Single Antigen Bead Assay Research Articles

Interrogating post-transplant donor HLA-specific antibody characteristics and effector functions using clinical bead assays

Single antigen bead (SAB) assays are the most common and sensitive method used to detect and monitor post-transplant donor specific HLA antibodies (DSA). However, a direct comparison across traditional and modified SAB assays to improve routine DSA monitoring using pre-treated IgG sera to eliminate interference has not been performed. We performed a technical comparison of 251 post-transplant DSA from n = 91 serum samples tested neat (pre-treated, undiluted), at a single 1:16 dilution, in the C1q bead assay, and for IgG subclasses (IgG1, IgG2, IgG3, IgG4) with IgG-enriched sera. We found that DSAs that are detectable by 1:16 dilution and/or C1q are associated with higher IgG MFI values and results could be predicted by testing neat sera. DSA detected at 1:16 dilution correlated with >7000 IgG MFI in neat sera and identified DSA that exceeded the SAB linear range for semiquantitative measurements. C1q positive DSA correlated with >15,000 IgG MFI in neat sera. C1q binding correlated most strongly with total IgG MFI (Spearman r = 0.82, p = 0.002) and not specific subclasses, demonstrating that DSA C1q binding capacity in this cohort is driven by HLA-specific IgG concentration. Evaluation of engineered pan-HLA class I-specific human IgG1 and IgG2 subclass monoclonal antibodies by SAB C1q and C3d assays revealed that IgG2 antibodies can bind complement at higher concentrations. The strengths and limitations of modified SAB assays must be considered to optimize efficient testing and accurate clinical interpretation.

A new strategy for systematically classifying HLA alleles into serological specificities: Update and refinement.

HLA antigens were historically defined according to the unique reactivity pattern of cells expressing HLA molecules with distinctive clusters of allo-antisera and/or monoclonal antibodies. Subsequently, amino acid residues determining epitopes (DEP) in the HLA molecule were correlated with reactivity patterns. In current clinical practice, the presence of allo-antibodies is assessed using Luminex-based solid phase single antigen bead (SAB) assays for transplantation. Recently, novel antigens were proposed for HLA molecules with DEP patterns that do not match any serologically defined antigens recognised by the WHO Nomenclature Committee. To validate the antigens, mean fluorescence intensity values of SABs tested on >13,000 patients' sera were extracted from clinical databases and analysed by scatter plots using a linear regression model. We found that when two proteins were considered as the same antigen in the original study, for example, HLA-A*02:01 and -A*02:06, their correlation ranked among the highest values at each locus. In contrast, discrete asymmetric outliers were observed when there were different antigens, for example, HLA-A*30:01 and -A*30:02, allowing validation and confirmation of 20 novel antigens for HLA-A, -B, -C and -DR. The outliers were confirmed to be true or false by flow cytometric crossmatches. In addition to the previously defined residues for antigen assignments, findings suggest that further distinction should be made for common antigens by including the substitutions at residue 67 of HLA-B, 67 and 74 of -DR. These serologic analyses can be applied systematically to identify and confirm novel antigens. These developments will lead to designing optimal SAB panels and further improving virtual donor-specific antibodies assessment.

Can median fluorescence intensity of HLA antibody detected by LABScreen Single Antigen Bead assay absolutely predict C1q activity?

Can median fluorescence intensity of HLA antibody detected by LABScreen Single Antigen Bead assay absolutely predict C1q activity?

PreSorb is superior to Adsorb Out in reducing high background and false positive reactivity in HLA single antigen bead assay

PreSorb is superior to Adsorb Out in reducing high background and false positive reactivity in HLA single antigen bead assay

Inverse correlation between MFI and bead counts in single antigen bead assay for anti-HLA antibody testing

Inverse correlation between MFI and bead counts in single antigen bead assay for anti-HLA antibody testing

221.4: Antibodies against Eplet mismatches can help identify the cause of ABMR in absence of DSAs on single antigen bead assay in transplantation.

221.4: Antibodies against Eplet mismatches can help identify the cause of ABMR in absence of DSAs on single antigen bead assay in transplantation.

V-110.7: Prevalence of non- representation of HLA antigens and alleles in Single Antigen Bead assay panels in patients at a referral lab.

V-110.7: Prevalence of non- representation of HLA antigens and alleles in Single Antigen Bead assay panels in patients at a referral lab.

Calcium-dependent HLA-DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay.

Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl2). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl2. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.

Sensitive HLA antibody testing and the risk of antibody-mediated rejection and graft failure.

Solid phase detection and identification of HLA antibodies in kidney transplantation currently relies on single antigen bead (Luminex®) assays, which is more sensitive than the previously used enzyme-linked immunosorbent assays (ELISA). To evaluate the impact of more sensitive HLA testing on antibody-mediated rejection (AMR) occurrence and allograft survival, we analysed 1818 renal allograft recipients transplanted between March 2004 and May 2021. In 2008, solid phase testing switched from ELISA to Luminex. We included 393 (21.6%) transplantations before and 1425 (78.4%) transplantations after transition from ELISA- to Luminex-based testing. For this study, bio-banked ELISA era samples were tested retrospectively with Luminex. Significantly less pretransplant DSA were found in patients transplanted with pre-existing HLA antibodies in the Luminex (109/387) versus the ELISA period (43/90) (28% vs. 48%, p < 0.01). Throughout histological follow-up, 169 of 1818 (9.3%) patients developed AMR. After implementing Luminex-based testing, the rate of AMR significantly decreased (p = 0.003). However, incidence of graft failure did not significantly differ between both eras. In conclusion, less patients with pretransplant DSA were transplanted since the implementation of Luminex HLA testing. Transition from ELISA- to Luminex-based HLA testing was associated with a significant decrease in AMR occurrence post-transplantation. Since the decline of AMR did not translate into improved graft survival, Luminex-based testing has the added value of preventing low-risk AMR cases. Therefore, Luminex' high sensitivity must be balanced against waiting time for a suitable organ.

#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 &amp;gt;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 &amp;lt; 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 &amp;lt; 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&amp;gt;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&amp;gt;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.

Prevalence of anti-HLA antibodies in COVID-19 convalescent plasma donors: an Indian experience

BackgroundCOVID-19 convalescent plasma is one of the experimental therapies used widely in moderately sick COVID-19 patients. However, there are a few risks involved in plasma transfusion; notably, transfusion-related acute lung injury (TRALI) caused by antibodies against human leukocyte antigens (HLA). This study was designed to assess the prevalence of anti-HLA antibodies in convalescent plasma donors using the single antigen bead method. Study design and methodsThis was a hospital-based observational study of consecutive plasma donors. A total of 252 samples were screened for anti-HLA Class I and Class II antibodies using the microbead assay with the identification of anti-HLA Ab in positive samples being performed using a single antigen bead assay. Luminex-based normalized background cutoff ratios of 10.8 for Class I and 6.9 for Class II and mean fluorescence intensity cutoffs of 2500 for Class I and 1500 for Class II were used for screening and the single bead assay, respectively. ResultsOf 252 screened samples, 28 (11.1 %) were positive for Class I, Class II or both Class I and Class II anti-HLA antibodies in donors with no history of a previous immunizing event. Moreover, 20/252 (7.9%) donors without any history of prior immunization had specific anti-HLA antibodies of Class I or Class II or both by the single bead assay. ConclusionsThe high prevalence of anti-HLA antibodies in our cohort of donors raises an urgent and immediate need for anti-HLA antibody screening in all convalescent plasma donors for safe therapy of COVID-19 patients.

Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study.

Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

New approaches to kidney transplantation in Ukraine

The number of patients with end-stage renal disease in Ukraine, as in the whole world, increases. Kidney transplantation remains the most effective method of treatment for such patients. After 1933, when in Ukraine the world’s first kidney transplantation was performed, the number of there surgeries remained insignificant (up to 130 operations per year). In 2018–2020, the reform of transplantation in Ukraine began due to the change of some laws. And as a result, the number of such operations is gro­wing every year. Control over the transplantation process is carried out by a newly created body of the Ministry of Health of Ukraine, the Ukrainian Transplant Coordination Center. New transplant centers are being created, immunological support of transplant couples is being improved using polymerase chain reaction, flow cytometers and solid-phase methods due to HLA typing of the donor and recipient according to A, B, C, DR, DQ, DP loci, detection of anti-HLA antibodies, allele-specific anti-HLA antibodies using single antigen bead assay. Examinations of patients are carried out at the pre-transplantation stage and as part of monitoring after transplantation. Given the military actions, the lack of aviation and the long time of getting to the clinic, it became possible to preserve organs by methods of hypothermic or nomothermic perfusion. The number of organ transplantations is increasing. In 2023, several successful simultaneous heart and kidney, kidney and pancreas transplantations were performed. The problems of overcoming tissue incompatibility, choosing the optimal immunosuppressive therapy regimen for a given patient, training of nephrologists, wider and more effective use of perfusion machines, new protocols of immunosuppressive therapy, studying the peculiarities of kidney transplantation from marginal donors (the number of which in Ukraine prevails), kidney transplantation in highly sensitized recipients are still actual in Ukraine. The cooperation with our European and American colleagues is very important to improve the long-term results of such operations.

HLA class I peptide polymorphisms contribute to class II DQβ0603:DQα0103 antibody specificity

Antibodies reactive to human leukocyte antigens (HLA) represent a barrier for patients awaiting transplantation. Based on reactivity patterns in single-antigen bead (SAB) assays, various epitope matching algorithms have been proposed to improve transplant outcomes. However, some antibody reactivities cannot be explained by amino acid motifs, leading to uncertainty about their clinical relevance. Antibodies against the HLA class II molecule, DQβ0603:DQα0103, present in some candidates, represent one such example. Here, we show that peptides derived from amino acids 119-148 of the HLA class I heavy chain are bound to DQβ0603:DQα0103 proteins and contribute to antibody reactivity through an HLA-DM-dependent process. Moreover, antibody reactivity is impacted by the specific amino acid sequence presented. In summary, we demonstrate that polymorphic HLA class I peptides, bound to HLA class II proteins, can directly or indirectly be part of the antibody binding epitope. Our findings have potential important implications for the field of transplant immunology and for our understanding of adaptive immunity.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

Complement and Non-Complement Binding Anti-HLA Antibodies Are Differentially Detected with Different Antigen Bead Assays in Renal Transplant Recipients.

Two semi-quantitative, Luminex-based, single-antigen bead (SAB) assays are available to detect anti-HLA antibodies and evaluate their reactivity with complement binding. Sera from 97 patients with positive panel reactive antibody tests (>5%) were analyzed with two SAB tests, Immucor (IC) and One-Lambda (OL), for anti-HLA antibody detection and the evaluation of their complement-binding capacity. IC detected 1608/8148 (mean fluorescent intensity (MFI) 4195 (1995-11,272)) and 1136/7275 (MFI 6706 (2647-13,184)) positive anti-HLA class I and II specificities, respectively. Accordingly, OL detected 1942/8148 (MFI 6185 (2855-12,099)) and 1247/7275 (MFI 9498 (3630-17,702)) positive anti-HLA class I and II specificities, respectively. For the IC assay, 428/1608 (MFI 13,900 (9540-17,999)) and 409/1136 (MFI 11,832 (7128-16,531)) positive class I and II specificities bound C3d, respectively. Similarly, OL detected 485/1942 (MFI 15,452 (9369-23,095)) and 298/1247 (MFI18,852 (14,415-24,707)) C1q-binding class I and II specificities. OL was more sensitive in detecting class I and II anti-HLA antibodies than IC was, although there was no significant difference in the number of class II specificities per case. MFI was higher for complement vs. non-complement-binding anti-HLA antibodies in both assays. Both methods were equal in detecting complement-binding anti-HLA class I antibodies, whereas the C3d assay was more sensitive in detecting complement-binding anti-HLA class II antibodies.

Evaluation of different microbead volumes in a single antigen bead assay for HLA antibody detection

Abstract OBJECTIVE S: The multiplexed single antigen bead (SAB) assay is now commonly used for detection of antibodies to human leukocyte antigens (HLA) in the management of solid organ transplantation. Serum is incubated with microbeads individually coated with unique HLA antigens and the presence of antibody is detected using a secondary antibody. Due to its cost, laboratories may use a lower-than-recommended volume of reagent microbeads, although the impact of microbead volume on assay performance has not been reported. The present study seeks to determine the concordance of an SAB assay when different microbead volumes were used. Although this is also a required laboratory practice when modifications are made to an FDA-cleared assay, requirements for the validation of a multiplexed assay are currently not well-standardized. Hence, this study may also serve as an example framework for validating a clinical multiplexed assay. METHODS SAB (LabScreen, One Lambda) was performed on 14 patient specimens [Mei San T1] selected to provide a range of class I and II HLA antibody levels. Each specimen was tested using three microbead volumes of 5 μL (manufacturer-recommended), 3 μL and 2.5 μL. All reactions were tested in a single session to minimize sources of variability. A mean fluorescence intensity (MFI) of ≥ 2000 was used to define positive beads, which is also the institutional threshold for defining incompatible antigens in solid organ transplantation. Concordance between different bead volumes was calculated using Cohen’s Kappa. Bland-Altman plots were used to visualize bias. All analyses were performed at individual bead level. RESULTS Kappa values were: 0.91 (class I) and 0.95 (class II) for 3 μL vs. 5 μL; 0.90 (class I) and 0.93 (class II) for 2.5 μL vs. 5 µL; 0.97 (class I) and 0.93 (class II) for 2.5 μL vs. 3 μL. Positive bias in MFI values was observed with lower bead volumes and was most pronounced in the 2.5 μL vs. 5 μL comparison; in contrast, MFI values between 2.5 µL and 3 µL were more similar. CONCLUSION Lower-than-recommended microbead volume may be used in the SAB assay without significant impact on classification of incompatible antigens. Although positive bias in MFI values was observed with lower microbead volumes, the impact on positive vs. negative beads assignment was minimal. Since the tested microbead volumes represented 12.5-20% of the total reaction volume, such a change could have caused a matrix effect and impacted antibody-bead binding, leading to differences in MFI values. Hence, individual laboratories modifying the SAB assay should validate such modifications before implementation to ensure the accuracy of HLA antibody assignment.

Donor-Specific Antibody Mean Fluorescence Intensity Threshold Predicts Platelet Transfusion Response in HLA-Alloimmunized Patients

Introduction:Patients with platelet transfusion refractoriness (PTR) due to HLA-alloimmunization respond to HLA-matched platelets. However, 4/4 HLA-matched platelets or platelets without donor-recipient antigenic mismatches are rarely available. Further, in severe HLA-alloimmunization, it is difficult to find donor products which do not contain antigens to which the recipient has developed antibodies ( i.e. antibody specificity prediction, ASP method). Hence, studies have evaluated platelet transfusion responsiveness in the presence of ≥1 donor specific antibodies (DSAs) to widen donor pools, a strategy termed permissive mismatching (PERMM). Using established semi-quantitative techniques for HLA-antibody detection, we sought to identify a cumulative DSA threshold which predicted platelet corrected count increment (CCI) failure despite PERMM. Methods:We retrospectively reviewed platelet transfusions among patients with HLA-alloimmunization and suspected PTR at the University of Washington/Fred Hutchinson Cancer Center from 2021-2023. HLA Class I antibodies were detected using a solid-phase single antigen bead assay (One Lambda) on the Luminex platform with individual antibody strengths measured by the mean fluorescence intensity (MFI). The calculated panel reactive antibodies (cPRA) were defined as the proportion of donors against whom recipients had antibodies. Transfusions were categorized as HLA matched, PERMM (DSA MFI&amp;gt;0), or random donor platelets (HLA-type unknown). CCI was calculated as described by Cohn ASH 2020. Results: Of 420 patients suspected to have PTR, 75 had cPRA &amp;gt; 50% and received ≥3 HLA-selected platelet transfusions. Of the 2726 transfusions these patients received, 1198 were excluded due to unavailability of post-transfusion platelet counts within 2 hours or due to multiple transfusions occurring between platelet counts. Ultimately, 1528 transfusions were analyzed (Panel A). In 1031 PERMM transfusions, the cumulative MFI was a stronger predictor of CCI at 2 hours than cPRA or the number of mismatched DSA (coefficients of determination or R2 of 7.3%, 2%, and 0.4%, respectively). We then examined the spectrum of cumulative MFIs to identify a threshold MFI predicting transfusion success/failure among the PERMM products. PERMM platelet transfusions in recipients with DSA MFIs of up to 5000 generated CCIs similar to HLA-matched platelets. However, CCI was significantly different between MFI &amp;lt;10,000 (median CCI 17,783) and MFI ≥10,000 (median CCI 3753) (Panel B). We then stratified data by splenomegaly, a significant non-immunologic cause of PTR (23.2% of transfusions, R2 15.8% for CCI prediction). Of the transfusions not affected by splenomegaly, 9.6% did not generate an adequate platelet response (defined as a CCI &amp;gt;5000) across all MFIs. This failure rate increased to 33.3%, 40.8%, and 52.8% among transfusions in recipients with MFIs &amp;gt;5000, &amp;gt;7500, and &amp;gt;10,000, respectively. After multiple testing correction, MFI of several individual DSAs (A1, A2, A24, A68, B8, B37, B39, B44, B51, B57, and B62) were also found to be significant predictors of CCI using linear regression ( n ranging 32-193, R2 ranging 6-33%). Conclusions: This is the largest study to date evaluating optimal MFI thresholds of DSAs for permissive HLA mismatching in the context of platelet transfusions. Cumulative DSA MFIs demonstrated a dose response effect with lower MFIs generating better CCIs. In our patient cohort overall, and when stratified by splenomegaly, we identified a threshold for cumulative DSA MFIs of 10,000, under which &amp;gt;50% of transfusions resulted in an adequate CCI. This MFI threshold (&amp;gt;10,000) has been implicated in HLA mismatched solid organ transplant rejections (Kallon, Transf. Med. 2020) as well as in a pooled analysis of smaller HLA-selected platelet transfusion studies conducted by our group (unpublished). If incorporated into a transfusion strategy, this MFI threshold may perform better than units chosen based on the number of mismatched DSAs alone or random donor platelets and widen the donor pool. Identifying the impact of specific HLA antibodies on CCIs needs further study. Studies are also underway to correlate cumulative DSA MFIs with complement activation (C1q levels) as a possible mechanistic explanation for the differences noted across the MFI threshold of 10,000 in our study.

Adaptation of HLA testing to characterize the cynomolgus macaque MHC polymorphisms and alloantibody signatures.

Nonhuman primates are the closest animal models to humans with respect to genetics and physiology. Consequently, a critical component of immunogenetics research relies on drawing inferences from the cynomolgus macaque to inform human trials. Despite the conserved organization of the Major Histocompatibility Complex (MHC) between cynomolgus macaques and humans, MHC genotyping of cynomolgus macaques is challenging due to high rates of copy number variants, duplications, and rearrangements, particularly at the MHC class I loci. Furthermore, the limited availability of commercial reagents specific to cynomolgus macaques that can be used to characterize anti-MHC class I and class II antibody (Ab) specificities in cynomolgus macaques presents a major bottleneck in translational research. Here we successfully characterized cynomolgus macaque Mafa class I and class II serologic specificities in 86 animals originating from various geographical regions using the complement dependent cytotoxicity (CDC) assay with human HLA class I and class II monoclonal antibody (mAb) typing trays. Further, we successfully induced and characterized anti-Mafa class I and class II alloantibody specificity using HLA single antigen bead assays. We also subsequently tracked the alloAb burden in the animals during treatment with anti-B lymphocyte stimulator (BLyS) treatment. Altogether, these methods can be easily used in translational research to serotype MHC class I and class II specificity in macaques, characterize their alloAb specificity, and evaluate the efficacy of novel therapeutic modalities in depleting circulating alloAbs in these animals.

IgM marks persistent IgG anti-human leukocyte antigen antibodies in highly sensitized heart transplant patients

Sensitization to human leukocyte antigens (HLA) is a persistent problem in heart transplant (HT) candidates. We sought to characterize the anti-HLA antibody and circulating B cell repertoire in a cohort of highly sensitized HT candidates. We assessed immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-HLA antibodies using Luminex single antigen bead assays in a cohort of 11 highly sensitized (HS; calculated panel reactive antibody≥90%) and 3 mildly sensitized (MS) candidates. We also performed B cell receptor repertoire sequencing (BCRseq) in HS candidates and 33 non-candidate controls. HLA antibody strength was measured by mean fluorescence intensity (MFI). We found that IgM anti-HLA antibodies were present in all HS candidates, but with a lower breadth and strength as compared to IgG. When anti-HLA IgG specificities intersected with IgM, binding strength was higher. In contrast, there were IgM but no intersecting IgG specificities for the MS group. In four candidates in the HS group, IgG anti-HLA antibodies decreased in both breadth and strength after HT, but the decrease in strength was smaller if the IgG possessed a specificity that intersected with pre-transplant IgM. BCRseq revealed larger B cell clonotypes in HS candidates but similar diversity as compared to controls. IgM marks IgG anti-HLA antibodies with higher strength before HT and persistence after HT. The presence of IgM intersecting IgG for an anti-HLA specificity may be a useful approach to determine which donor HLA should be avoided for a sensitized candidate.