Serum HLA Antibodies Research Articles

#2402 Reproducibility of memory B-cell and HLA antibody detection in patients on kidney transplant waiting list

Abstract Background and Aims Detection of HLA antibodies (HLA-Abs) impacts on the access and outcomes of kidney transplantation (KT). Currently, evaluation of HLA-Abs in serum is performed using single antigen bead assays (SAB). The analysis of circulating HLA-specific memory B cells (mBc), capable of differentiating to HLA-Abs secreting cells, was conceived with the aim of complementing the study of the HLA-Abs found in serum, but there is debate regarding the representativeness and reproducibility of the test. Thus, we studied HLA-Abs generated by mBc from patients on the waiting list (WL) at two different time points without sensitizing events in between and compared them with HLA-Abs detected in serum. Method We selected 7 patients on the WL for a first KT with a cPRA of 3%-100%: 4 had pregnancies and 2 had blood transfusions as known sensitizing events. We collected blood and serum samples at 2 time points separated by an average of 3.5 months (T1, T2). We cultured polyclonally activated PBMCs and isolated and concentrated IgG from the supernatant (SN) to study HLA-Abs. We tested SN and sera with SAB assays. Beads with MFI >750 were considered positive. Results SAB serum study (Fig. 1a). Eighty-two percent of the specificities found at T1 were also detected at T2, and 97% of those identified at T2 were also observed at T1. Thus, there was good concordance between T1 and T2 samples (κ = 0.869, Cohen) and the MFI the specificities correlated (p < 0.0001, Spearman). SAB SN study (Fig. 1b). Forty-one percent of the positive specificities detected at T1 were also identified at T2, and 59% of the positive specificities found at T2 had been seen at T1. Thus, there was a moderate concordance between T1 and T2 samples (κ = 0.454, Cohen) and the MFI of the specificities still correlated (p < 0.0001, Spearman). Only 42% and 35% of the positivities found in serum were also detected in the SN at T1 and T2, respectively. However, we found 9 positivities unique to the SN T1 (100% HLA-ABC) in 2 patients with cPRA>80% and we also identified a total of 20 positivities unique to the SN at T2 (45% HLA-ABC, 55% HLA-DPDQDR) in 3 patients with cPRA>80%. Conclusion The study of the degree of sensitization with SAB serum analysis remains stable between samples along time, while the mBc analysis shows less reproducibility. We identified a higher number of HLA-Ab in the study of serum compared with the study of the SN of mBc; however, we found some HLA-Abs unique to the SN. We suggest that, probably due to the small number of circulating mBC, increasing the number of samples tested for mBc improves the representativeness of the test.

416.5: Pregnancy-Induced Sensitization in Female Transplant Candidates: HLA-Specific Memory B Cells and Paternal HLA Epitope Mismatch

Background: Access to kidney transplantation (KT) may be limited by HLA antibodies (HLA-Ab) induced by sensitizing events. Pregnancy is a frequent sensitizing event in female candidates, through which they are exposed to foreign paternal HLA antigens. Consequently, they can generate specific HLA-Ab and memory B cells (mBc), which can be re-stimulated upon antigen reencounter after KT. We aimed to study the existence of mBc able to produce specific HLA-Ab against paternal antigens in female KT candidates sensitised by previous pregnancies, to compare them with HLA-Ab found in their serum and to analyse these HLA-Ab in the context of the molecular HLA mismatch between mother and father. Methods: We selected 8 HLA sensitised women awaiting KT with at least one successful pregnancy from a single partner, detectable serum HLA-Ab (MFI≥1000) and available paternal DNA for full HLA typing. All women and their partners were typed by next-generation sequencing for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1, DPB1. To study mBc, we used peripheral blood mononuclear cells cryopreserved before KT and cultured them with a polyclonal stimulation cocktail consisting of a toll-like receptor agonist and an interleukin for ten days. We examined concentrated culture supernatants (SN) derived from activated mBc, purified IgG and identified HLA-Abs using single antigen beads on Luminex®. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA mismatch with HLAMatchmaker® software. Results: After 10-day polyclonal stimulation, mBc increased from 29.9% (day 0) to 65.3% (day- 10), and antibody-secreting cells (ASC) from 0.1% (day-0) to 29.4% (day-10) (Figure 1). SAB analysis revealed 211 HLA-Abs in serum and 116 in SN: 98/211 (46,5%) HLA-Ab were serum-exclusive (79,6% class I and 20,4% class II) and only 3/116 (2,3%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34,6%) HLA-Ab shared by serum and SN: 65/113 (57,5%) class I and 48/113 (42,5%) class II.Pregnancy-Induced Antibodies (PIA) were present in serum in 6/8 women (21 HLA-Abs: 10 class I, 11 class II) and in 4/8 women in SN (8 HLA-Abs: 2 class I, 6 class II). The HLAMatchmaker analysis showed that those specific HLA-ab were reactive against incompatible paternal epitopes: in 3/6 women for class I & 3/6 women for class II in serum; in 2/4 women for class I & 3/4 women for class II in SN (Table 1).Conclusion: We describe the study of the memory B cell compartment and the HLA-Abs produced with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by memory B cells followed a restricted pattern compared with serum HLA-Ab. The study of paternal HLA molecular mismatch explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitised patients awaiting TK is currently a matter of study.

FC 112: HLA-Specific Memory B Cells and Paternal HLA Molecular Mismatch in Female Transplant Candidates Sensitized by Pregnancy

Abstract BACKGROUND AND AIMS Access to kidney transplantation (KT) may be limited by HLA sensitization. It is clinically relevant to know both level of HLA-immunization and potential sensitizing events in the history transplant candidates [1–3]. Pregnancy is a frequent sensitizing event in female transplant candidates. Women exposed to foreign paternal HLA can generate specific HLA antibodies (HLA-Ab) and memory B cells (mBc) [4, 5], which can be re-stimulated upon antigen reencounter after TR. We aimed to study the existence of mBc able to produce HLA-Ab in female KT candidates with previous pregnancies, to compare them with HLA-Ab in their serum and to analyse them in the context of molecular HLA mismatch between mother and father. METHOD We selected eight HLA sensitized women awaiting KT, who fulfilled these criteria: minimum of one successful pregnancy before KT (12–44 years before) from a single partner, HLA-Ab (HLA class I or II) and availability of a DNA sample from the father of their children. All women in the study cohort and the father of their children were HLA typed by next-generation sequencing (NGS®) for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1 and DPB1. We used peripheral blood mononuclear cells cryopreserved before KT. We cultured them with a polyclonal stimulation cocktail consisting of a Toll-like receptor 7/8 agonist and a proleukin for 10 days. We examined concentrated culture supernatants (SN) derived from activated cells, purified IgG and identified HLA-Abs using single antigen beads (SAB) on Luminex. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA incompatibilities with the HLAMatchmaker software. RESULTS After 10-day polyclonal stimulation, mBc increased from 29.90% (day 0) to 65.29% (day 10) (Figure 1), and antibody-secreting cells (ASC) from 0.08% (day 0) to 29.37% (day 10). SAB analysis revealed 211 HLA-Abs in serum and 116 HLA-Abs in SN: 98/211 (46.5%) of total HLA-Ab were serum-exclusive [78/98 (52.9%) class I and 25/98 (34.3%) class II] and only 3/116 (2.59%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34.6%) HLA-Ab shared by serum and SN: 65/113 (57.5%) class I and 48/113 (42.5%) class II. Seven of eight women had Pregnancy-Induced Antibodies (PIA) in serum (20 HLA-Abs: 10 HLA class I and 10 HLA class II) and 4/8 women had PIA in SN (8 HLA-Abs: 2 HLA class I and 6 HLA class II) (Table 1). The HLAMatchmaker analysis showed that antibodies were reacting against incompatible paternal epitopes: in 6/8 women for HLA class I and 5/8 women for class HLA II in serum, and in 2/8 women for HLA class I and 4/8 women for HLA class II in SN. CONCLUSION We describe the study of the memory B cell compartment and the specific HLA-Abs produced by them with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by these memory B cells followed a restricted pattern in number and intensity compared with serum HLA-Ab. The study of paternal HLA molecular mismatch incompatibilities explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitized patients awaiting TK is currently a matter of study.

Sarcolemmal Staining by Anti-C4d/C3d in Cardiac Transplant Endomyocardial Biopsies and Associated Serum HLA Antibodies and Pathologic Rejection

<h3>Purpose</h3> Immunologic evidence of complement fixation in endomyocardial biopsies (EMB) is a feature used to assess pathologic antibody-mediated rejection in cardiac transplants. Uncommonly, sarcolemmal staining (SS) of myocytes is observed. The significance of this is unclear and under-investigated. This study aims to temporally correlate SS with changes in HLA antibodies and pathologic rejection. <h3>Methods</h3> All EMBs that had SS by anti-C4d and/or C3d on immunofluorescence or immunohistochemistry (Figure 1) were identified via the laboratory information system between 10/1/2016 and 10/1/2019. The HLA antibodies and all other EMB results since time of transplant for each case were reviewed and temporally correlated to the biopsy that showed SS. <h3>Results</h3> 28 patients (mean: 31 yrs; range: 6-73 yrs) with 31 SS events were identified. Associated features are summarized in Table 1. Of note, 29/31 events of SS had HLA antibodies measured within a 2 week period before/after the time of biopsy. A temporally associated expansion of HLA Class I/II antibodies was noted in 12 (39%) cases. 21 (68%) cases had prior donor specific antibodies before EMB showing SS, with less immediate temporal association. <h3>Conclusion</h3> Sarcolemmal staining of complement split products in EMBs was associated with a concurrent expansion in strength and/or breadth of HLA antibodies in 39% of cases. This is a novel observation that may partially explain the appearance of SS by immunologic assays. Further investigation is required to fully elucidate the significance of SS in EMB.

Free Airway C4d after Lung Transplantation - A Quantitative Analysis of Bronchoalveolar Lavage Fluid

In recent years, the utility of vascular complement factor 4d (C4d) deposition as diagnostic tool for antibody mediated rejection (AMR) after lung transplantation, has become a controversial issue. We aimed to pinpoint the problematic nature of C4d as biomarker with a simple experiment.We quantified C4d in broncho-alveolar lavage (BAL) of lung transplant patients with diverse post-transplant complications in 3 different settings of clinically clear cases of: 1/ chronic lung allograft dysfunction (CLAD); 2/ acute complications acute rejection (AR), lymphocytic bronchiolitis (LB), antibody-mediated rejection (AMR) and respiratory infection (INF); 3/ patients with parallel C4d immunostaining and Anti-HLA. All groups were compared to BAL of stable patients. C4d was measured via standard ELISA.C4d was increased in CLAD, predominantly in RAS (p = 0.0026) but not in BOS (p = 0.89). C4d was increased in all acute events, AR (p = 0.0025), LB (p < 0.0001), AMR (p = 0.0034), infections (p < 0.0001). In patients with parallel C4d immunostaining and serum HLA antibodies, C4d was increased in C4d-/HLA- (p = 0.0011); C4d-/HLA+ (p = 0.013); HLA+/C4d + (p = 0.0081). A correlation of systemic C-reactive protein (CRP) with C4d was found in all patients (r = 0.49; p < 0.0001). We hypothesize that free C4d in BAL may only be representative of a general immune response in the transplanted lung.

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method.

HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method. Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results. Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-Mpos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g≥1 + ptc≥1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%). Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA.

Sarcolemmal Staining by Anti-C4d/C3d in Cardiac Transplant Endomyocardial Biopsies and Associated Serum HLA Antibodies and Pathologic Rejection

Immunologic evidence of complement fixation in endomyocardial biopsies (EMB) is a feature used to assess pathologic antibody-mediated rejection in cardiac transplants. Uncommonly, sarcolemmal staining (SS) of myocytes is observed. The significance of this is unclear and under-investigated. This study aims to temporally correlate SS with changes in HLA antibodies and pathologic rejection. All EMBs that had SS by anti-C4d and/or C3d on immunofluorescence or immunohistochemistry (Figure 1) were identified via the laboratory information system between 10/1/2016 and 10/1/2019. The HLA antibodies and all other EMB results since time of transplant for each case were reviewed and temporally correlated to the biopsy that showed SS. 28 patients (mean: 31 yrs; range: 6-73 yrs) with 31 SS events were identified. Associated features are summarized in Table 1. Of note, 29/31 events of SS had HLA antibodies measured within a 2 week period before/after the time of biopsy. A temporally associated expansion of HLA Class I/II antibodies was noted in 12 (39%) cases. 21 (68%) cases had prior donor specific antibodies before EMB showing SS, with less immediate temporal association. Sarcolemmal staining of complement split products in EMBs was associated with a concurrent expansion in strength and/or breadth of HLA antibodies in 39% of cases. This is a novel observation that may partially explain the appearance of SS by immunologic assays. Further investigation is required to fully elucidate the significance of SS in EMB.

Elimination of HLA antibodies by platelet adsorption

The presence of HLA antibodies in patient serum or plasma may make antibody identification difficult. These HLA antibodies may mask the presence of clinically significant red blood cell (RBC) alloantibodies. Because platelets strongly express HLA antigens, it is possible to remove HLA antibodies by adsorbing the patient's serum or plasma using a platelet pool. Large numbers of random platelets are pooled to ensure a wide variety of HLA types are present. Elimination of the reactivity after adsorption suggests the presence of HLA antibodies in the patient's serum or plasma. The adsorbed patient sample may then be used to evaluate RBC alloantibodies without HLA antibody interference.

An Easy and Sensitive Method to Profile the Antibody Specificities of HLA-specific Memory B Cells.

Pretransplant immunological risk assessment is currently based on donor-specific HLA antibodies in serum. Despite being an excellent source for antibodies produced by bone marrow-residing plasma cells, serum analysis does not provide information on the memory B-cell compartment. Although B-cell culture supernatants can be used to detect memory B cell-derived HLA antibodies, low IgG concentrations can preclude detectability of HLA antibodies in luminex single-antigen bead (SAB) assays. Culture supernatants of polyclonally activated B cells from alloantigen exposed (n = 13) or nonexposed (n = 10) individuals were either concentrated 10-fold, or IgG was isolated by using a protein G affinity purification method to increase the IgG concentration. These processed culture supernatants, as well as paired serum samples were tested for the presence of HLA antibodies using luminex SAB analysis. In immunized individuals, 64% were found to have HLA-specific B-cell memory in concentrated supernatants, whereas 82% showed HLA-specific B-cell memory when IgG isolated supernatants were used for HLA antibody detection. IgG-isolated supernatants showed higher mean fluorescence intensity values compared with concentrated supernatants without increased background. In some individuals, HLA-specific B-cell memory was detected in the absence of accompanying serum antibody specificities. We developed a novel, highly sensitive method to assess the HLA-specific memory B-cell compartment using luminex SAB technology. This assay allows direct comparison to the serum compartment and may therefore provide a more complete picture of the humoral alloimmune response in patients with a history of alloantigen exposure.

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.

Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols

The flow cytometric crossmatch (FCXM) assay, which detects the presence of donor specific HLA antibodies in patient sera, is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time, we developed and validated two modified crossmatch procedures, namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60% and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification, employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63% vs 69 ± 19.06%), reduce cell isolation time (by ∼40%) and conserve FCXM assay reagents. Importantly, linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98–0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally, a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7% and 96.8% specificity and sensitivity, respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care.

P150 Utilization of two different Luminex single antigen beads assays (SAB) platforms in patients with unusual reactivity patterns

Luminex Single Antigen Beads Assay (SAB) are currently the most widely used means to identify HLA antibodies in sera from sensitized transplant candidates. Although these assays greatly enhance solid organ transplantation practices in terms of transplantig highly sensitized patients and the ability to perform virtual (electronic) crossmatch, issues concerning identifying unusual patterns or reactivity in some patients have emerged. This unusual pattern can be seen in male non-sensitized patients, patients with underlying auto-immune diseases, and normally sensitized patients. In order to investigate this pattern further, our lab utilized two primary testing platform. The One Lambda SAB Class I and Class II, was utilized as our primary testing patform. The LifeCodes SAB Class I and Class II was used as the secondary testing platform. Patterns of unusual reactivity were also examined by performing a flow cytometry crossmatch with surrogate donors. As control group, sera from 40 sensitized and non-sensitized patients were utilized to perfom the initial comparative study. Our results showed the HLA antibodies pattern of reactivity were identical between the two platforms. For patients with unusual HLA antibodies patterns, there was minimal correlation between the two platforms. False positive patterns identified by One Lambda SAB assays, were re-tested with LifeCodes SAB assays and results either in none or completely different false positive reactive patterns (see graph below). The absence of correlation between the two testing platforms in terms of false positive patterns might suggest that the patterns of reactivity might be directed to non-HLA IgG. In order to address that, we performed total IgG purification from two sera with this false positive pattern utilizing Protein A magnetic beads. SAB assay performed on purified IgG fraction eluted from the beads resulted in significant increase in the MFI values of these samples without any effect on the reactivity. In conclusion, interefence in SAB assays could be due to IgG fraction. Additional studies utilizing further blocking might be needed to eliminate this interfering patterns. Download : Download high-res image (237KB) Download : Download full-size image

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss.

Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain-binding polyclonal F(ab')2 (IgHPolyFab) is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG (FcMonoIgG). The fluorescent signal from the 2nd-Ab is measured as mean florescence intensity (MFI). When IgHPolyFab is used, the signal is amplified as a result of the binding of multiple polyclonal Fabs to the C region of primary IgH. The reliability of such amplification for Ab measurements was not validated, nor were MFIs compared with 1:1 binding of FcMonoIgG to primary Abs. Comparing the MFIs of anti-HLA Abs obtained with IgHPolyFab and FcMonoIgG against normal human sera, IVIg, and allograft recipients' sera, it was observed that the number of HLA-Abs was notably higher with IgHPolyFab than with FcMonoIgG The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. Indeed, MFIs of the de novo allo-HLA Abs were markedly higher with FcMonoIgG than with IgHPolyFab. Serum titration established the superiority of FcMonoIgG for monitoring MFIs of de novo allo-HLA Abs in allograft recipients. Avoiding false amplifications of the number and MFIs of anti-HLA IgG with FcMonoIgG may minimize immunosuppressive therapies, maximize the number of donors for patients waiting for allografts, and enable better prediction of graft rejection.

A Memory B Cell Crossmatch Assay for Quantification of Donor-Specific Memory B Cells in the Peripheral Blood of HLA-Immunized Individuals.

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow-residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA-specific memory B cells are scarce and insufficient in quantifying the complete donor-specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor-specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme-linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II-specific memory B cells per million IgG-producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor-specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross-match assay to quantify donor-specific memory B cells in patients with a history of sensitizing events.

Should epitope-based HLA compatibility be used in the kidney allocation system?

The new kidney allocation system (KAS) still applies donor-recipient HLA compatibility mostly at the antigen level and although some four-digit alleles have been included. This system is used to record unacceptable mismatches for sensitized transplant candidates with serum HLA antibodies. Since the reactivities of such antibodies are specifically associated with epitopes rather than HLA antigens, a more scientifically accurate assessment of mismatch acceptability could be based on epitopes. HLA class I and class II epitope specificity analyses can now be readily performed with serum antibody assays with single allele panels. This report describes an epitope-based HLA compatibility system for KAS and involves recipient and donor HLA typing at the four-digit allele level. It focuses on sensitized patients who have serum antibodies specific for HLA epitopes that can be entered as unacceptable mismatches in the transplant candidate database. Newly developed software programs could readily identify compatible HLA types.

P028 Uncovering the invisible; pre-treatment of sera with EDTA relieves prozone effect

Aim High titer HLA antibodies in patient sera can be falsely assessed due to interfering factors, also known as the prozone phenomenon, causing the masking of such antibodies. In this study we assessed the elimination of prozone by treatment of sera with DTT, EDTA and dilution. Methods Sera from 10 patients were treated with DTT, 6% EDTA, and 1:10 dilution. Antibody screening was performed by Luminex single antigen bead (SAB) assay. Effect of each treatment on the negative and positive controls, false-positive, and false-negative beads were compared to one another as well as to the untreated serum. Results Patient sera treated with DTT, EDTA and diluted at 1:10, all showed elimination of interfering factors and resolved the prozone effect. Comparison of the three treatment approaches showed that, DTT resolved the prozone, however it appeared to cause a slight increase in the mean fluorescent intensity (MFI) of the Luminex negative control (NC) bead. EDTA treatment eliminates prozone effect and has no effect on the NC bead. The 1:10 dilution also can resolve prozone, however the sensitivity can be compromised especially for the lesser strength antibodies. Conclusion In circumstances where prozone phenomenon is suspected, such as in the pre-transplant evaluation of highly sensitized patients, or during post-transplant monitoring for detection of donor specific antibodies, DTT, EDTA, and dilution are all effective strategies in elimination of interfering factors. However in addition to a higher NC, DTT-treatment, unlike EDTA, requires an additional incubation step with the sera (45 min) which affects the overall turnaround time. Dilution of serum can affect the sensitivity of the assay and in addition, often one dilution might not be sufficient for an accurate assessment of prozone, and performing multiple dilutions can prove to be costly. Routine treatment of sera with EDTA can accurately assess antibody specificities without affecting the sensitivity, turn around time and test cost.

OR3 The strength of positive linear correlations between donor-specific HLA antibodies and flow-cytometry crossmatch is locus-dependent

To determine correlation between the mean fluorescence intensity (MFI) of donor specific antibodies (DSA) and the median channel shift (MCS) of the flow crossmatch (FXM). HLA antibodies (Ab) in DTT-treated sera were analyzed using One Lambda (OL) single antigen beads (SAB). T cell FXM (51 pronase & 120 untreated) with single class I DSA and B cell FXM (41 pronase & 101 untreated) with single class II DSA were analyzed. MCS values were plotted against the MFI of the DSA and Pearson’s correlations (R2 were assessed. Positive correlations between T-FXM and DSA to HLA-A (R2 = 0.86) or HLA-B (R2 = 0.83) were strong, but low for Cw DSA (R2 = 0.28) [Figure]. B-FXM and DSA to DR (R2 = 0.54) and DQ (R2 = 0.55) had medium degree of +ve correlations while DP (R2 = 0.48) correlation was low. Pronase enhanced MCS but did not improve XM-DSA correlations. DSA with MFI < 2K to a single HLA-A/B or to a single DR/DQ did not cause +ve T or B FXM respectively. For Cw, +ve T-FXM was not observed when DSA was <8K MFI. Similarly, +ve B-FXM was not observed for DP when DSA MFI was <10K. XM reactivity of Cw Ab can be serum dependent. Pronase T-FXM between 3 Cw4+ donors and 2 sera with comparable Ab profiles (Ab to KIR2DL1-binding C2 epitope) on both OL and Immucor SAB panels produced conflicting results. Serum 1 (Cw4 MFI = 8081) was +ve with all donors (MCS = 78, 109, 155) while serum 2 (Cw4 MFI=9610) yielded all T-FXM -ve (MCS ⩽ 10). Sera can be false +ve with OL Cw beads, i.e., −ve with Immucor SAB and −ve T-FXM. Some displayed 10–20K MFI reactivity to certain Cw beads (Cw1, 12, 15) and others reacted to all Cw beads except to self. Continuing correlation studies between DSA and XM are critical for the precise virtual XM prediction. Standalone Cw Ab require additional scrutiny including screening by another reagent and T-FXM. Compared to HLA-A/B/DR/DQ DSAs, 4 or 5 times more Ab are required to yield +ve XM by Cw and DP DSAs respectively, and thus elevating MFI cutoff for Cw and DP unacceptable antigen listing will draw an enhanced donor offer for broadly sensitized patients.Download : Download high-res image (262KB)Download : Download full-size image

Detecting the humoral alloimmune response: we need more than serum antibody screening.

Whereas many techniques exist to detect HLA antibodies in the sera of immunized individuals, assays to detect and quantify HLA-specific B cells are only just emerging. The need for such assays is becoming clear, as in some patients, HLA-specific memory B cells have been shown to be present in the absence of the accompanying serum HLA antibodies. Because HLA-specific B cells in the peripheral blood of immunized individuals are present at only a very low frequency, assays with high sensitivity are required. In this review, we discuss the currently available methods to detect and/or quantify HLA-specific B cells, as well as their promises and limitations. We also discuss scenarios in which quantification of HLA-specific B cells may be of additional value, besides classical serum HLA antibody detection.

37-P

The introduction of Luminex-Single Antigen bead (LSA) assay significantly improved identification of HLA antibodies in sera from highly sensitized transplant candidates. Variants of this assay allowed to detect IgM antibodies and complement-fixing antibodies. Recently, the LSA assay was shown to be prone to the complement-mediated prozone effect leading to false-negative results. In this study we performed a comprehensive analysis of the HLA class I and class II antibodies present in sera from 16 highly sensitized kidney transplant candidates (%FlowPRA class I and II: 96 ± 4 and 95 ± 5 respectively) using four variants of the LSA technique: IgG-LSA, IgM-LSA, C1q-LSA and also EDTA-IgG-LSA to overcome the complement-mediated prozone effect. In 61% of EDTA-IgG-LSA tests we evidenced a strong increment of several antibody row-value (13,709 ± 5,863) respect to IgG-LSA results. This method was also able to reveal additional HLA class I and II antibodies (A2, A25, A68, A69, B42, B57, B59, B67, B77,DR17, DQ7, DQA1∗05:01) in 33% of the performed tests. Correlating EDTA-IgG-LSA and C1q-LSA results we found that all but one sera contained both complement-fixing and non complement-fixing antibodies; in the remaining serum we only detected anti-DP antibodies unable to fix complement. In one case, EDTA-IgG-LSA assay revealed the presence of a wide antibody pattern (anti-DQ2, -DQ4, -DQ7, -DQ8, -DQ9) specific for 84-87QLEL/89-90TT epitopes while C1q-LSA assay identified a similar but smaller antibody pattern (anti-DQ7, -DQ8, -DQ9) due to the recognition of 55P epitope. In 5 sera, IgM-LSA assay was able to detect HLA antibodies not revealed by IgG-LSA technique; unexpectedly several of these antibodies were HLA-Cw specific. In conclusion our findings evidenced the importance to make use of all LSA potentiality for characterization of HLA pre-sensitization status of transplant candidates in order to assess the real immunological risk of a potential kidney transplant.

174-P: VIRTUAL CROSSMATCH IN A PATIENT WITH HLA ALLELE SPECIFIC ANTIBODY

Aim HLA typing at low/intermediate resolution levels is acceptable for the characterization of HLA genes of solid organ recipients and donors. However, the interpretation of virtual crossmatch may be problematic for transplant candidates with allele specific antibodies. We present here a case report and a possible strategy to alleviate this problem. Methods HLA typing was performed using SSO for low/intermediate resolution and SSP for high resolution. One Lambda Single Antigen Bead assay was used for the detection and identification of serum HLA antibodies. Crossmatch was performed using CDC and flow cytometry. Results The patient is a 52-year old Caucasian male, previously sensitized to HLA antigens, listed for heart transplantation. Due to the urgency of the transplant, an organ offer from a 28-year old donor was accepted even though the patient displayed an allele specific, potentially donor specific antibody (DSA) directed to DRB1∗13:03 antigen ( Table 1 ). [table1] The available donor HLA class II type was: DR13, DR15, DQ6. This donor was accepted based on the known frequency of HLA-DR13 alleles (13:03 allele is 5-fold less frequent than 13:01) and the presumed decreased risk of anti-HLA class II DSA compared to class I DSA in heart transplantation. CDC and flow cytometry crossmatches were performed at the time of transplant and yielded negative results. Extended typing of the donor revealed that the donor carried the HLA-DRB1∗13:01 gene. Thus, the allele specific DRB1∗13:03 antibody was not a DSA. Post-transplant outcome was favorable. C4d staining was negative in serial allograft biopsies. Conclusions In patients with allele specific antibodies, the frequency of related HLA alleles may guide the interpretation of the virtual crossmatch. Whenever possible, extended HLA typing of the donor should be performed to confirm the presence or absence of DSA.