Luminex Single Antigen Bead Assay 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.

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.

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.

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.

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.

Highly sensitised patients awaiting deceased donor renal transplants are disadvantaged by the presence of denatured HLA antibody detected in routine HLA antibody testing.

Luminex single antigen bead (SAB) assays used to detect HLA antibodies may artificially increase sensitisation in highly sensitised patients (HSP). The presence of denatured HLA (dHLA) within the assay enables antibodies specific to cryptic HLA epitopes to bind, such antibodies are not clinically relevant. We sought to exclude dHLA reactivity in a cohort of very HSP, calculated reaction frequency (cRF) 95%-100% and determine the effect upon sensitisation. Such patients have limited access to suitable donors and small changes in their HLA antibody profile, particularly where their cRF is 100%, can increase their opportunity of a transplant. We determined the presence of dHLA by aligning antibody reactivity which did not correspond to known HLA class I epitope mismatches with the results of assays modified to detect class I dHLA. 130 class I dHLA reactions were identified within 11 HSP, all of whom had clear sensitising events. cRF was corrected for dHLA, mean cRF 98.2% (93-100) pre and 95.5% (87-100) post correction (p= 0.0156). An increase in the number of predicted compatible donors (p= 0.0078) after dHLA correction was demonstrated. Two manufacturers SAB assays were used. A reduction of patients with 100% cRF was observed for both manufactures. dHLA is contributing to sensitisation in HSP and is detrimental to their chances of receiving a compatible transplant. The observed dHLA reactivity varied according to kit manufacturers (p= 0.0001), this is potentially a useful finding for laboratories wishing to discriminate between nHLA and dHLA, but without the resources required to regularly perform dHLA assay and epitope analyses.

A Comprehensive Evaluation of the Antibody-Verified Status of Eplets Listed in the HLA Epitope Registry.

Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of de novo donor-specific antibody formation. However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data.

Desensitization Therapy in Kidney Transplantation Cases with Positive Baseline Complement-Dependent Cytotoxicity Crossmatch and High Donor-Specific Antibodies

Objective: The objective of the study is to assess the graft and patient outcome after desensitization in human leukocyte antigen incompatible kidney transplantation (KT) with positive baseline complement-dependent cytotoxic (CDC) crossmatch and high mean fluorescein intensity (MFI) of donor-specific antibodies (DSA). Methods: This was a retrospective study conducted at Jaypee Hospital, Noida. This study included highly sensitized patients who were transplanted with positive CDC and DSA &gt;10,000 MFI for single antigen or &gt;5000 MFI for multiple donor antigens. The patient's renal outcomes were documented. The desensitization protocol consisted of rituximab, therapeutic plasma exchanges (TPE), and thymoglobulin. Results: A total of five patients who had positive CDC crossmatch with very high level of preformed DSA underwent KT. Three patients had end-stage renal disease due to diabetic kidney disease while other two due to autosomal dominant polycystic kidney disease and chronic glomerulonephritis. All the patients were on dialysis. The MFI by Luminex single antigen bead assay for Class I varied from 1657 to 23440 and for Class II varied from undetectable to 11120. The mean number of pretransplant TPE sessions given per patient was 7.8 ± 2.68 and posttransplant TPE sessions per patient was 0.8 ± 0.45. The mean follow-up period was 308.2 days. Mean creatinine on the day of discharge was 0.58 ± 0.17 mg/dL. None of the patients had any postoperative infections or rejections. Conclusion: The current report showed favorable short-term patient and graft outcomes post-KT without any postoperative infections or rejections with desensitization therapy comprising of rituximab, TPE, and thymoglobulin induction.

Prospective De Novo Donor-Specific Antibody Monitoring in Renal Transplant Patients

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection leading to kidney transplant failure. However, many transplant patients are stable with no signs of graft dysfunction at the time of dnDSA detection at screening test. We prospectively studied 26 kidney transplant patients for 3 years, with dnDSA detected using the Luminex single-antigen bead assay in a routine test. Proteinuria and estimated glomerular filtration rate were evaluated along with dnDSA monitoring at least annually. Graft loss associated with dnDSA occurred in 8 (31%) patients, 14 ± 11 months after the initial detection of dnDSA. These patients had more frequently multiple dnDSA (P=.004) and remarkable proteinuria, more than 1 g daily (P < .001). The remaining 18 patients were followed for 38 ± 15 months and considered stable as there was no deterioration regarding estimated glomerular filtration rate and proteinuria. In 7 (39%) of these patients, dnDSA waned and were not detected anymore at follow-up. Antibodies against HLA class I had a significantly (P < .001) lower mean fluorescence intensity (MFI) (2603 ± 1098) compared with those against HLA class II (11,109 ± 6414). In regression analysis, they were predictive of dnDSA wane (P=.043; odds ratio, 0.18; 95% confidence interval, 0.04-0.94). Despite fluctuations during follow-up, there was no significant change in MFI for those who retained the dnDSA. The presence of dnDSA is transient in a significant proportion of stable renal transplant patients, especially in those with antibodies against HLA class I and a low MFI. Multiple dnDSA and severe proteinuria adversely affect renal graft survival.

Intensity of de novo DSA detected by Immucor Lifecodes assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and allograft loss. We tested Immucor* (IM) Luminex Single-antigen beads (LSAB) assay and C3d-fixing antibodies in the setting of dnDSA and subclinical (s) ABMR. This retrospective multicentric study included 123 patients biopsied because of the presence of subclinical de novo DSA detected by One Lamda* Labscreen (MFI > 1000). In 112 patients, sera of the day of the biopsy were available and tested in a central lab with IM Lifecodes LSAB and C3d fixing antibodies assays. In 16 patients (14.3%), no DSA was detected using Immucor test. In 96 patients, at least one DSA was determined with IM. Systematic biopsies showed active sABMR in 30 patients (31.2%), chronic active sABMR in 17 patients (17.7%) and no lesions of sABMR in 49 KT recipients (51%). Intensitity criteria (BCM, BCR and AD-BCR) of DSA were not statistically different between these 3 histological groups. The proportion of patients with C3d-fixing DSA was not statistically different between the 3 groups and did not offer any prognostic value regarding graft survival. Performing biopsy for dnDSA could not be guided by the intensity criteria of IM LSAB assay. C3d-fixing DSA do not offer added value.

Allosensitization in Heart Transplantation: The Importance of a Comprehensive Approach

<h3>Introduction</h3> HLA sensitization presents a major barrier to transplantation yet sensitive techniques such as the Luminex Single Antigen Bead (SAB) assay, when used in isolation, may inappropriately limit the donor pool. We report a case of apparent robust HLA sensitization following HBV vaccination and describe our approach to the assessment of HLA reactivity leading to successful heart transplantation (HTx). <h3>Case Report</h3> A 50 year old Caucasian male with long standing ARVD developed recurrent VT and worsening RV dysfunction leading to HTx evaluation. There was no history of transfusion, surgery, or mechanical circulatory support. The Luminex SAB assay at listing and serially thereafter was negative. Unexpectedly, broad class II HLA sensitization (cPRA 98%) was suddenly detected by SAB assay after HBV vaccination concerning for either a heterologous immune response or false reactivity to the beads. Multiple antibodies (Abs) persisted at 1:8 dilution and included Abs against the recipients own HLA (autologous flow XM negative). At the time when a donor became available, 4 DSA were identified on the most recent Luminex SAB. Given his high cPRA in the absence of a strong allosensitizing event, we combined assessment with an alternative solid phase assay (LABScreen PRA), 4-digit donor typing, and prospective crossmatches by Flow Cytometry (FC) and Complement Dependent Cytotoxicity (CDC) to determine the safety of proceeding with HTx (Figure 1). Notably, no HLA Abs were detected using the LABScreen PRA and both FC and CDC crossmatches were negative. The patient underwent successful HTx with no rejection and normal graft function. <h3>Summary</h3> HLA allosensitization portends worse outcomes after transplant. However, the high sensitivity of the Luminex SAB and potential for false positivity may result in inappropriately limiting access to HTx. This case highlights the importance of comprehensive assessment of HLA sensitization in situations of uncertainty to help identify pathogenic HLA Abs and allow for successful HTx.

Location, Location, Location - Does Epitope Matching Matter in Pediatric Heart Transplantation?

<h3>Purpose</h3> Recent data in adult solid organ transplantation suggest that epitope-based human leukocyte antigen (HLA) matching may permit better risk assessment of <i>de novo</i> donor‐specific antibody (dnDSA) development compared to antigen-based HLA matching, but data in pediatric heart transplantation (PHTx) are scarce. We evaluated the association between epitope mismatches and dnDSA in PHTx. <h3>Methods</h3> We performed high resolution HLA typing and epitope mismatch analysis (HLA Matchmaker) in children undergoing primary PHTx at a single center from 2014 - 2020. DSA >1000 MFI (Luminex Single Antigen Bead Assay; One Lambda) were considered positive. Data are presented as median (interquartile range). <h3>Results</h3> Epitope and dnDSA data were available in 109 transplants (age 5.8 years [1-12], 66% male, 32% non-Hispanic White, follow-up 2.5 years [1.3 - 4.7]). There was significant diversity in the number of epitope mismatches within each HLA locus (Fig 1A), and the number of mismatches were: A 20 (13-28), B 17 (12-22), C 10 (6-15), DR 16 (8-30), DQ 12 (8-16), and DP 8 (3-13). The number of epitope mismatches did not differ among any donor-recipient race combination (not shown). The development of dnDSA was not associated with a higher number of epitope mismatches at any HLA-loci (Fig 1B). However, patients with antibody mediated rejection (AMR) had more DQ mismatches (15 vs 11; p=0.04) than those without AMR, and there were more anti-DQB1 DSA than any other DSA in the cohort (Figure 1C). <h3>Conclusion</h3> There is significant variability in the number of HLA epitope mismatches within each HLA locus. These data suggest that it may not be the total number of epitope mismatches but rather epitope mismatches at specific loci that vary in location and function that are of immunogenic importance. Further investigation of epitope-matching in PHTx may permit a more personalized immunosuppressive approach based on individualized alloimmune risk assessment in the future.

Comparison Between Phenotypic Bead Assay and Single Antigen Bead Assay for Determining Specificity of HLA Antibodies in Kidney Transplant Waiting List

ObjectivesThe determination of unacceptable antigens in patients on kidney transplant waiting list is a critical laboratory investigation in sensitized patients. The Luminex single antigen bead (SAB) assay has high sensitivity and accuracy. However, several countries have not yet implemented SAB testing for waitlisted patients because of limited financial resources. In Thailand, specificities of HLA antibodies are identified by using a phenotypic bead assay. The aim of this study was to evaluate the performance of the phenotypic bead assay for determining HLA antibody specificities when compared with the SAB assay. MethodsA total of 254 sera from patients awaiting kidney transplantation were included. Of 254 sera, 206 and 171 were positive for HLA class I and II antibodies, respectively. Antibody specificities of sera that were tested with both phenotypic and SAB assay were analyzed. The performances of the phenotypic bead assay were compared with those of the SAB assay as the gold standard by using estimation of pooling sensitivity, specificity, and accuracy. ResultsThe sensitivity, specificity, and accuracy of the phenotypic bead assay for determining HLA class I antibodies was 53.9%, 93.0%, and 78.1%, respectively. The sensitivity, specificity, and accuracy of the phenotypic bead assay for determining HLA class II antibodies were 57.3%, 94.9%, and 81.4% respectively. ConclusionIn waitlisted kidney transplant patients, the phenotypic bead assay had high specificity and moderate accuracy when compared with the SAB assay. However, the low sensitivity of the test suggests that the use of the phenotypic assay for determining HLA specificities should be applied with caution in sensitized patients.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Significance of Luminex-based single antigen class II bead assay and its mean fluorescence intensity in renal transplant cases; a retrospective observation in 97 cases

ABSTRACTThe objective of this study was to determine the mean fluorescence intensity (MFI) values of class II Luminex single antigen bead (L-SAB) assay and compare these MFI values with cell-based complement-dependent cytotoxicity crossmatch with anti-human globulin (CDC-AHG-XM) and IgG-B-cell flow cytometry crossmatch (FC-XM) results and explore the near-accurate MFI-cutoff values of positive cell-based crossmatch results. This retrospective study was an analysis in 97 renal transplant recipients, who were tested for the presence of DSA by CDC-AHG-XM and IgG-B-cell-FC-XM methods with their corresponding donor as well as for anti-human leukocyte antigen (HLA) antibody detection using a sensitive L-SAB assay. In the group having DSA MFI values <1000, none of the patients showed positivity for FC-XM and CDC-AHG-XM; in the group having MFI values between 1000 and 3000, 35.48% showed positivity for FC-XM but none by the CDC-AHG-XM method. However, in the group having MFI values >3000, 83.33% of cases were positive for FC-XM. Further, in those groups with MFI values between 3000 and 6000, 38.09% were positive for CDC-AHG-XM, while 86.66% showed positivity in the group with MFI >6000. Our results indicated that Luminex-DSA MFI value >1995 (P < .0001) significantly correlated with IgG-B-cell-FC-XM positivity while Luminex-DSA MFI value of >4247 (P < .0006) was significantly correlated with positive CDC-AHG-XM. MFI cutoff of 1995 exhibited a diagnostic sensitivity of 97.56% and specificity of 89.29% for predicting positive IgG B-cell FC-XM and MFI cutoff of 4247 exhibited a diagnostic sensitivity of 90.48% and specificity of 97.37% for predicting positive CDC-AHG-XM. However, a cutoff MFI of >5000 and >7000 for SAB assay had a sensitivity and specificity of 100% in detecting a positive IgG B-cell FC-XM and CDC-AHG-XM, respectively.