Single Antigen Bead Research Articles

Abstract 4144583: Beyond Acute Rejection Screening Following Pediatric Heart Transplant: In Patients Negative for Rejection, Elevated Donor-Derived Cell-Free DNA is Associated with Cardiac Allograft Vasculopathy (CAV) and Donor Specific Antibodies (DSA)

Donor-derived cell-free DNA (dd-cfDNA) has been increasingly used to detect acute rejection (AR). We aimed to compare our institutional dd-cfDNA results to previously published adult and pediatric dd-cfDNA AR cutoffs. We also hypothesized that in the absence of AR, elevated dd-cfDNA would be associated with CAV and positive DSA. Patients (pt) < 18 years at transplant with > 1 dd-cfDNA between 2021-2023 were included. Using dd-cfDNA levels from this cohort, sensitivity, specificity, NPV, and PPV were calculated. False positives and false negatives (FN) were determined using published dd-cfDNA thresholds. AR was defined as decision-to-treat with increased immunosuppression, which was independent of dd-cfDNA in our cohort. In pt without AR, t -test was used to compare the means of dd-cfDNA levels in pt with and without DSA. χ 2 testing was then performed to evaluate the association between dd-cfDNA levels above and below 0.2% and the presence/absence of DSA and CAV. DSA was defined as allele-specific DSA identified by single antigen bead with mean fluorescence intensity >1000, and CAV as any disease by angiography. There were 379 samples among 163 pt, a median of 2 samples per pt, and 32 samples obtained at time of AR. Performance of dd-cfDNA in our cohort vs published dd-cfDNA thresholds is shown in Table 1. The FN rate ranged from 16 to 37% as the dd-cfDNA threshold increased. Mean dd-cfDNA was higher in patients with positive DSA versus those without (0.83% vs 0.19%, p<0.001). In patients that did not meet AR criteria, dd-cfDNA levels > 0.2% were associated with a higher prevalence of positive DSA (n=66) (48% vs 13%, p<0.001), and trended towards a higher prevalence of CAV (n=20) (15% vs 11%, p=0.08). Dd-cfDNA had high sensitivity and NPV for AR but the FN rate using published dd-cfDNA cutoffs was high in our validation cohort. The clinical utility of dd-cfDNA may extend beyond AR screening given increased frequency of DSA and CAV in pt with elevated levels that are negative for AR. We speculate that monitoring intra-pt dd-cfDNA variability will reduce the FN rate when screening for AR in addition to enhancing its clinical utility when monitoring for CAV and DSA.

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

Statistical modeling to assess variation between tests in single antigen bead analysis

Statistical modeling to assess variation between tests in single antigen bead analysis

Use of single antigen beads coated with engineered variants for mapping a functional epitope of an HLA-DQA1*01 specific antibody

Use of single antigen beads coated with engineered variants for mapping a functional epitope of an HLA-DQA1*01 specific antibody

Evaluating the shared epitope phenomenon using expanded single antigen bead panels

Evaluating the shared epitope phenomenon using expanded single antigen bead panels

HLA A10 spurious reactivity with single antigen beads investigated

HLA A10 spurious reactivity with single antigen beads investigated

Human leukocyte antigen compatibility and incidence of donor-specific antibodies in pediatric liver transplant recipients.

Antibody-mediated rejection following liver transplantation (LT) has been increasingly recognized, particularly with respect to the emergence of de novo donor-specific antibodies (DSAs) and their impact on graft longevity. While substantial evidence for adult populations exists, research focusing on pediatric LT outcomes remains limited. To investigate the prevalence of human leukocyte antigen (HLA) mismatches and DSA and evaluate their association with rejection episodes after pediatric LT. A cohort of pediatric LT recipients underwent HLA testing at Santa Casa de Porto Alegre, Brazil, between December 2013 and December 2023. Only patients who survived for > 30 days after LT with at least one DSA analysis were included. DSA classes I and II and cross-matches were analyzed. The presence of de novo DSA (dnDSA) was evaluated at least 3 months after LT using the Luminex® single antigen bead method, with a positive reaction threshold set at 1000 MFI. Rejection episodes were confirmed by liver biopsy. Overall, 67 transplanted children were analyzed; 61 received grafts from living donors, 85% of whom were related to recipients. Pre-transplant DSA (class I or II) was detected in 28.3% of patients, and dnDSA was detected in 48.4%. The median time to DSA detection after LT was 19.7 [interquartile range (IQR): 4.3-35.6] months. Biopsy-proven rejection occurred in 13 patients at follow-up, with C4d positivity observed in 5/13 Liver biopsies. The median time to rejection was 7.8 (IQR: 5.7-12.8) months. The presence of dnDSA was significantly associated with rejection (36% vs 3%, P < 0.001). The rejection-free survival rates at 12 and 24 months were 76% vs 100% and 58% vs 95% for patients with dnDSA anti-DQ vs those without, respectively. Our findings highlight the importance of incorporating DSA assessment into pre- and post-transplantation protocols for pediatric LT recipients. Future implications may include immunosuppression minimization strategies based on this analysis in pediatric LT recipients.

260.11: Impact of HLA donor specific antibody vs non-donor specific antibodies by single antigen bead method in predicting outcome of living related renal allograft recipients.

260.11: Impact of HLA donor specific antibody vs non-donor specific antibodies by single antigen bead method in predicting outcome of living related renal allograft recipients.

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.

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.

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.

#1390 Differences in the detection of anti-HLA antibodies between two single antigen bead methods in kidney transplantation patients

Abstract Background and Aims Two semiquantitative single-antigen bead (SAB) methods are currently available for the detection of anti-HLA antibodies and the estimation of their activity, depending on complement binding. However, data on accordance of the two methods on specificities detection and their reactivity evaluation are scarce. The purpose of this study is to compare the two methods in terms of anti-HLA antibodies specificities detection and of their complement binding capacity Method Sera from 97 patients with a positive Panel Reactive Antibody Test (&amp;gt;5%) was tested by two SAB methods, Immucor (IC) and One-lambda (OL), for class I and II anti-HLA antibody specificities and their ability to bind complement. The patients were either on a waiting list for kidney transplantation or transplanted patients tested routinely or after a clinical event. Statistical analysis of the results and comparison of the two methods followed. Results OL detected more class I positive specificities per patient [25 (9-34) vs. 18 (7-28) with IC, p &amp;lt; 0.001], while the two methods showed no difference in class II anti-HLA antibodies. IC detected 1608 out of 8148 and 1136 out of 7275 totally examined, positive anti-HLA class I and II antibody specificities respectively, with a median mean fluorescence intensity (MFI) of 4195 (1995-11272) and 6706 (2647-13184) respectively. Similarly, OL detected 1942/8148 and 1247/7275 positive specificities of anti-HLA class I and II antibodies respectively, with MFI 6185 (2855-12099) and 9498 (3630-17702)] respectively. In the IC method, 428/1608 [MFI 13900 (9540-17999)] and 409/1136 [MFI 11832 (7128-16531)] positive class I and II specificities, respectively, bound the C3d fraction of complement. Similarly, OL detected 485/1942 [MFI 15452 (9369-23095)] and 298/1247 [MFI 18852 (14415-24707)] class I and II specificities respectively that bound the C1q fraction of complement. In the analysis of the specificities that were positive by both methods, MFI showed a strong positive linear correlation between the two methods (r = 0.61, p &amp;lt; 0.001 and r = 0.54, p = 0.001 for classes I and II respectively). Accordingly, data were analyzed with Principal Components Analysis, in which negative results were also included. In the projection of the plane defined by the first two principal components the vector corresponding to the IC method for class I antibodies was congruent with the vectors of the complement binding detection methods, C3d and C1q. In contrast, the OL vector was nearly perpendicular to the other vectors, indicating the existence of non-complement-binding specificities that are not detected by IC. A similar picture was found for antibodies against HLA class II (Figure). Conclusion OL was more sensitive for detecting class I and II anti-HLA antibodies despite the fact that there was no difference in the number of class II specificities per patient. However, the additional specificities detected by OL, potentially do not bind complement. The MFI of the complement binding specificities were higher in both methods. The two methods were equivalent in detecting complement-binding class I specificities, however the C3d IC method was more sensitive in detecting complement-binding class II anti-HLA antibodies.

#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.

Safety and Immunogenicity of SARS-CoV-2 mRNA Vaccine Booster Doses in Kidney Transplant Recipients: Results of a 12-mo Follow-up From a Prospective Observational Study.

Booster doses of SARS-CoV-2 mRNA vaccines are commonly used in kidney transplant recipients (KTRs). However, there is uncertainty regarding the waning of vaccination responses and immunological safety in KTRs. A total of 123 KTRs were included in the final analysis of this prospective observational cohort study. The aim was to evaluate the immunogenicity and immunological safety. SARS-CoV-2 antispike IgG antibodies and anti-HLA antibodies were measured at baseline and then at months 3, 6, and 12 after vaccination with the first booster dose (ie, the third vaccine dose). Antibodies against S1 and S2 subunits of SARS-CoV-2 were evaluated using an immunochemiluminescent assay (cutoff 9.5 AU/mL, sensitivity 91.2%, and specificity 90.2%). Anti-HLA antibodies were analyzed using single-antigen bead technology. Seroconversion was reached in 65% of KTRs previously nonresponding to 2-dose mRNA vaccination; the overall seroconversion rate 3 mo after the first booster dose was 83%. Vaccination induced a durable humoral response, and the antibody levels were stable during the 12-mo study follow-up. Higher age (exponentiated beta coefficient [eβ] 0.97; 95% confidence interval [CI], 0.943-0.997) and a full dose of mycophenolate (eβ 0.296; 95% CI, 0.089-0.984) were negatively associated with SARS-CoV-2 IgG antibody levels, whereas better graft function (eβ1.021; 95% CI, 1.005-1.037) was associated positively. There were no systematic signs of anti-HLA antibody development after vaccination. However, during the follow-up, there was a nonsignificant signal of an increase in anti-HLA antibodies in those who developed COVID-19. Additional booster doses of SARS-CoV-2 mRNA vaccines induce durable antibody response even in a large subset of previous nonresponders and are not associated with the risk of allosensitization. Furthermore, a signal linking COVID-19 to the development of anti-HLA antibodies was observed, and this should be confirmed and further examined (NCT05483725).

DONOR-SPECIFIC ANTIBODIES AS A PREDICTOR OF GRAFT REJECTION AFTER LIVER TRANSPLANTATION

The main reason for graft loss is the rejection of the donor organ, which may occur at different time after transplantation and may be caused by the recipient’s organism reaction against donor’s human leukocyte antigen (HLA) proteins. Donor-specific antibodies (DSA) are produced in patient’s organism as a response to foreign HLA antigens. Aim. The purpose of our study was to evaluate the effects of already existed and/or de novo generated DSAs in liver transplantation as predictors of graft rejection and to establish an interconnection between blood biochemical parameters (Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin level) with the level of DSA in patients with liver transplant. Methods. xMAP-Luminex next generation flow cytometry technology and LABScreen Single antigen beads reagent (Onelambda, USA) were used for antiHLA determination. Total bilirubin level was detected photometrically. The activity of ALT and AST was determined spectrophotometrically on the automatic analyzer COBAS C 111 (Roche, Switzerland) in accordance with the manufacture’s instruction. Results. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol. Conclusions. The DSA and PRA levels as well as total bilirubin and ALT and AST activity corresponded to each other and could be used for comprehensive both pre- and post-transplantation screening of patients requiring liver transplantation or re-transplantation. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol.