HLA Antibody Screening Research Articles

New Therapies for Highly Sensitized Patients on the Waiting List.

Exposure to HLA alloantigens through pregnancy, blood products, and previous transplantations induce powerful immunologic responses that create an immunologic barrier to successful transplantation. This is commonly detected through screening for HLA antibodies using Luminex beads coated with HLA antigens at transplant evaluation. Currently accepted approaches to desensitization include plasmapheresis/low-dose or high-dose intravenous Ig plus anti-CD20. However, these approaches are often unsuccessful because of the inability to remove high titer circulating HLA antibodies and limit rebound responses by long-lived anti-HLA antibody secreting plasma cells (PCs) and memory B cells (B MEM ). This is especially significant for patients with a calculated panel reactive antibody of 99%-100%. Newer desensitization approaches, such as imlifidase (IgG endopeptidase), rapidly inactivate IgG molecules and create an antibody-free zone by cleaving IgG into F(ab'2) and Fc fragments, thus eliminating complement and cell-mediated injury to the graft. This represents an important advancement in desensitization. However, the efficacy of imlifidase is limited by pathogenic antibody rebound, increasing the potential for antibody-mediated rejection. Controlling antibody rebound requires new strategies that address the issues of antibody depletion and inhibition of B MEM and PC responses. This will likely require a combination of agents that effectively and rapidly deplete pathogenic antibodies and prevent immune cell activation pathways responsible for antibody rebound. Here, using anti-IL-6 receptor (tocilizumab) or anti-IL-6 (clazakizumab) could offer long-term control of B MEM and PC donor-specific HLA antibody responses. Agents aimed at eliminating long-lived PCs (anti-CD38 and anti-B-cell maturation antigen×CD3) are likely to benefit highly HLA sensitized patients. Complement inhibitors and novel agents aimed at inhibiting Fc neonatal receptor IgG recycling will be important in desensitization. Administering these agents alone or in combination will advance our ability to effectively desensitize patients and maintain durable suppression post-transplant. After many years of limited options, advanced therapeutics will likely improve efficacy of desensitization and improve access to kidney transplantation for highly HLA sensitized patients.

(777) - Routine Screening for HLA Antibodies in Heart Transplant Patients - Does It Affect Clinical Decision Making?

(777) - Routine Screening for HLA Antibodies in Heart Transplant Patients - Does It Affect Clinical Decision Making?

Routine screening for HLA Antibodies in Heart Transplant patients-Does it affect clinical decision making?

We aimed to assess outcomes in patients with and without donor specific antibodies (DSA) and to evaluate the relationship between DSA presence and graft function, cardiac allograft vasculopathy (CAV), and mortality. The study population comprises 193 consecutive long-term heart transplanted (HTx) patients who underwent DSA surveillance between 2016 and 2022. The patients were prospectively screened for CAV through serial coronary angiograms, graft function impairment through serial echocardiograms, and cardiac biomarkers. The patients were followed from the first DSA measurement until death, 5 years follow-up or right censuring on the 30th of June 2023. DSAs were detected in 50 patients using a cut-off at MFI ≥1000 and 45 patients using a cut-off at ≥2000 MFI. The median time since HTx was 9.0 years [3.0-14.4]. DSA positive patients had poorer graft function and higher values of NT-proBNP and troponin T, and more prevalent CAV than DSA negative patients. In total, 25 patients underwent endomyocardial biopsies due to DSA presence while another eight patients underwent endomyocardial biopsies for other reasons. Histological antibody mediated rejection (AMR) signs were seen in three biopsies. During a median follow-up of five years [4.7-5], a total of 41 patients died. Mortality rates did not differ between DSA positive and DSA negative patients (HR 1.2, 95% CI.6-2.4). DSA positive patients were more likely to experience CAV progression than DSA negative patients (HR 2.7, 95% CI 1.5-4.8) CONCLUSIONS: Routine screening reveals DSA in approximately 25% of long-term HTx patients but is rarely related to histopathological AMR signs. DSA presence was associated with poorer graft function and more prevalent and progressive CAV. However, DSA positive patients had similar survival rates to DSA negative patients.

A-150 Abo Subgroup Incompatibility Leading to Red Cell Engraftment Failure and Fatal Outcome: A Rare But Preventable Complication in Related Allogeneic HSCT

Abstract Background Pre-allogeneic hematopoietic stem cell transplant (HCST) testing includes, among others, ABO and HLA typing and testing for anti-HLA antibodies. ABO subgroup typing is not routinely performed, as A2 subgroup representes 1% of the total group A population and only 0.4% of A2 individuals have anti-A1 in their serum. ABO antigens are also found on the platelet surface. We describe a case of alloHSCT performed between siblings, the donor A1 and the recipient A2, with a fatal outcome caused by red cell and platelet engraftment failure due to anti-A1 antibodies, and emphasize the need to routinely investigate subgroup incompatibility in the pre-alloHSCT workup. Methods Patient data was collected from hospital and laboratory electronic records. Blood typing and immunohematological investigation was performed at the hospital blood bank using gel cards for ABO typing, DAT and antibody screening panels (Grifols, Barcelona). Tube method was used for A subgroup typing (Fresenius, Germany). Bone marrow analysis and other blood tests were performed at local hospital laboratory. HLA typing and antibody screening (Luminex, USA) were sent to a specialized laboratory. Results A 51 year-old male with Non-Hodgkin lymphoma underwent a full-match ABO compatible allogeneic HSCT, both brother/donor and recipient group A. Pre-transplant anti-HLA or ABO antibodies were negative. WBC recovery was achieved on D + 22. The patient had a catheter infection on D + 15 (Permcath removed), CMV infection on D + 24 (treated with gancyclovir) and received several blood transfusions. On D + 22, he had a hemolytic transfusion reaction with fever (38.5°C), chills and a decline in hemoglobin (7.3 g/dL to 6.6 g/dL in the following day), low haptoglobin and increased indirect bilirrubin levels. Immunohematological investigation revealed a positive DAT with an anti-A antibody in the eluate. Subgroup incompatibility was suspected and the patient was found to be subgroup A2, whereas the donor typed A1. Three days later the DAT became negative and anti-A1 was no longer detected. Other RBC antibodies were also excluded. Only group O RBCs and A / AB platelets were transfused thereafter to prevent further hemolysis. On D + 35 the patient was diagnosed with GI-GVHD, which responded to corticosteroids. He never achieved transfusion independence and received RBC and platelet transfusions on a regular basis. Failure of RBC and platelet engraftment was confirmed by a bone marrow aspirate on D + 45, showing only 20% erythroid lineage and absence of megakaryocytes. Marrow chimerism revealed 83% of donor origin. Sepsis by S. aureus ensued on D + 57 and three days later he had fever (38.2°C) and dyspnea after a platelet transfusion. Despite entubation and mechanical ventilation, he evolved to RCP and died on D + 60. Conclusion ABO subgroup typing is a simple and low-cost technique routinely available in the blood bank. Detection of A subgroup incompatibility impacts on key decisions, such as donor choice (if more than one candidate) and the need for erythrocyte depletion of donor stem cells and recipient plasmapheresis if the anti-A1 titer is high. Since graft failure due to ABO subgroup incompatibility can be hazardous, we recommend routinely ABO subgroup typing in the pre-alloHSCT workup to increase donor safety.

The impact of pretransplant suspected HLA antibody on the long-term outcome of the graft kidney: A retrospective cohort study

IntroductionThe preoperative examination of kidney transplantation includes HLA antibody screening to initially determine the presence of preexisting donor-specific antibody (DSA) that mediates hyperacute rejection. Recipients with positive HLA antibodies require further HLA specificity analysis to type the antigen and determine the antigen mismatches between the donor and recipient. However, recipients with suspected antibodies would have no further HLA specificity analysis. It is unclear whether suspected HLA antibodies would affect renal graft function. This study aimed to explore the impact of pretransplant suspected HLA antibody on the long-term outcome of the graft kidney and thus determine the necessity of routinely performing the HLA specificity analysis in recipients with suspected HLA antibodies preoperatively. MethodsThis is a single-center retrospective cohort study. 179 kidney transplant recipients (KTRs) were included and further divided into HLA antibody-negative group (Group 1) and HLA antibody-suspected groups (Group 2) based on the result of the pretransplant HLA antibody screen test. And the antibody-suspected group was further divided into a low-mismatched group (Group A) and a high-mismatched group (Group B) according to the HLA specificity analysis. We tracked the renal function indexes, biochemical indexes, and posttransplant adverse events within 5 years after transplantation and explored the necessity of further HLA specificity analysis in recipients with pretransplant suspected HLA antibodies. ResultsThere was no statistically significant difference in demographics between HLA antibody-negative group and HLA antibody-suspected groups. At 5 years of follow-up, the KTRs in HLA antibody-negative group had significantly higher eGFR levels, lower serum creatinine levels, and less urinary protein compared to those in antibody-suspected group. Meanwhile, the KTRs in low-mismatched group also had significantly higher eGFR levels, lower serum creatinine levels, and less proteinuria compared to those in high-mismatched group. Correlation analysis showed that the age of KTRs, urinary protein levels and the load capacity of HLA mismatches were associated with eGFR levels of KTRs at 5 year posttransplant. ConclusionKTRs with suspected HLA antibodies before kidney transplantation have worse graft function than the preoperative HLA antibody-negative recipients in the long-term posttransplant follow-up. The specific load capacity of HLA mismatches, the age of the recipient and the urinary protein was found to be negatively correlated with long-term posttransplant renal outcomes. It is necessary to undergo further HLA specificity analysis for recipients with suspected HLA antibodies in HLA antibody screen test to explicit HLA mismatches and improve long-term posttransplant outcomes.

Intercontinental Deceased-donor Kidney Transplantation in Dutch Antilleans: Favorable Outcomes Despite Prolonged Ischemia.

Intercontinental Deceased-donor Kidney Transplantation in Dutch Antilleans: Favorable Outcomes Despite Prolonged Ischemia.

P8.129: Description of the Process of Inclusion and Kidney Transplantation Waiting List - Uruguay

Introduction: In Uruguay with 3,286,314 inhabitants (2011 census), renal transplantation is performed in 3 transplant centers, is regulated by the Instituto Nacional de Donación y Trasplante (INDT) and financed by the Fondo Nacional de Recursos. The allocation is made on a national algorithm and it is an INDT´s responsibity. In the last 5 years, including the Health Emergency, an average of 139 kidney transplants were performed per year. The average rate for these 5 years of renal transplantation is 42.33 pmp. Objective: Describe the renal waiting list (WL) admission process and the recipient population. Methods: The study algorithm for admission to the WL include: □ABO and Rh blood group typing. □HLA typing by PCR-SSO (Luminex) for A, B, C, DRB1, DQA1 and DQB1 locus. □HLA antibody screening (Luminex), class I and/or II. If positive, a specificity search is performed. □Search for autoantibodies by microlymphocytotoxicity technique (MLCT). □Patient’s serum is preserved (serum collection). □PRA (wall reactive antibodies) by MLCT, defining hyperimmunized PRA>80%. During the period in WL the following procedures are performed: □serum collection update every 3 months or after sensitizing events. Failure to comply with this requirement excludes recipients from the allocation. □HLA antibody screening and specificity analysis are performed annually and PRA every six months. Results: There are currently 434 patients in the WL, including one vascular emergency. Distribution - by age: 1.8% <18 years old, 85% 18-64 years old and 13.2%>65 years old; by sex: 43.8% female, 56.2% male. Blood group distribution: 54% are 0.35% A, 8% B and 2% AB. The highest immunological risk group: 18%, who aspire to be retransplanted, 1.4% hyperimmunized. There are 26.5% with positive HLA antibody screening, see table and distribution in graphs. Conclusions: A single national protocol is applied at the inclusion on the WL. In the renal WL, the adult population (18 to 65 years old), blood group 0, with a male/female ratio of 1.3 prevails. The immune risk group does not present significant changes with respect to previous evaluations. The prevalence of sensitized to both classes and hyperimmunized is maintained.

Effects of different sensitization events on HLA alloimmunization in renal transplant cases; a retrospective observation in 1066 cases

BackgroundPatients awaiting solid organ transplantation may develop anti-HLA antibodies after sensitization events such as transfusions, pregnancies, or previous transplantations. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using cell-based assays and solid-phase assays. In this study, we evaluated and compare how different sensitization events affect the HLA antibody screening (HLA-Ab) and donor specific antibody (DSA) status in solid renal organ transplantation patients. MethodsHLA antibody (HLA-Ab) screening tests like complement-dependent cytotoxicity crossmatch (CDC-XM), flow cytometry crossmatch (FC-XM) and Luminex panel-reactive antibody (L-PRA) were performed in all 1066 patients (635 males and 431 females). If any of these tests turned out to be positive, a Luminex single antigen bead (L-SAB) assay was performed for DSA identification. Test positive rates and antibody strengths were analyzed according to the different sensitization events and gender. ResultsIn this study, HLA-Ab screening tests positive rates (L-PRA, FC-XM and CDC-XM) were significantly higher in patients with previous transplantation (73.91%, 100% and 56.52% p < 0.001), previous pregnancy (57.46%, 70.14% and 18.85% p < 0.001) or blood transfusion (27.33%, 35.55% and 7.33% p < 0.001) compared with patients without a sensitizing event (6.17%, 13.58% and 1.09). In this study, re-transplantation group showed significantly stronger antibody strength (DSA) than non sensitized group (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 2659, 3329; P < 0.001) and those with single sensitization events of transfusion (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 5790.26, 6004.16; P < 0.001) or pregnancy (class I & II MFI 11418, 17,837 vs class I and II MFI 8631.71, 7253.29; P < 0.001). ConclusionsPregnancy and blood transfused had high allo-immunization rate for class I HLA antigens. While re-transplantation patients had high allo-immunization rate for both the HLA classes (HLA- class I and HLA- class II). Re-transplantation group showed significantly stronger antibody strength, followed by pregnancy and then transfusion.

Human leukocyte antigen antibody sensitization, lung transplantation, and health equity

Women with advanced lung disease, particularly Black and Hispanic women, are more likely than other patients to have anti-human leukocyte (HLA) antibodies against potential donors. Sensitized patients, especially those who are highly sensitized, are less likely to be listed for lung transplant or to be considered candidates for mechanical circulatory support. They are also at higher risk for waitlist death. Institutional variability in approach to HLA antibody screening and pre-transplant management creates barriers to transplant that disproportionately impact Black and Hispanic women. At the same time, our understanding of the clinical significance of pre-transplant antibodies lags behind the sophistication of our screening assays. The lack of national data on pre- and post-transplant HLA antibody characteristics hinders research into strategies to mitigate concerns about these antibodies and to improve access to lung transplant among sensitized patients. Ongoing work should be done to identify clinically higher risk antibodies, to develop better strategies for safely crossing antibodies at the time of transplant, and to model changes in lung allocation to give priority to sensitized patients for a HLA antibody-antigen compatible donors. These priorities mandate a commitment to collaborative, multicenter research and to real time translation of results to clinical practice and allocation policy.

Preemptive HLA Antibody Screening Prior to Episodic Transplant Renal Biopsy Enables Early Diagnosis and Therapeutic Response in Asymptomatic Chronically Active Antibody-Related Rejection: A Case Report

Common management of renal transplant recipients includes episodic renal biopsy based on clinical findings such as an increase in proteinuria or serum creatinine. When antibody-related rejection is suspected from the renal biopsy, subsequent testing for donor-specific antibodies (DSAs) is performed. We instead performed preemptive screening of asymptomatic post–renal transplant recipients for DSAs prior to renal biopsy. In this case, a 30-year-old woman with a secondary transplant was positive for 61 anti-HLA antibodies of class I and class II, among which DQ2 was a DSA with a mean fluorescence index of 2039. The patient had a living kidney transplant 9 years earlier. She had never been diagnosed with rejection, her serum creatinine was around 1.0 mg/dL, and her proteinuria was negative. Following the positive DSA result, a renal biopsy was performed, and she was diagnosed as C4d-negative chronic-active antibody-mediated rejection (CAABMR) with a Banff score of cg1b, (g + ptc) ≥ 2, and C4d 0. Intravenous steroid pulse, deoxyspagarin, antithymocyte globulin, rituximab, and oral everolimus were administrated. The treatment resulted in a gradual decrease in the DSA, which became negative 1 year later. The patient’s serum creatinine remains around 1.0 mg/dL, and proteinuria remains negative. Treatments for advanced CAABMR are often expensive and ineffective. Our present case suggests that early detection and treatment through preemptive HLA antibody screening could improve the prognosis of renal transplants.

Pre-Formed Donor-Specific Antibodies Impact Long Term Survival of Heart Transplants

We previously reported that heart transplant (HTx) recipients with pre-formed, anti-HLA antibodies at low titer had one-year survival that was comparable to patients without any anti-HLA antibodies. As a result, we defined unacceptable HLA antigens by MFI>1000 in serum at 1:16 dilution. The current study evaluates how pre-formed, low-titer, donor-specific antibodies (DSA) and positive crossmatch affect long term survival of HTx recipients. Total 495 adult recipients transplanted at our center between Jan. 2007 and Dec. 2016 were studied. Prior to HTx, recipients had HLA antibody screening (FlowPRA) and specificity testing (LABScreen). Unacceptable HLA antigens were defined by MFI>1000 in serum at 1:16 dilution. Acceptable donors were selected by virtual crossmatch using the criteria above, and flow cytometry crossmatch (FCXM) was performed peri- or post-HTx. Recipients transplanted across low titer DSA, positive in undiluted serum but negative at 1:16 dilution, received peri-operative plasmapheresis. We used log-rank testing to evaluate survival in the following groups compared to their controls: (1) presence of any pre-formed HLA antibodies; (2) presence of pre-formed DSA; and (3) positive FCXM. Of 495 HTx recipients, 179 (36%) had pre-existing HLA antibodies, and their overall survival was not different from patients without HLA antibodies (p=0.883). However, median survival time was reduced in patients with pre-formed DSA (n=62), compared to DSA-negative recipients (n=117): 9.1 years (95% CI 3.52 to 14.65) vs. 10.9 years (95% CI 6.13 to 15.70); p=0.02. The survival rates for 1-, 3-, 5-year of DSA-negative recipients were 91.5%, 85.4% and 79.5%, and the DSA-positive recipients were 83.9%, 66.1% and 61.7%, respectively. Total 62 recipients had pre-HTx DSA. Among them, 26 had a positive FCXM (46%). The survival rates of FCXM-positive cases were lower than FCXM-negative cases (n=36) at 1, 3, and 5 years: 80.8%, 57.9%, 52.1% vs. 86.1%, 71.4%, 67.6%. Our findings suggest that pre-formed DSA, even at low titer, negatively impacted HTx survival. Cases with positive FXCM had the worst outcomes. Avoiding all pre-formed DSA may improve survival for HTx patients, but may increase waitlist mortality. To improve the HTx survival, close post-transplant monitoring and proactive immunological management may be required when pre-formed DSA are present.

Evalution of electronic dp epitype profiling to determine dp donor-specific antibodies during virtual crossmatch

Our aim is to evaluate the utility of incorporating epitype analysis into the computerized virtual crossmatch (vXM) workflow for donors with DP alleles that are not covered by a recipient’s HLA antibody screening and identification assay. Our lab has used vXM to select deceased donors for transplantations for more than ten years. If the donor DP antigens are covered by the assay, the DSA can be determined directly by vXM. If the donor antigen is not included in the assay but is covered by an epitype (EDP) group which includes a DP antigen covered by the assay, the DSA can be determined based on the positivity of the EDP antigen using manual epitype profiling. A new epitype profiling system was deployed by informatics vendor HLA Data Systems, developers of the mTilda Lab Management System and VxMatch, to ensure that DP alleles not covered by HLA antibody tests are incorporated into an electronic vXM workflow. The epitype profiling system contains a data table of the highly polymorphic DP hypervariable regions (HVR) for DPB1 alleles. There are a total of six HVR regions (HVR-A, B, C, D, E, F) containing the unique amino acid profile per allele and 291 epitype groups determined so far which correspond to alleles belonging to the same HVR motif. The system returns the HVR motifs for donor and alleles of the recipient antibodies and then sorts alleles of recipient antibodies by increasing reactivity. Total negative and positive HVR matches are based on a configurable positivity cutoff value. Matches are color-coded for investigation of possible DSA when there is a positive match that is accompanied by zero negative matches. If a DP antigen bead is negative, presence of antibodies to polymorphic motifs carried by the DP antigen can be excluded. The computerized epitype profiling system was validated against our manual process for donors with alleles not contained within the assay, DPB1*16:01 and DPB1*85:01, and HVR motif profiling performed without error. With the increasing discovery of DP alleles, the system can cross-reference G-groups to determine HVR motifs for new alleles and can be configured to profile other HLA genes. Our evaluation suggests that extending vXM to include computerized epitype profiling is an efficient and reliable means to evaluate DP alleles not covered by antibody assays.

Minimal data reporting standards for serological testing for histocompatibility

Histocompatibility testing, and HLA antibody screening in particular, varies in practice among laboratories. Currently, standards are lacking regarding the reporting of testing methods in publications. It is essential that scientific methods are rigorously and transparently described upon publication, so that results can be accurately interpreted and independently corroborated. Additionally, this would allow work groups to compile diverse data to achieve clinically significant conclusions from meta-analyses. These efforts are hindered when there is a paucity of method descriptions and where variability in serum treatment, protocol modifications, and assay thresholds affecting assay interpretation are known to exist. Thus, the ASHI Science and Technology Initiatives Committee (ASHI STIC) undertook the task of formulating recommendations for reporting HLA antibody testing by solid phase assays, and the associated HLA typing required for interpretation, in scientific publications. Herein we put forth standards for minimum information about HLA antibody testing methods reported in histocompatibility publications.

Humoral Rejection in Lung Transplantation

Introduction The diagnosis of humoral rejection in lung transplant recipients is sometimes elusive, involving histopathologic (capillaritis), immunopathologic (C4d staining), and serologic (donor-specific anti-HLA antibodies, DSA) parameters. These criteria are not always present in cases and, moreover, the involvement of non-HLA antibodies in lung-allograft humoral rejection remains largely unclear. The aim of the study was to assess the involvement of HLA and non-HLA antibodies in humoral rejection diagnosis after lung transplantation. Materials and Methods A total of 60 consecutive lung transplant recipients were followed in our Institution from 2015. Protocol biopsy at day 21 post transplantation and screening for serum HLA and non-HLA antibodies by Luminex, LABScreen Autoantibody (One Lambda), before lung transplant and at the moment of the biopsy, were performed. Results and Discussion A total of 5 lung transplant recipients (8.33%) were C4d+ on biopsy but only one of them developed de novo DSA without evidence of non-HLA antibodies. The remaining 4 patients C4d+, were serum positive for non-HLA antibodies (2 against LG3 and other 2 against myosin) prior lung transplantation (6.67%) but negative at the moment of the biopsy. The incidence observed of preformed non-HLA antibodies in patients awaiting for lung transplantation was significant and potentially could drive C4d deposition on lung tissue with subsequent humoral rejection. Conclusion The screening of non-HLA and HLA antibodies could help to identify patient at risk of lung-allograft rejection.

Repeated Freezing and Thawing Does Not Affect HLA Antibody Titer

Introduction For deceased donor organ transplantations, we are not currently using virtual crossmatches (XMs) and preliminary negative T cell XMs are mandatory for kidney and pancreas allocation in Korea. Korean Network for Organ Sharing (KONOS) distributes transplant candidates’ sera to 21 OPO laboratories and KONOS preliminary XMs are performed in these laboratories using frozen stored sera. We are performing more than 40% of nationwide KONOS XMs in the Korea Organ Donation Agency (KODA) laboratory, and we had difficulties in keeping the frozen sera from more than 20,000 transplant candidates in small aliquots to avoid HLA antibody deterioration. We investigated the extent of change of antibody titer after repeated freezing and thawing. Materials and Methods Two different sera (serum A and B), which were tested positive for both class I and class II HLA antibodies were selected for the investigation. Sodium azide (0.1% in final concentration) was added to the sera as preservative, and freezing-thawing cycles were repeated for 20 times. A total of 7 serum samples (1, 3, 6, 9, 12, 15, and 20 times frozen-thawed; labeled #1, #3, #6, #9, #12, #15, and #20) were used for HLA antibody screening test (One Lambda, USA), and 3 samples (1, 9, and 20 times frozen-thawed) were used for HLA antibody single antigen assay (One Lambda, USA) and T cell flow cytometry XM (FCXM) test. To avoid prozone phenomenon, 1:1 (neat), 1:5, and 1:20 diluted sera were tested by screening assay and 1:20 sera for both serum A and B were used for the investigation. HLA antibody strength in mean fluorescence intensity (MFI) values and FCXM result in MFI ratios were compared. Test results of #20 sample compared to #1 sample were calculated as percentage values and variabilities of the test results among different samples of repeated freezing-thawing were calculated as coefficient of variation (CV) values. Results and Discussion The results are summarized in Table 1. There was little change in MFI values after 20 times of repeated freezing-thawing cycles: percentage of #20 vs # 1 sample as baseline (100%) was in the range of 99-110% for screening assay and 90-136% for single antigen assay. For serum A, change of MFI values of 5 class I and 5 class II antibodies of the #1, #9 and #20 samples are shown (Fig. 1 and Fig. 2). Variability of the test results among different samples of repeated freezing-thawing was low, and most of the CV values (7 out of 8) were <10% (2.2-6.9%). T cell FCXM results (3 XMs) also showed little change in MFI ratios (103-107%) and low CV values (4.2-6.7%). Unlike the notion that antibodies are deteriorated after repeated freezing-thawing, the strength of HLA antibodies was little affected after 20 times of freezing-thawing cycles.Conclusion HLA antibody strength is little affected by up to 20 times of repeated freezing and thawing. For preliminary HLA XM tests for deceased donor organ transplantation, serum samples from transplant candidates can be stored frozen without aliquoting.

The good, the bad, and the ugly of luminex donor-specific crossmatch.

The presence of donor-specific antibodies directed against human leukocyte antigen significantly influences renal transplant because of antibody-mediated rejection. We performed the screening of pre-renal transplant patients for preformed anti-HLA antibodies using anti-human globulin augmented-complement-dependent lymphocytotoxicity crossmatch (AHG-CDCXm), luminex donor-specific crossmatch (LumXm) and HLA antibody screening. Seven hundred and fifty-four patients were assessed for LumXm. HLA antibody screening was possible in 325 out of 754 cases. All the three investigations viz. CDCXm, HLA antibody screening and LumXm was performed in 325 patients. All CDCXm positive patients (10/325, 3.08%) were also positive with LumXm and HLA antibody screen whereas 14 cases (4.31%) with CDCXm negative were positive with luminex-based assays. LumXm and HLA antibody screening were both positive in 24 (7.38%) cases, LumXm and HLA antibody screening were both negative in 275 (84.63%) cases and LumXm negative and HLA antibody screening was positive in 22 (6.76%) cases. However, there were four cases (1.23%) which were positive in LumXm in spite of being negative in HLA antibody testing. Single Antigen Bead (SAB) assay was performed in all patients positive for HLA antibody test. We suggest that LumXm is a useful and sensitive technique for the detection of anti-HLA antibodies in pre-transplant renal patients. However, other measures such as luminex antibody screen, SAB assay, history of the donor, and the class of antibodies involved should be taken into consideration for pre-transplant work up of renal patients.

Flow Cytometry Panel-Reactive Antibody Screening of Anti-HLA Antibodies in the Waiting List Significantly Reduces the Occurrence of Acute Rejection After Kidney Transplantation

IntroductionThe presence of preformed HLA-reactive antibodies in recipient serum before transplantation has long been recognized as a prominent risk factor for a generally worse graft outcome. Screening and identification of HLA antibodies can be used to stratify patients into high- and low-risk categories. Materials and methodsWe determined patients' anti-HLA antibodies using flow cytometry panel-reactive antibody (flowPRA) screening, specifying more than 5% after positive screening. According to the results of the screening test, patients were allocated to the induction immunosuppressive protocol according to the actual immunologic risk. ResultsIn the group of 78 patients, screening with flowPRA of anti-HLA antibodies was done twice a year. Patients were divided into 2 groups of immunologic risk (low or medium), and we chose the induction immunosuppressive protocol according to the risk. Stratification of the risk was correct, because the only predictor for development of acute rejection in the monitored period of 12 months was delayed graft function (odds ratio 33.2501; 95% confidence interval 10.0095–110.4508; P < .0001). The occurrence of acute rejection upon implementing the screening was reduced in our transplant center from 44% to 19% (P < .0001). No difference was recorded in the 12-month survival of grafts and patients according to the applied induction immunosuppressive protocol. ConclusionWe confirmed significantly reduced occurrence of acute rejection in the follow-up period of 12 months by using individualized induction according to flowPRA screening of anti-HLA antibodies. FlowPRA screening represents a suitable alternative for screening and specification of anti-HLA antibodies in case the Luminex methodology is unavailable.

OR34 Platelet refractoriness in a patient with 0% PRA due to a rare anti-CD36(NAKa) antibody

Platelet transfusion refractoriness in patients with hematological malignancies ranges from 7% to 34%. The etiology of platelet refractoriness in this patient population is typically attributable to due to formation of HLA antibodies (Abs) from pregnancy or previous transfusion. We report platelet refractoriness in the setting of induction chemotherapy for AML from allo-Abs to NAK a antigen/CD36/glycoprotein (GP) IV. A 66yo AAF presented with transfusion dependent AML with 70% circulating peripheral blasts and started on 7 + 3 regimen. She developed profound thrombocytopenia and her platelet count was 0 by day 13. As part of her evaluation for refractoriness, HLA antibody screening was done, but was negative. An Immucor ELISA assay was then performed and revealed positive Abs to GPIV (NAK a antigen/CD36). Abs against GPIIb/IIIa, GPIa/IIa, GPIb/IX, and HLA Class I were all negative. GPIV Abs were confirmed by Blood Center of Wisconsin using monoclonal Ab MBC 131.7 against CD36. Cross-matching was performed at our institution with multiple single donor apheresis platelets, all showing strong positive reactivity. Because CD36 in these units may be soluble, an incompatible leukoreduced, irradiated and washed single donor platelet unit was given which was tolerated well with a resultant increase to 14,000/ μ L. Despite strong reactivity of this unit in the crossmatch, the patient’s platelet count increased and 1 week later was 16,000/μL despite no transfusion support. Platelet refractoriness in the setting of hematological malignancy is well documented. Abs develop from prior pregnancy and transfusion in the setting of prolonged pancytopenia in hematological malignancies. Typically, allo-Abs develop against platelet membrane structures including human platelet antigens, HLA antigens, and rarely NAK a antigen on the CD36 glycoprotein molecule. CD36 is expressed in almost 100% of white Europeans and is not detectably expressed by 2% of sub-Saharan Africans and 10% of Asians. CD36 is a soluble antigen and is expressed on various cells such as erythroblasts and monocytes. We propose that washing platelets may be a useful strategy in treating platelet refractoriness in patients with abs to NAK a antigen as finding CD36 negative donors is extremely difficult and few blood banks in North America test donors for this antigen.

P164 Role of anti-mica antibodies in graft survival of renal transplant recipients of India: A single centre experience

Aim To find the incidence of MHC class I chain-related chain A (MICA) alloantibodies in patients undergoing live related donor (LRD) renal transplantation and their association with short-term graft survival. Methods Pre-transplant, consecutive, serum samples of ESRD patients undergoing first renal allograft transplantation were tested for anti-MICA antibodies. Association of various demographics, HLA-A + B + DRB1 mismatches and HLA antibody screen with MICA antibodies was assessed using Pearson’s Chi-square test. A multivariate analysis was also performed to check for potential confounders. The effect of pre-transplant blood transfusions on antibodies against HLA Class 1 & class II, or MICA was studied. Results Out of 646 serum samples for MICA antibodies, 94 (14.6%) were positive and 552(85.4%) negative for MICA antibodies. Patients with MICA antibody had a graft survival 89.3% as compared to 94.7% in patients without MICA antibody (P = 0.01). The hazards ratio for all patients was 3.744 (P = 0.01). MANOVA effect was determined for three factors (HLA antibody, MICA antibody & HLA mismatch) and Graft survival which was statistically significant. Two of the three ANOVA’s were statistically significant viz. MICA antibody and presence of HLA antibody. Conclusions MICA antibodies were present in 14.6% of the patients. Preformed MICA antibodies independently increase the risk of kidney rejection and therefore a guideline should be made for mandatory testing of these antibodies prior to transplant.

Caractérisation de l’allo-immunisation anti-HLA et impact clinique en transfusion et en transplantation d’organe

Allo-immunizations against HLA antigens are known to be deleterious in transfusion and organ transplantation. The development of new tests based on solid phase assays for screening and identification of HLA antibodies in particular those using Luminex® bead based technology has completely changed the way of allo-immunization monitoring because of their extreme sensitivity. They allow a better characterization of these antibodies, identification of acceptable antigens and the use of virtual cross-matches. All these new possibilities improve the managing of patients before and after platelets transfusion or organ transplantation. However, this technology displays some limits that should be known in order to interpret correctly the results. Beside these bead based assays, cellular cross-matches based on Complement Dependent Cytotoxicity (CDC) and flow cytometry are still used and useful in organ transplantation since beads are produced in vitro and do not reflected exactly what happens physiologically. Moreover, differences of sensitivity between these methods make results interpretation and decision making difficult in some cases.