Luminex Single Antigen Bead Research Articles

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.

Exploring the immunological relevance of pre-transplant donor-specific antibody in intestinal transplantation, with special consideration to the liver.

Despite recent advances that have improved outcomes following intestinal transplantation (ITx), achieving long-term patient survival and rejection-free survival is still challenging. Understanding the relevance of pre-transplant human leukocyte antigen (HLA) donor-specific antibody (DSA) in ITx and the immunomodulatory potential of the liver within the allograft is crucial to providing an accurate assessment of pre-transplant immunological risk, which could influence and improve post-transplant outcomes further. This was the primary objective of this retrospective study of 95 adult ITx transplants which took place at Cambridge University Hospitals (United Kingdom) between 2007 and 2019. Two novel programs were developed and validated to identify DSA (tested by Luminex single antigen beads) in this dataset. Data analysis utilised Kaplan-Meier survival methods, and statistical analysis was performed using log-rank tests and adjusted Cox models. Fifty-four (57%) ITx cases contained a liver, and 36 (38%) were sensitised to HLA. Pre-transplant DSA >500 mean fluorescent intensity appeared to negatively affect post-ITx patient survival and rejection outcomes. Additionally, liver-inclusive allografts seemed to show particular resistance to HLA class I DSA. Our data hints towards consistency with other ITx studies where deleterious effects of DSA have been demonstrated, and where liver inclusion is protective from HLA class I DSA. This is in line with current national guidelines for immunological risk. Our publicly available research programs could support future large or multicentre studies where statistically relevant data might be gained.

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.

Association between blood transfusion after kidney transplantation and risk for the development of de novo HLA donor-specific antibodies and poor clinical outcomes: A single-center retrospective study

BackgroundBlood transfusion after kidney transplantation may increase the risk of sensitization and development of de novo human leukocyte antigen (HLA) donor-specific antibodies (DSAs). This study aimed to evaluate whether blood transfusion during the first year after kidney transplantation influences the development of de novo DSAs and clinical outcomes of kidney transplantation recipients. MethodsThis retrospective cohort study included nonsensitized first-time kidney transplantation recipients at Tianjin First Central Hospital from 2010 to 2022. The incidence of de novo DSA development and clinical outcomes between the groups were compared. Luminex single antigen beads were used to monitor DSAs. ResultsOf the 538 non-HLA-sensitized kidney transplantation recipients included in the study, 164 patients who received at least one unit of leukoreduced red blood cell transfusion within the first year (the transfused group), whereas the remaining 374 patients received no blood transfusion (the non-transfused group). Our analysis showed that there was a significant difference in the development of de novo DSAs and de novo anti-class I HLA-Ab between the two groups. Indeed, the transfused recipients had a higher serum creatinine and lower estimated glomerular filtration rate (eGFR) at 1-, 6-, and 12-month (all p > 0.05) after transplantation. Futhermore, a higher incidence of CMV infection, antibody-mediated rejection (AMR), hyper acute rejection (HAR), and delayed graft function (DGF) was identified in the transfused group (all p < 0.05). The graft survival was lower in the transfused group compared with patients in the non-transfused group (P = 0.002). Blood transfusion post-transplantation was a risk factor for de novo DSAs development but not an independent predictive factor for AMR and graft loss (odds ratio = 2.064 [1.243–3.429], p = 0.005). ConclusionsOur study showed that blood transfusion after transplantation is associated with the occurrence of de novo DSAs increasing an immunological risk for poor clinical outcomes for kidney transplantation recipients.

P516 A case of false positive Class II antibody reactivity consistent in both the One Lambda and Immucor Luminex single-antigen bead (SAB) assays

P516 A case of false positive Class II antibody reactivity consistent in both the One Lambda and Immucor Luminex single-antigen bead (SAB) assays

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells.

HLA Class I and II expression are known to differ locus-to-locus, however, HLA expression on the cell-surface is frequently reported as the total amount of HLA Class I or II antigens. This is despite evidence that indicates the differential expression of HLA can influence patient outcomes post-transplantation. Although numerous commercially available HLA monoclonal antibodies (mAbs) exist to characterize HLA expression, there is currently a lack of detailed information regarding their reactivities to HLA specificities. The specificities of locus-specific HLA mAbs (nine Class I and four Class II mAbs) were evaluated by two solid-phase Luminex single antigen bead assays. The reactivity patterns of these mAbs were then confirmed by flow cytometry using lymphocytes and PBSCs (peripheral blood stem cells). Out of the 13 HLA mAbs tested, only four (one Class I and three Class II mAbs) displayed intra-locus reactivity without also reacting to inter-locus specificities. Epitope analysis revealed the presence of shared epitopes across numerous HLA loci, explaining much of the observed inter-locus reactivity. The specificity of the HLA mAbs seen in solid-phase assays was confirmed against PBSCs and lymphocytes by flow cytometry. Using this method, we observed differences in the cell surface expression of HLA-C, HLA-DR, HLA-DQ, and HLA-DP between PBSCs and lymphocytes. Our results emphasize the need to characterize the reactivity patterns of HLA mAbs using solid-phase assays before their use on cells. Through understanding the reactivity of these HLA mAbs, the cellular expression of HLA can be more accurately assessed in downstream assays.

A retrospective observational study to estimate the risk of HLA alloimmunization with blood transfusion: Can the risk be reduced by leucodepletion?

BackgroundIn this retrospective study, our aim was to find the effect of leucodepleted (LD) blood transfusions on the formation of anti-HLA-antibodies when compared to non-leucodepleted (non-LD) transfusions using Luminex-based method. MethodsIn this study, Luminex single antigen bead assay (L-SAB) and HLA typing were performed on 310 patients. Test positivity rates (as MFI - Mean florescence intensity) were analyzed according to the different sensitization events and gender. ResultsOf the 310 patients included in the study, 58.06% (180) patients were male and 41.93% (130) were female. The average age of the patients was 42.86 (±12.37) years. In this study, test positivity rates were significantly lower in the patients who received LD RBC units than in those who received non-LD RBC units (28.43% = 29 of 102 Vs 55.22% = 74 of 134, p < 0.05). In our study, transfusion combined with a history of pregnancy had higher number of significant HLA antibodies compared to cases where transfusion was the only sensitization event (81.81% = 18/22 Vs 39.71% = 85/214, p < 0.05). In addition, anti-HLA-antibodies-MFI were significantly (p < 0.01) higher in non-LD patients compared to LD patients. ConclusionPatients who received LD RBC units had a significantly lower rate of transfusion-associated alloimmunization compared to those who received non-LD RBC units. Multiparous women had a high risk for transfusion-related alloimmunization compared to both nulliparous women and male patient. Furthermore, class I-anti-HLA-antibodies (HLA-B and HLA-A + B) were significantly associated with pregnancy sensitization and/or blood transfusion as a single sensitization.

Luminex Crossmatch in Kidney Transplantation.

The introduction of the Luminex Crossmatch assay (LumXm) which uses Luminex bead technology, consists of extracting the donor's Human Leukocyte Antigen (HLA) molecules from their lymphocytes, and binding them to fluorescent beads that are put in contact with recipient's serum. HLA donor-specific antibodies (DSA) are detected using a fluorescent conjugate. The goal of our study is to determine the benefits of using LumXm in a renal transplantation algorithm. We tested 78 recipients' sera using the LumXm, and the results were compared with the Luminex single antigen bead assay (SAB) for all sera, as well as the Flow Cytometry Crossmatch (FCXM) for 46 sera. We compared our results with those of SAB using 3 cutoffs, the first being the manufacturer's criteria where sensitivity and specificity were at 62.5% and 91.3% respectively for HLA class 1, and 88.5% and 50.0% respectively for HLA class 2. When using the third cutoff criteria ( ≥2 Adjusted values + MFI (Mean fluorescence intensity) >500 + Neg MFI < 500), the sensitivity increased to 69.0% for HLA class 1 and decreased to 84.0% for HLA class 2, while the specificity increased for HLA class 1 and 2. When comparing with FCXM, the 3 assays agreed in 55.8% of results for class 1 and 2 alike. However, major discrepancies were found for two groups in HLA class 1 and one in HLA class 2. The LumXm when used with other techniques to overcome its' weak points, can provide an interesting insight into the patient's HLA-DSA profile.

A modeling approach for mean fluorescence intensity value harmonization and cutoff prediction for luminex single antigen bead assays of two different vendors.

Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r2 : 0.946, class II r2 : 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r2 : 0.952, class II r2 : 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended.

C3d-binding assay for the detection of complement activating HLA antibodies: A useful tool for allocation to highly sensitised recipients in the post-CDC era?

The CDC crossmatch test is being phased out in solid organ donor allocation, and standard luminex single antigen bead assays do not differentiate complement activating function of HLA antibodies. The current study investigated the LIFECODES C3d-binding assay to determine if it could accurately predict actual T and B cell CDC results in a cohort of highly sensitised patients. Nineteen serum samples from different highly sensitised solid organ patients were crossmatched against cells from 62 unique donors, with 174 total T and B cell crossmatches performed. The sera also underwent SAB assay using OLI and LC platforms, and C3d-binding assay. Complement activating ability of each unique HLA antibody specificity detected using SAB was assigned based on the actual CDC results, which was then used to determine the accuracy of the C3d-binding assay. The C3d-binding assay was found to be highly accurate, with sensitivity of 95%, specificity 89% and negative predictive value 97% for class I DSA and the T cell CDC crossmatch results. Furthermore, we found 100% accuracy for prediction of the complement activating function of HLA-C antibodies. Negative predictive value of above 90% was also found for HLA class II DSA. C3d-binding proved more accurate than virtual crossmatch alone to predict CDC results. This study confirms that the C3d-binding assay predicts actual CDC crossmatch results accurately. In particular, the high negative predictive value of the C3d-binding assay may be extremely useful to define HLA antibodies that do not activate complement in highly sensitised recipients.

RE: Imlifidase-generated Single-cleaved IgG: Implications for Transplantation.

With great interest, we read the article by Bockermann et al1 on crossmatch assays after the administration of the novel desensitization drug Imlifidase. Proteolysis by Imlifidase generates single-cleaved immunoglobulin G (IgG) in a rapid first reaction and subsequently 2 F(ab’)2 fragments and a fully separated Fc fragment in a longer second reaction.2 Bockermann et al1 demonstrated that Luminex single antigen bead (SAB) testing, and crossmatching with both flowcytometry (FCXM) and the anti-human IgG-amplified complement-dependent cytotoxicity crossmatch (CDCXM) do not fully distinguish single-cleaved IgG from intact IgG. Bockermann et al1 suggest that the nonamplified CDCXM is best capable of demonstrating relevant residual intact IgG. Our center performed the first Imlifidase reimbursed HLA-incompatible kidney transplantation in March 2022. Contrary to Bockermann et al,1 we had difficulties in the interpretation of nonamplified CDCXM after Imlifidase treatment. A 29-y-old female with 6-y dialysis vintage after 2 failed kidney transplants was running out of vascular access. Despite high-urgency status and priority allocation via the Eurotransplant Acceptable Mismatch program, she remained without kidney offers. This was caused by her virtual panel-reactive antibody status of 100%. Her historical SAB mean fluorescence intensity (MFI) levels were high with broad reactivity in B-cell memory, with current MFI levels in the lower ranges. To enable for a timely HLA-incompatible kidney transplant, we delisted all unacceptable antigens with corresponding MFI levels of <3000, provided that CDC screening was negative. By this delisting, we increased her donor frequency of 0.000%–3.95%. Eight days later, this resulted in an HLA 1-2-2 mismatched kidney offer, with 2 “delisted DR-unacceptables.” Imlifidase was administered based on a virtual crossmatch. Serum was sampled 2 and 4 h after Imlifidase administration. Nonamplified CDCXMs with donor spleen cells were performed with preblood simultaneously tested with post 2-h and post 4-h sampling. Pre-CDCXM without dithiothreitol (DTT) treatment (which dissolves the disulfide bridges of IgM tetramers) was positive, indicating autoreactivity. DTT treatment revealed a negative pre-Imlifidase CDCXM. To our surprise, both the 2- and 4-h Imlifidase CDCXMs with DTT were positive (value 35 of 100). We had to decide whether or not to proceed with transplantation based on the nonamplified CDCXMs alone, as additional FCXM and SAB testing were scheduled for the next day. Because our patient had not recently received blood transfusions, was without infectious episodes, and had a negative pre-Imlifidase CDCXM, we expected assay interference by Imlifidase and decided to proceed with transplantation. The retrospectively performed pre-Imlifidase FCXM was negative for T cells and was 1.94 MCS for B cells. Luminex SAB was positive on 2 donor DR loci with 2500 and 2200, and became negative after Imlifidase. We investigated CDCXM on frozen donor spleen cells retrospectively and noticed CDC-positivity 2 and 4 h after Imlifidase, with negative CDCXMs on day 3. The patient did experience a vascular rejection after 1 wk, without signs of AMR. SAB testing revealed HLA antibody rebound from day 4 onward; however, DSA remained negative until current follow-up of 4 mo. This case demonstrates that even in nonamplified CDCXM, an interference by Imlifidase may occur. We advise to combine different assays when interpreting desensitization results after Imlifidase treatment.

Recipient-Specific Antibodies (RSA) in Mismatched Related Transplants May Induce Transplant-Associated Thrombotic Microangiopathy (TA-TMA)

HLA incompatibility increases the risk of transplant-related complications. Specifically, donor-specific antibodies (DSA) in mismatched related transplants (mm-HCT) increase the risk of graft failure. Less is known about the role of recipient-specific antibodies (RSA) detected in donors, as the immune status of the donor is not routinely examined before transplant with regard to anti-HLA antibodies. These antibodies may be transferred together with transplanted cells or produced by memory donor cells. We hypothesize that donor-derived RSA may induce immune-mediated complement activation in the classical pathway (CH50), leading to overproduction of membrane attack complex (MAC) and exacerbation of endothelial damage. Here we present preliminary epidemiological data regarding the presence of DSA and RSA, and the impact of RSA antibodies on transplant-related complications, especially transplant-associated thrombotic microangiopathy (TA-TMA) and acute graft-versus-host disease (aGvHD). Methods We prospectively analyzed the presence of anti-HLA antibodies in 20 pairs of donors and recipients qualified for mm-HCT. All recipients suffered from hematological malignancies and received a myeloablative conditioning regimen and immunosuppression consisting of post-transplant cyclophosphamide, tacrolimus, and mycophenolate mofetil. Patients’ and donors’ immunization status were tested with the solid phase method. Tests were carried out using a flow fluorometer across the screening range (Luminex Screen, One Lambda, USA). In the group of positive samples for anti-HLA class I and/or II antibodies, identification of the specificity of the HLA antibodies was performed (Luminex Single Antigen Beads, One Lambda, USA). Reagents enabling the determination of anti-HLA antibodies in the IgG class were used. All results were presented as mean fluorescence intensity (MFI) values. Monitoring of complement activation on days 30, 60, and 100 post-transplant was started after completing epidemiological data in the first 10 pairs. All patients were checked for TA-TMA and GvHD development during routine post-transplant visits. TA-TMA screening included TA-TMA triad: hypertension or kidney injury, LDH increase and decrease in number of platelets or refractoriness to transfusions, and additionally decrease in hemoglobin concentration, proteinuria, and clinical presentation with TA-TMA involved organ damage. Schistocytes as non-objective and nonrepeatable markers were excluded from diagnostic criteria. Results Anti-HLA antibodies were detected in 9/20 (45%) of recipients, and in 9/20 (45%) of the donors despite the lack of any transfusion history in donor group. Neither the presence of DSA nor RSA was affected by gender. RSAs were present more often in donors (7/9; 77%) than DSAs in recipients (2/9; 22%); p=0.052. Data summarizing MFI values and type of antibodies are presented in table 1. Laboratory diagnosis of TA-TMA met 12/20 (60%) patients, with a significant clinical presentation including severe hypertension (3) pericarditis (1), gastrointestinal (5), or neurological (6) impairment in 8/12 (66%). All patients with RSA (7/7; 100%) developed TA-TMA compared to no-RSA group (5/13; 38%) (p<0.01). TA-TMA-mediated organ damage was noticed more often in the RSA population (5/7; 71%) compared to the no-RSA group (3/13, 23%) (p<0.01); (Table 2). A trend for higher activation of CH50 and MAC in RSA compared to a no-RSA group was noted with a mean CH50 93 vs. 82 (p=0,16), and MAC 1428 vs. 1007 (p=0,24) respectively. A trend for higher maximum concentrations of tacrolimus 14 vs 10.7 (p=0.07) was observed in patients with TA-TMA. There was no impact of ABO incompatibility on TA-TMA development. During the time of observation 6 patients (30%) developed aGvHD - grades I, II, and III were diagnosed in 3, 1, and 2 patients respectively. Only 1 patient with RSA developed aGvHD. There was no correlation between RSA and GvHD occurrence (RSA 1/7, no-RSA 5/13, p=0.26) and between aGvHD and TA-TMA. CMV reactivation was documented in 13/20 (65%) patients, with no differences between the RSA and no-RSA groups (p=0.65). Conclusions RSAs occur surprisingly often in the donor population, irrespectively to the donor`s gender and the lack of transfusions history. The exact role of these antibodies is not fully understood; however, our data may suggest their impact on complement activation and TA-TMA induction. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

315.11: Tacrolimus Withdrawal After Mesenchymal Stromal Cell Therapy Is Associated With Donor-Specific Antibody Formation in Kidney Transplant Recipients

Introduction: Recently, the clinical phase II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy is a safe method to facilitate tacrolimus withdrawal in kidney transplant recipients (KTRs). Here, we analyzed de novo donor-specific antibody (dnDSA) formation and HLA molecular mismatch in MSC-treated KTRs. Methods: All patients underwent a first, living-donor kidney transplantation and received alemtuzumab induction, steroids, tacrolimus and everolimus. Patients in the MSC arm of the study received two infusions of autologous MSC at week 6 and 7 post-transplantation. Subsequently, tacrolimus was tapered and completely withdrawn at week 8. Patient sera were tested by luminex single antigen bead (SAB) assays (Immucor) for dnDSA assignment; positivity was defined as MFI > 1000. DSA were further characterized using a modified SAB assay to detect IgG1, IgG2, IgG3 and IgG4 subclasses. Complement binding was assessed with a C3d assay (Immucor). Patients and donors were HLA typed on 11 loci by next-generation sequencing. Eplet mismatches were calculated using HLAMatchmaker 2.0. Results: In total, 3 out of 27 patients (11%) in the control group and 11 out of 29 patients (38%) in the MSC group developed dnDSA, of which the majority was directed against HLA-DQ. The majority of DSA were complement binding and had different IgG subclasses. Although patients in the MSC group had an increased rate of dnDSA formation, the degree of antigen mismatch was similar to the control group (Figure 1). Cox regression showed that both MSC treatment group (HR=4.247, P=0.032) and HLA class II antibody-verified eplet mismatch (HR=1.176 per single eplet mismatch, P=0.001) were independently associated with dnDSA development. Despite the increased dnDSA formation in MSC-treated patients, incidence of rejection or graft loss was not increased and eGFR was stable at 2 years follow-up (Figure 2). Conclusion: Tacrolimus withdrawal after MSC therapy is associated with an increased rate of dnDSA formation in KTRs. However, no increased risk of rejection or inferior graft function was found, raising the question on the pathogenicity of these DSA, despite the presence of complement fixing IgG subclasses. We found that patients with a high HLA class II eplet mismatch load were less likely to tolerate tacrolimus withdrawal without developing dnDSA, which is in line with previous findings that HLA eplet mismatch load modulates tacrolimus through levels required to prevent dnDSA formation. Further research is warranted to explore HLA molecular mismatch load as a biomarker to guide personalized immunosuppression in transplantation. Jon J van Rood Transplantatiefonds.

P8.012: The Impact of a Positive B-Cell Flow Crossmatch in Living Donor Renal Transplant Recipients

Background: The aim of this study is to determine the incidence of early acute rejection and graft outcomes in living donor renal transplants with an isolated positive B-Cell Flow-Cytometry Crossmatch (B-FCXM). Methods: We performed a retrospective analysis of graft outcomes in 152 adults who received living donor renal transplants between January 2019 and March 2021 at Mount Sinai Hospital in New York CIty. Pre-transplant histocompatibility was assessed with flow cytometric crossmatch (FCXM) and Luminex single-antigen beads. Positive B- FCXM is was defined as median channel shift of ≥ 40. A mean fluorescence intensity (MFI) ≥ 1400 defined positive donor-specific HLA Class I and/or II antibody (DSA). Most patients received induction immunosuppression consisting of rabbit anti-thymocyte globulin, steroids and intravenous immunoglobulin. Estimated glomerular filtration rate (eGFR) and biopsy-proven rejection at six months’ post-transplant were compared between patients with and without a positive B-FCXM, and positive and negative DSA. Results: Of 152 patients, 89 (59%) were males and 63 (41%) females; the mean age was 44 years (SD ± 13.8). 45 donor B-FCXMs (30%) were positive and 107 (70%) were negative. 42 (28%) positive donor B-FCXM had no DSA and 3 (2%) had DSA. 91 (85%) negative donor B-FCXMs did not have DSA, and 16 (15%) had DSA. Thirty-six (80%) out of 45 patients with positive donor B-FCXM, including the 3 patients with DSA, received intravenous-immunoglobulin (IVIG) as part of induction immunosuppression. None of the patients with both positive donor B-FCXM and DSA had rejection. eGFR and rejection rate at 6 months were similar between patients with positive and negative donor B-FCXM [median 58.9 (IQR= 50.2-70.8) vs 57.6 (48.8-76.6), p=0.88; 6.8% vs 5.6%, p=0.7, respectively]. Comparison by combination of donor B-FCXM and DSA status (B-FCXM- DSA-, B-FCXM- DSA+, B-FCXM+ DSA-, B-FCXM+ DSA+) did not show statistically significant differences in eGFR or rejection rate at 6 months [median 57.4 (IQR= 48.5-74.3) vs 59.3 (52.6-83.3) vs 58.2 (50.1-70.8) vs 76.6 (58.3-102.5) p=0.4; 3.3% vs 18.7% vs 7.3% vs 0% p=0.09, respectively]. Conclusion: In our cohort, there was no statistical difference in early acute rejection and graft function at 6 months between living donor transplant recipients with positive and negative donor B-FCXM even after accounting for DSA status. Peri-transplant treatment with IVIG for a positive donor B-FCXM might be a helpful adjunct in this setting.

Clinical utility of serial serum dilutions for HLA antibody interpretation.

Luminex single antigen bead (SAB) testing has increased the sensitivity and specificity of accurately identifying HLA antibodies, in support of all organ transplantation. However, as described in manufacturers' recommendation, the output of the assay, using mean fluorescence intensity (MFI) units, is only semi-quantitative. Therefore, the ability to use MFI values to compare between different assays, to accurately guide clinical practice, or be used as an endpoint measure in clinical trials, is limited. To improve potential quantification, one must circumvent inherent limitations of SAB assays such as interference and saturation phenomena. In this review, we discuss how measurement of pre-transplant serum dilutions can be used to determine unacceptable antigens for wait-listing, determine the likelihood for successful HLA antibody reduction with desensitization, and compare degree of HLA (in)compatibility among various living donors. We also discuss how serum dilutions are optimal for measuring and comparing the efficacy of antibody depletion therapies for desensitization or antibody mediated rejection treatment post-transplant. Historically, one of the main criticisms for the use of serum dilutions and titer has been the potential labor and cost associated with additional testing. Here, we show how only one or two dilutions can add major value in most circumstances. In summary, the practical use of serum dilutions and titer determination are important methods that can be used before and after transplantation of all organs to quantify antibody accurately and reliably in routine practice and in clinical trials.

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.

Impact of catch-up vaccinations on anti-HLA antibody response in pediatric kidney transplant candidates.

Efforts have been concentrated on improving vaccination administration during the pretransplant evaluation period. However, concern for human leukocyte antigen (HLA) sensitization subsequent to vaccination exists. A retrospective review of pediatric kidney transplant candidates (PKTCs) ≤18 years old who had received vaccinations between February 1, 2017 and November 30, 2019 was conducted. Emergence of de novo anti-HLA antibody (HLA-Ab) 3-4 weeks postvaccinations detected by the Luminex single antigen bead assay (SAB) was evaluated. Outcomes assessed included change in the HLA-Ab mean fluorescence intensity (MFI) ≥25% from baseline, and change in preexisting HLA-Ab MFI strength, categorized as weak: 1000-2999; moderate: 3000-9999; and strong: ≥10 000. Sixty vaccinations were administered to 14 patients. Forty-one potential de novo HLA-Ab were detected in five patients. After additional antibody panel testing, 5/41 potential de novo HLA-Ab were determined to be HLA specific; the remaining 36 were deemed nonspecific. The 5 de novo HLA-Ab were observed in three patients and were deemed weak antibody (Ab). Median MFI showed a significant increase for nonspecific Ab, but not de novo HLA-Ab. Median MFI values were deemed transient at 7-10 week follow-up. No HLA-donor-specific Ab developed posttransplant in the patients who developed de novo HLA-Ab. Vaccination resulted in a transient increase in non-HLA-specific Ab. The majority of responses were non-HLA specific, hypothesized to be related to denatured antigens on single antigen beads. These data suggest limited clinical impact of vaccinations on the emergence of de novo HLA-Ab.

The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.

Development of De Novo Donor Specific Antibodies Following COVID-19 Vaccination in Cardiac Transplant

PurposeSome transplant patients mount an insufficient response to standard COVID-19 vaccine (COVAX) dosing and a booster dose has now been approved. Vaccination carries the theoretical risk of stimulating antibody production to the donor. There are no prior studies surveying de novo donor specific HLA antibodies (dnDSA) after COVAX. HLA-DQ is the most common dnDSA post cardiac transplant and is associated with worse outcomes. We reviewed the development of dnDSA to HLA-DQ in cardiac transplant patients after COVAX.MethodsDSA testing (luminex single antigen bead) was performed routinely at the time of surveillance endomyocardial biopsy. We retrospectively recorded any dnDSA to HLA-DQ with MFI >1000 after COVAX. We further identified a historical cohort (pre vaccination) of 32 patients who had DSA testing a minimum of 5 months after transplant.Results32 adult patients completed COVAX (16 Pfizer-BioNTech, 8 Moderna, 8 Johnson and Johnson) 3-79 mths after transplantation. 16 patients had DSA testing 1-5 mths post COVAX (4-84 mths post transplant) (Table 1). Three patients (18.8 %) demonstrated dnDSA to HLA-DQ (MFI 6360-25,824) 10-20 weeks post COVAX and all had episodes of high grade acute cellular rejection. One additional patient had rejection post COVAX without dnDSA. All 3 patients with dnDSA had robust response to the vaccine with antibodies to spike protein RBD of > 200 U/ml (Elecsys®). Eight vaccinated patients without dnDSA had anti-RBD testing, half had titres > 200 U/ml. In the historical pre COVAX cohort, 3 patients (9 %) demonstrated dnDSA (2022-25480 MFI), one of these had cellular rejection and one developed antibody mediated rejection.ConclusionOne fifth of cardiac transplant recipients developed dnDSA in the weeks following COVAX, all associated with rejection. While no definitive conclusions may be drawn, we believe these data suggest the need for immunological surveillance after COVAX. This may be even more important after a booster dose.

Significant Reduction of Donor Specific Antibodies in Heart Transplant Recipients Treated with Proteasome Inhibitors for Antibody Mediated Rejection

<h3>Purpose</h3> Antibody-mediated rejection (AMR) results in deleterious outcomes, including coronary artery vasculopathy (CAV), graft failure, and death. Proteasome inhibitors (PI) were used in pre-transplant desensitization to reduce antibodies to Human Leucocytes Antigen (HLA). We report our experience treating heart transplant recipients with AMR using PI, plasmapheresis (PLEX), and intravenous immunoglobulin (IVIG). <h3>Methods</h3> We retrospectively analyzed all patients treated with PI, either bortezomib (BTZ) or carfilzomib (CFZ), for AMR at our institution from 2018-present. CFZ (20mg/m2) or BTZ (1.3mg/m2) was given in combination with PLEX on days 1,2, 8, 9, 15 and 16, followed with IVIG 2g/kg. Mean fluorescence intensity (MFI) for donor-specific antibody (DSA) was evaluated by Luminex single antigen beads (One Lambda). Graft dysfunction (GD) was defined by left ventricular ejection fraction (LVEF) < 40%. <h3>Results</h3> Eleven patients were treated with PI for AMR (10 with CFZ, 1 with BTZ). Patients received 1-3 cycles of PI (1 cycle n=4; 2 cycles n=4; 3 cycles n=3), based upon the response in DSA. Before treatment, all patients except one had one or more strong DSAs (MFI> 8000) at neat serum. Most of the 22 strong DSAs observed were specific for HLA class II (DQ, n=14, 64%; DR, n=5, 23%). The treatment reduced MFI by 47+/- 46% at neat serum and by 70+/- 31% at 1:16 dilution (Figure 1). Four DSAs from 3 patients did not respond to treatment at 1:16 dilutions: one patient had high-titer DSA before treatment (MFI=14027 at 1:1024 dilution), another had rebound DSA from pre-transplantation. 5/11 patients presented with GD at onset (median EF reduction by 67%); 3/5 recovered LVEF to >40% after treatment. 9/11 (81.8%) patients survived to 1 year after AMR diagnosis. <h3>Conclusion</h3> Using PI in AMR treatment resulted in significant DSA reduction in 8/11 patients. Large studies are needed to further evaluate PI in AMR treatment.