature. Our data suggest that there is no conclusive proof of an association between HLA-B27 and Whipple's disease.