HLA Antibody Screening Research Articles

Peri-Transplant Blood Transfusion Is Associated With Significant Formation of HLA Antibodies Despite Immunosuppression.

Blood transfusion is a recognised source of allo-sensitization in transplantation. Despite pre-treatment with immunosuppressive therapy transfusion in the perioperative period may potentially increase the risk of sensitization. We studied the prevalence of transfusion during the transplantation period in our centre and analysed the impact on HLA antibody status. We retrospectively analysed all adult kidney recipients transfused with leuko-depleted red blood cells and platelets during the first 48 hours of transplantation surgery from January 2010-April 2013. HLA antibody screening was performed by single antigen Luminex® bead assay (GenProbe). HLA antibodies and donor specific antibodies (DSA) pre- and post-transplantation (between days 14-31) were analysed. All patients (n=187) received corticosteroid, tacrolimus and mycophenolic acid based immunosuppression. We also examined the occurrence of Delayed Graft Function (DGF) and incidence of acute rejection in the first 2 months post transplant compared with an un-transfused control group. Among 187 patients included in the study, 67 patients (36%) received blood transfusion (median 2 units) and 120 patients (64%) were not transfused. 18 patients (29%) among the transfused group developed HLA antibodies compared to 12 (10.4%) in the non-transfused group. The risk factors for sensitization with transfusion were female recipients, second graft recipients, primary polycystic kidney disease and non-Caucasian populations- all were more likely to develop HLA Antibodies. Transfused kidney transplant recipients developed clinically significant HLA antibodies (MFI>300) [ Odds ratio 3.37] in 26% of cases. A higher rate of DGF was observed in the transfused group compared to the un-transfused group. Conclusion: Despite immunosuppressive drug therapy with tacrolimus, mycophenolic acid, cortico-steroid and IL2Receptor blockade, blood transfusion in the peri-operative period is associated with significant development of HLA antibodies. DISCLOSURES:Coates, P.: Speaker's Bureau, Alexion, Other, Novartis, Advisory Board.

Evaluation of Donor-Specific Antibodies Through 5 Years With Belatacept in BENEFIT and BENEFIT-EXT.

Background: Donor-specific antibodies (DSA) are associated with negative post-transplant outcomes, including increased risk of antibody-mediated rejection and graft failure. De novo (DN) DSA occur in ˜11 % of kidney transplant recipients by 1 year and in ˜20% by 5 years (Everly et al, Transplantation 2013;95(3):410-7). The 3 year ITT results from BENEFIT and BENEFIT-EXT showed that belatacept was associated with lower rates of DN DSA than CsA (3% vs. 8%, Larsen C et al, ESOT 2011 [presentation O244]). Here we report the incidence of DN DSA through Year 5 in the long-term extensions (LTE) of these studies. Methods: In BENEFIT, patients received living donor or SCD kidneys, and in BENEFIT-EXT, patients received ECD kidneys. Patients received more (MI) or less intensive (LI) belatacept or CsA and could enter the LTE after 3 years. The presence of DSA was established centrally by comparing the HLA type of donors and recipients, identifying the recipient’s HLA antibodies, and then determining if any of those HLA antibodies were donor directed. HLA antibody screening was performed by solid phase flow cytometry (FLowPRATM), and specificity (Class I and II) assessed by LabScreenTM single antigen beads (One Lambda, Inc.). Results: From baseline through Year 5 in the LTE cohort of both studies, numerically fewer belatacept- vs. CsA-treated patients developed DN DSA (Table): in BENEFIT, 3 (1.9%) MI, 6 (3.6%) LI, and 16 (11.8%) CsA, and in BENEFIT-EXT, 7 (6.7%) MI, 1(0.9) LI, and 9 (10.3%) CsA-treated patients developed DN DSA. Conclusions: These data demonstrate a reduced rate of DN DSA with belatacept vs. CsA from baseline through 5 years, consistent with the findings observed through 3 years.Table: No Caption available.DISCLOSURE:Ganguly, B.: Employee, Bristol-Myers Squibb, Stockholder, Bristol-Myers Squibb. Townsend, R.: Employee, Bristol-Myers Squibb. Meier-Kriesche, U.: Employee, Bristol-Myers Squibb. Harler, M.: Employee, Bristol-Myers Squibb.

Successful Unintentional ABO-Incompatible Renal Transplantation

To report a successful unintentional transplantation of a deceased donor kidney from an "incompatible" A1B donor into a recipient who was blood group A2B with unsuspected preformed anti-A1 antibodies. The donor and recipient were both typed for ABO antigens. The recipient was tested for ABO and non-ABO antibodies. The recipient was typed for HLA class I and class II antigens, including HLA antibody screen. The T-and B-flow cytometry crossmatch test was performed using standard protocol. The donor-recipient pair was a complete six-antigen human leukocyte antigen mismatch, but final T- and B-flow cytometry cross-match tests were compatible. The recipient was a 65-year-old woman with a medical history of end-stage renal disease secondary to diabetic nephropathy who underwent kidney transplantation from a 46-year-old brain-dead standard criteria donor. The recipient's RBCs were negative with A1 lectin, and the recipient was thus typed as an A2 subgroup. Anti-A1 could be demonstrated in the recipient's plasma. The donor's RBCs were positive with A1 lectin, thereby conferring an A1 blood type. It is safe to transplant across the A1/A2 blood group barrier provided that the preformed antibodies are not reactive at 37°C and with anti-human globulin.

Persistence of recipient human leucocyte antigen (HLA) antibodies and production of donor HLA antibodies following reduced intensity allogeneic haematopoietic stem cell transplantation.

The effects of reduced intensity conditioning (RIC) on human leucocyte antigen (HLA)-alloimmunization and platelet transfusion refractoriness (PTR) following allogeneic haematopoietic stem cell transplantation (Allo-HSCT) are unknown. We studied HLA-alloantibodies in a cohort of 16 patients (eight HLA-alloimmunized with pre-transplant histories of PTR and eight non-alloimmunized controls) undergoing Allo-HSCT using fludarabine/cyclophosphamide-based RIC. Pre- and post-transplant serum samples were analysed for HLA-antibodies and compared to myeloid, T-cell and bone marrow plasma cell chimaerism. Among alloimmunized patients, the duration that HLA-antibodies persisted post-transplant correlated strongly with pre-transplant HLA-antibody mean fluorescence intensity (MFI) and PRA levels (Spearman's rank correlation = 0·954 (P = 0·0048) and 0·865 (P = 0·0083) respectively). Pre-transplant MFI >10,000 was associated with post-transplant HLA antibody persistence >100 d (P = 0·029). HLA-antibodies persisted ≥100 d in 3/8 patients despite recipient chimaerism being undetectable in all lympho-haematopoietic lineages including plasma cells. Post-transplant de-novo HLA-antibodies developed in three control patients with two developing PTR; the donors for two of these patients demonstrated pre-existing HLA-antibodies of equivalent specificity to those in the patient, confirming donor origin. These data show HLA-antibodies may persist for prolonged periods following RIC. Further study is needed to determine the incidence of post-transplant PTR as a consequence of donor-derived HLA alloimmunization before recommendations on donor HLA-antibody screening can be made.

Acute Antibody-mediated Rejection Possibly Due to Anti–human Leukocyte Antigen DQB1 Antibodies after Renal Transplantation – Case Report

A 61-year-old Japanese woman, who had undergone hemodialysis because of chronic glomerulonephritis, received a living renal transplant from her ABO blood type–compatible spouse. HLA typing of A, B and DRB showed 3/6 mismatches. Complement-dependent cytotoxicity crossmatches, HLA antibody screening with the use of flow panel reactive antibody (PRA), and flow cytometry crossmatches (FCXM) were all negative. Tacrolimus, mycophenolate mofetil, methylprednisolone (MP), and basiliximab induction were used as the standard immunosuppressive therapy. After renal transplantation, her serum creatinine level favorably decreased, but urine output was not sufficiently obtained, contrary to our expectations. Doppler sonography revealed disappearance of diastolic arterial flow on postoperative day 2. The episode biopsy showed acute antibody-mediated rejection (AMR) based on the current Banff classification, although FCXM and flow PRA were still negative. To determine the cause of acute AMR, we expanded the HLA typing at high resolution levels to Cw, DQB1, and DPB1. Retrospective analysis of perioperative sera demonstrated the presence of low levels of donor-specific HLA IgG and moderate levels of IgM antibody against DQB1 before transplantation. There was an elevation of IgM antibody at the time of rejection, whereas IgG antibody showed no remarkable change. AMR was successfully treated with plasma exchange, low-dose intravenous immunoglobulin, high-dose intravenous MP pulse, and rituximab.

Pitfalls in interpreting anti-HLA antibodies by Luminex technology

The Luminex technology has become an important tool for HLA antibody screening and identification. This is the most sensitive technology to detect HLA antibodies for transplant patients and patients on awaiting list, and it has ushered a new strategy to determine HLA compatibility between donor and recipient. Moreover, the clinical relevance of all detected anti-HLA antibodies is not well understood, because this technique was shown to be prone to many artefacts or interferences, leading to a complicated interpretation for biologists and clinicians. Our objective in this article is to provide a careful consideration about this solid phase assay, and to focus attention on raised questions about technical performance and interpretation of the results. We should keep in mind that our results could change the clinical management of sensitized patients, their aptitude to receive a graft, and their follow-up.

14-P

The trend to HLA antibody testing by multiplex immunoassays has led us to question the current role of the ELISA test in our HLA antibody screening. HLA antibody testing algorithms are used for efficiency and for standardization. The ELISA test has been part of the screening route. Routine HLA antibody screening is the first step of the algorithm. Initially, our lab used cytotoxicity and ELISA tests for HLA antibody screening. We currently screen renal candidates’sera by ELISA Class I alternating with the Luminex Class I/II screen methods. The conflicting results require follow-up tests. In the ASHI Proficiency survey, less than 10 labs are reporting results by the ELISA method, thus the results are not graded. The ELISA Class I Screen is cost effective for periodic PRA screening of our renal waitlist patients. We have observed results on some sera that tests positive on ELISA and then negative on a luminex screen. These patients(< 10% of all listed patients) exhibit high background on luminex platform. Conversely some patients’ sera test positive on the luminex screen and negative on the Elisa screen. The conflicting results could lead to confusing interpretation; however, in those patients with an established (3 to 6 succesive screens) negative, the change to positive or indeterminate has indicated a change in patient health or a sample discrepancy. A review of our results on ELISA screens over the past five years, (2007 through 2012) indeterminate or repeated tests were observed to be lot dependent, more than tech dependent. The current algorithm includes ELISA Class I screen, luminex screen (ClassI/II), and luminex single antigen tests. The elimination of this ELISA test is being considered as the more specific luminex screen result contributes information, including Class I and II antibody simultaneously. Although a reliable test methodology, the reduction in reagent cost, QC and proficiency on this less sensitive test would be a benefit of elimination.

99-P: HLA ALLOIMMUNIZATION FROM RED CELL TRANSFUSIONS IN NEWLY DIAGNOSED TREATED ACUTE MYELOID LEUKEMIA PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION

Aim HLA alloimmunization from leucodepleted RBC transfusions prior to hematopoietic stem cell transplantation (HSCT) is not well defined. Donor specific HLA antibodies (DSA) are associated with a 2- to -10 fold increase of graft failure of HLA mismatched HSCT, irrespective of the type of graft or patient conditioning. A less explored aspect is the presence of non-DSA antibodies prior to transplantation. Methods We reviewed 212 (114 [54%] M: 98 [46%] F; median age 58 [17-84] years) newly diagnosed AML patients of which 48 (22.6%) underwent HSCT following chemotherapy. All 48 [26 (54%) M: 22 (46%) F], median age 52 (17-73) years were screened for HLA antibodies (LABSCREEN, Luminex) prior to HSCT and received leucodepleted RBC and platelets transfusions. Of the 22 females (median age 48 [19-71] years), 8 had no history of a pregnancy and 14 had a median of 2 (1-3) pregnancies. Results HLA antibodies were found in 18 (37.5%) patients (5 [28%] M:13[72%] F) who received a median of 17 (6-44) RBC and 12 (4-69) platelet transfusions. In 30 (62.5%) patients (21 [70%] M: 9[30%] F) the HLA Antibody screen was negative and they received a median of 15 (5-44) RBC and 8 (2-28) platelet transfusions. The grafts were from 21 matched related/ 23 matched unrelated/ 4 double cords donors. 17 (35%) patients received HLA mismatched transplants (13-1DP mm/3-2 DPmm/1-C mm). The following HLA antibodies were found in males (A∗24:03; B37,57,58,72,78, DR1,4) and in females (A∗68:02, A1,2,3,4,6,11,23,25,29,30,31,33,A∗34:03,43,66,68,69; B∗44:03, B7,45,51,57,58,67,76,78,82; Cw4; DQ4, DQB1∗06:03,06:09,13:03,DQA1∗05; DR4,8,11,12,13,14,15,16,17,18,52, DRB3∗01:01,03:01,04:02, DP2,10,14,18, 28. Conclusions All of the patients engrafted excepted for one who received a 2 DP mismatched transplant with no pre-transplant HLA antibodies. From this review, HLA alloimmunization secondary to red cell transfusion is only significant in males (p = 0.03) in our patient population. This finding should be however be confirmed in a larger study.

14-OR

To determine the incidence and clinical significance of donor specific antibodies (DSA) in renal allograft recipients with functioning grafts after more than 10 years post-transplantation. HLA antibody screening and identification were performed using One Lambda Single Antigen Bead (SAB) assay. Antibody capacity to fix complement was tested using the C1q assay (One Lambda). Serum samples were obtained from 182 recipients (mean age 43, female 35%, re-transplants 10%, live donor transplants 52%) with functioning grafts after more than 10 years post-transplantation (median time from transplantation 14 yrs, range 10-32 years). All recipients had a negative CDC crossmatch at the time of transplantation. After >10 years post-transplantation, 47% of the patients showed no evidence of sensitization to HLA antigens (cPRA ⩽ 10%, Ab-), 26% displayed cPRA > 10% in the absence of DSA (Ab+DSA-), and 25% displayed cPRA > 10% with DSA (Ab+DSA+). DSA directed to HLA class I, class II or both were identified in 9%, 14% and 2% of patients, respectively. The antibodies most frequently observed were directed to donor DQ antigens (8% of patients). To determine whether DSA had the capacity to bind complement, DSA+ sera were tested using the C1q assay. C1q binding DSA were observed in 47% of the DSA containing sera. Current serum creatinine levels were significantly higher in the Ab+DSA+ group compared to Ab- or Ab+DSA- group. However, there was no difference between creatinine levels in patients with C1q versus non-C1q binding DSA [ Table 1 ]. These results indicate that there is a close correlation between DSA detected late post-transplantation and graft function. Thus, DSA monitoring may assist in the long term management of renal transplant recipients and identifies patients with DSA who should not be considered for reduction in immunosuppression.

64-P: HEPATITIS B VACCINATION MAY CAUSE HLA SENSITIZATION

Hepatitis B vaccination for dialysis patients is generally thought to be non-immunogenic. However, flu vaccination is a proposed mechanism of HLA sensitization [Ref 1-3] and other vaccines may also be implicated. We reviewed the Hep B vaccination history of the hemodialysis patients (HD pts) in our centre and correlated this with results of HLA antibody screening. We studied 68 HD pts awaiting kidney transplantation. Of these, 19 were patients awaiting re-transplantation (regraft patients). Our results demonstrated a temporal surge in panel reactive antibodies (PRA) in 3/32 HD pts after Hep B vaccination. The average change in PRA was at least 20% in Class I and 20% in Class II. Sensitization was more common in regraft patients (1 in 8). Two additional dialysis patients (∗) from outside of this cohort were also identified and one of them was still on immunosuppression (IS). Possible sensitization due to transfusion was ruled out during vaccination period except for 1 patient (#). [ Table 1 ] [ Table 2 ] i) HLA sensitization following Hep B vaccination appears to occur with a frequency comparable to that caused by flu vaccination [1-3]. ii) Regraft patients represent the group with the highest risk for sensitization. iii) Given the relatively low risk of Hep B infection in North America, we propose that the vaccination policy be reviewed particularly for regraft patients. Ref [1] AJT 2013;13(4):1026-33 [2] Transpl Infect Dis. 2012;14(6):575-83 [3] AJT 2011;11(8):1727-33.

3-OR

Current Hematopoeitic cell transplant (HCT) donor selection strategies do not include matching at the DP locus and this locus is rarely typed for in either the related or the unrelated potential donor setting. Since the advent of the solid phase assays for detection of anti-HLA antibodies, our ability to successfully detect and determine specificity of both Class I and II antibodies has been greatly improved, particularly following the introduction of the single antigen bead platforms. We report on 3 HCT candidates who developed strong antibodies to DP (DPA/B subunits) which were found to profoundly affect the donor selection process and hence the possibility for transplant. Single Antigen HLA antibody screening was performed using One Lambda LABScreen Single Antigen kits. Results were evaluated for the presence of DP antibodies. DPA/B typing was performed using either One Lambda LabType SSO kits and/or Micro SSP trays. At our center, all candidates for HCT are screened for the presence of anti-HLA antibodies. Antibody screening results become particularly important and are used to guide donor selection in instances where patients, due to the unavailability of matched related donors or URD are restricted to mismatched, haplotype matched or cord blood donors. We report the case of 3 patients who developed strong antibodies to both the DPA1 and DPB1 subunits of DP. The strength of reactivity of the DP single antigen beads ranged from 1K- > 15K MFI and resulted in positive B-cell CDC and Flow crossmatches with appropriately MM DP donors. Finding appropriate HCT donors, lacking the offending DPA/B antigens impacts the HCT donor search process significantly. 1 patient, with rarer HLA types was unable to proceed to transplant with a URD for 2.5 years. These 3 cases highlight the influence that the presence of DP antibodies can have on the donor selection process and on the possibility of transplant. Pereira: Linkage Biosciences: Consultant; Science Med Advisor; Stockholder; Scisco Genetics: Consultant; Science Med Advisor.

180-P: ANTI-HLA-CLASS-I ANTIBODIES AND PLATELET TRANSFUSION REFRACTORINESS

It is known that: 1) anti-class-I-HLA antibodies are responsible for platelet transfusion refractoriness, 2) leucocytes in transfusion products sensitise patients to produce anti HLA antibodies, 3) leucodepleted blood products reduce but do not eliminate HLA sensitisation. It is not clear what the level of platelet refractoriness risk is in HLA-sensitised patients, therefore it is not standard practice to screen for HLA antibodies in platelet transfusion. This is a pilot study to assess that risk and measure the value of HLA-antibody screening in platelet transfusion. This study looks at the relationship between HLA-class-I antibody status and the total number of units of transfused platelets is studied. Since platelets are transfused according to a standard transfusion trigger, the total number of platelets units transfused indirectly measures thrombocytopenia and platelet transfusion refractoriness. Leukaemia patients with anti-HLA-class-I antibodies use on average 36 more units of platelets than patients without antibodies. Sensitised patients: average = 120 units, sd = 120 unit, n = 82. Non-sensitised patients: average = 84 units, sd = 112 units, n = 73. Using a cutoff point of 105 units a 2x2 contingency table shows a likelihood ratio of 2.29 and a chi square of 10.8, statistically significant for a p < 0.005. Conditional probabilities. 72% of patients using more than 105 units of PLT will have anti-HLA Ab 44% of patients using less than 105 units of PLT will have anti-HLA Ab 44% of patients with anti-HLA Ab will use more than 105 units of PLT 19% of patients without anti-HLA Ab will use more than 105 units of PLT. 1) Ab screens reveal the risk of excessive platelet transfusion requirements. 2) Generalised leucodepletion is not by itself sufficient to address the widespread problem of platelet transfusion refractoriness. 3) Optimal platelet transfusion may have to take into account the patient’s Ab status and the HLA and PLT Ag phenotype of both donor and recipient.

Evaluation of a computerised clinical documentation system (CCDS) implementation in the transplant laboratory

Although many handwritten reports have been replaced by computer generated ones in clinical laboratories, they still remain a common feature in many transplant laboratories for two main reasons: either the laboratory information system (LIS) does not support report formats, or it is costly to purchase a specialised database. A time and motion study was conducted to evaluate one such clinical database, the IG database. Before and after the implementation of our IG database, we evaluated the: 1 stationary cost reduction from paper, toner cartridge, and other auxiliaries 2 turn-around time from the laboratory information system (MediTech) 3 time reduction on analysing report using the electronic reports on three main testing areas; HLA typing, antibody screening, and cross-matching. Approximately $22,035 is saved annually. The turn-around time (TAT) and time on analysing report (TAR) are statistically significant in all testing areas. The IG database demonstrated a significant reduction in cost, a decrease in turn-around time and time on analysing report, and an increase in overall efficiency.

HLA Antibodies in an Untransfused Male - Unexpected Reactivity Explained by Taking a Careful History

Introduction: HLA antibodies are stimulated primarily by exposure to non-self HLA through organ transplantation, blood transfusion, or pregnancy. The occurrence of HLA antibodies in untransfused, untransplanted male subjects has been assumed to be a naturally occurring phenomenon in the absence of exposure to HLA. Patient and methods: A 44 year old man presented to a renal unit with hypertension and progressive renal failure. Renal biopsy showed severe tubular atrophy and no primary glomerulonephritis. Despite control of hypertension his serum creatinine rose from 394 umol/l on presentation to 428 umol/l six months later and he was worked up for living donor renal transplantation from his brother. Results: On screening for HLA antibodies he was found to have donor specific antibodies detected by screen and single antigen beads (MFIs:- A1 2000u, C7 1500u, DQ7 500u) and also other HLA antibodies (A31 5500u, DP23 4000u). Review of the history indicated he had previously been fit and well, with no hospital admissions and no question of administration of therapeutic blood products at any time of his life. However, he had been active for some years as a bare knuckle cage fighter. Cage fighting is a form of ‘all in’ combat with an opponent in a boxing ring enclosed in a wire cage. During these contests it was apparently common to suffer cuts on the hands and for the hands to be covered with an opponent's blood. Transplantation was performed after 2 sessions of double filtration plasmapheresis, and there was good function with no rejection. Post-transplant antibody monitoring showed no changes in the antibodies to Class 1, but the donor specific DQ7 antibody rose from MFI 500u to 3250u. Conclusion: In this patient the only documented source of HLA sensitisation was exposure to blood through cage fighting, and this underlines the importance of taking a careful history in any patient who presents with unexpected HLA antibody reactivity. Sensitisation to HLA through this route has not, to our knowledge, been previously reported.

Pronase Treatment Interfering with T Cell Crossmatch

Introduction: Pronase was used to remove Fc receptor and/or CD20 on cell surface in order to reduce non-specific reactivity of B cell flow cytometry crossmatch. However, the pronase treatment may interfere with T cells resulting in false positive T cell crossmatch. Recently, we observed three crossmatches with unexpected positive T cell reactivity. Methods: Lymphocytes isolated from either peripheral blood samples or tissues (lymph nodes or spleens) were treated with pronase prior to crossmatch. The cells were incubated with serum samples of patients. PerCP-anti-CD3 and PE-anti-CD19 conjugates were added to distinguish T and B cells. Crossmatch reactivity on cells was marked by addition of FITC-anti-human IgG conjugate. The crossmatches were analyzed by flow cytometry. Pooled positive and negative control sera were included in each crossmatch run. HLA antibody screening and identification were performed by using Flow PRA and Luminex single antigen bead assay. Results: In crossmatches of 3 recipients (R1, R2 and R3) with 3 deceased donors (D1, D2 and D3), unexpected positive T flow cytometry crossmatches were observed. All 3 recipients had no detectable HLA antibodies. The crossmatches were repeated with pronased and non-pronased D1 and D2 cells versus DTT-treated and non-DTT-treated sera of R1 and R2. A wash step following pronase treatment was added in crossmatch of D2 versus R2. In the crossmatch of D1 versus R1, a negative T cell crossmatch was obtained with non-pronased cells regardless if the serum was treated with or without DTT. Interestingly, DTT-treated serum showed a negative T cell crossmatch regardless if the cells were treated with or without pronase. In crossmatch of D2 versus R2, non-pronased cell crossmatch was negative and pronased cell crossmatch was positive regardless if the serum was treated with or with DTT and with or without a wash step. A negative T cell crossmatch was obtained for R3 when cells of D3 were not treated with pronase, while a positive T cell crossmatch was detected when cells were pronased. High dose pronase treatment resulted in stronger crossmatch reactivity for T cell and B cell as well when compared with standard dose pronase treatment. Conclusions: Our findings suggest the unexpected positive T cell crossmatches in these 3 recipients were caused by pronase treatment. While pronase treatment removed Fc receptors and/or CD20, it might have changed the conformation of other antigens on cell surface resulting in accessibility to epitopes of denatured antigens. If the recipients had antibodies against denatured antigens, a positive crossmatch might be detected. The clinical relevance of a positive crossmatch caused by antibodies to denatured antigens may not be significant.

7-OR

Aim Maternal-fetal transmission of IgG antibody is well known. We present the case of a newborn with fetal cardiomyopathy and presence of an anti-HLA antibody to a self-HLA antigen. The antibody is believed to be transmitted by mother. Methods A female infant demonstrated severe biventricular dysfunction on fetal echocardiogram without associated hydrops, structural defect, or valve insufficiency. Transplant evaluation at 5 days of life demonstrated an HLA antibody screen by flow cytometry of 55% Class I and 5% Class II PRA. Specificities by Luminex demonstrated one of the antibodies was to an infant HLA antigen, Cw7. Maternal HLA antibody screen demonstrated the same specificities. Maternal history is significant for fetal demise two years prior. Paternal HLA typing demonstrated A1 and Cw7 antigens. Infant antibodies progressively diminished with no intervention. Ventricular function improved to normal. Evaluation for etiology of the cardiomyopathy demonstrated no abnormalities. Mother later demonstrated additional sensitization from this infant. Results Table 1 . Conclusions We report the transmission of maternal HLA antibodies to a newborn with fetal cardiomyopathy. Speculation is that presence of a transmitted maternal antibody to infant HLA antigen produced ventricular dysfunction, similar to antibody-mediated rejection. Sensitization most likely occurred from the prior pregnancy.

The complement‐mediated prozone effect in the Luminex single‐antigen bead assay and its impact on HLA antibody determination in patient sera

The Luminex xMAP system has become an important tool for HLA antibody screening and identification in sera of transplant patients. Recently, the Luminex single antigen bead assay was shown to be prone to an artefact, the so called prozone phenomenon: Sera with high titer HLA antibodies gave negative results when tested neat, but reacted strongly positive after 1:10 dilution. We also observed such a phenomenon and found that it was most likely caused by the complement component 1 (C1) by competitively displacing the detection antibodies. In this article we review the complement-mediated prozone effect and other mechanisms of interference with solid phase assays, and we discuss possible consequences for HLA antibody testing with the Luminex SAB assay.

Blood donors implicated in transfusion‐related acute lung injury with patient‐specific HLA antibodies are more broadly sensitized to HLA antigens compared to other blood donors

The prevalence of HLA antibodies in randomly surveyed blood donors was compared to the prevalence of antibody in donors who were associated with transfusion-related acute lung injury (TRALI) cases reported to Canadian Blood Services (CBS). Current operating procedure mandates that the CBS TRALI Medical Review Group (TMRG) refer possible TRALI cases to the (CBS) Platelet Immunology Laboratory for investigation. Donor samples from these TRALI cases were screened for HLA antibodies. In parallel, a survey was conducted to screen serum samples from blood donors who were not associated in TRALI cases. A comparison analysis of HLA antibody profiles in the two groups of donors was performed. We studied 121 TRALI-associated donors (TDs) who were recalled in a total of 44 cases reported to CBS and classified by TMRG. We also studied 149 survey donors (SDs) who were deferred for donation for varied reasons and consented to participate in a survey for HLA antibody screening. Twenty-two percent of SDs and 50.4% of TDs tested positive for HLA antibodies. In addition, TDs who were implicated in TRALI demonstrated broader sensitization and higher level of quantitative HLA antibody compared to nonimplicated TDs and SDs. Patient-specific Class I and II HLA antibodies are directly related to the risk of TRALI. Moreover, it supports the concept that HLA antibody strength is directly related to the risk of TRALI when the HLA antibody is patient specific; however, no clear cutoff as defined by mean fluorescence intensity is evident.

Prognostic value and cost‐effectiveness of different screening strategies for HLA antibodies prior to kidney transplantation

HLA antibody screening is conducted routinely prior to kidney transplantation, but the comparative prognostic value and cost-effectiveness of different methods are unclear. Pre-transplant sera of 141 patients transplanted between 1998 and 2000 were screened by ELISA and Luminex assays, and antibody specificities of reactive sera determined using bead array techniques. ELISA screening detected donor-specific antibodies (DSA) in 19 patients, who had a higher incidence of impaired graft function (60% vs. 20%, p = 0.04) and antibody-mediated rejection (AMR) within 90 d after transplantation (AMR, 35% vs. 5%, p = 0.02). Luminex screening detected eight additional patients with DSA, among those one with AMR. Six of eight patients with Luminex-only-DSA reported no prior immunizing events. Death-censored graft survival was shorter only in patients with DSA and AMR (median, 1.7 yr instead of between 9.5 and 11.0 yr for patients without DSA or patients with DSA but no AMR, p < 0.001). Material costs per detected clinically relevant DSA were about 57% higher for Luminex screening, but this increase could be avoided by modifying the cut-off recommended by the manufacturer. Conclusively, specification of antibodies only in sera reactive in screening tests was cost-effective to prevent shortened graft survival. Preformed DSA were only harmful if AMR was diagnosed within 90 d after transplantation.

TRALI‐risk‐reduction strategies: Japanese approach

Transfusion‐related acute lung injury (TRALI) is one of the most serious complications of blood transfusion. We have been gathering information about TRALl in Japan since 1997 through our national haemovigilance system. Recently, we have 20–40 TRALI cases per year (five million bags issued per year). Although TRALI is a rare complication of transfusion, it may become severe and may sometimes be fatal. A substantial number of TRALI cases have been caused by donor HLA or HNA antibodies, which were generated in alloimmunized, i.e. parous females. Considering this mechanism, TRALI mitigation strategies such as the use of male‐only‐plasma have been implemented in many blood centres or countries. Because of the availability of male plasma and the time limit for the production of fresh frozen plasma (FFP), we first tried to prepare FFP‐LR2 derived from 400 ml whole blood only from male donors. FFP‐LR2 accounts for 75% of FFP in Japan. (Six per cent of FFP products are from 200 ml whole blood and 19% from apheresis plasma.) A preliminary project on male‐predominant plasma was started at several core blood centres. To minimize the burden on nurses regarding marking bags collected from males or females, which might lead to serious errors in collection sites, we updated the computer system that allows the staff members in the production department to sort male and female blood automatically. In a month after the system implementation nationwide, we successfully increased the production rate of male plasma for FFP‐LR2 product by up to more than 98%. Regarding FFP‐LR1 derived from 200 ml whole blood, the percentage of female donors is approximately 75%. One of the core blood centres has successfully implemented the same strategy for producing FFP‐LR1, but others have not done so yet. Although male‐only‐plasma strategies achieved good results without any expensive screening tests to reduce TRALI incidence in some countries, the standard measures for other plasma rich blood products such as platelet concentrates or apheresis plasma have not been established yet. There are some measures to consider reducing the risk of TRALI caused by other plasma‐rich products – recruiting only males as apheresis platelet donors, testing a certain number of donors for HLA antibodies, replacing the supernatant of a platelet pool with male plasma and excluding female donors from apheresis donations. Some blood centres adopted some of these measures, but so far there is little evidence for the efficacy of these measures for reducing the risk of TRALI. We have recently conducted research about the relationship between strength of HLA antibodies and TRALI development. On the basis of the results of the research, we can set the cut‐off level in HLA antibody tests, which can reduce the risk of TRALI by eliminating strong HLA antibodies that presumably cause TRALI development if the specificity of an antibody matches the patient’s antigen. Preliminary screening for donor HLA antibodies is ongoing in three core blood centres in Japan, results of which enable us to establish effective and feasible screening strategies for HLA antibodies in the near future.