Solid-phase Single Antigen Bead Research Articles

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.

Virtual crossmatching and epitope analysis in kidney transplantation: What the physician involved in kidney transplantation should know?

Solid-phase single antigen bead (SAB) assay for detection of anti-human leukocyte antigen (HLA) antibodies and high-resolution HLA typing have enabled tremendous progress in virtual crossmatch (VXM) technology in recent years. However, misinterpretation of the SAB assay may result in detrimental consequences after kidney transplantation. Meanwhile, epitope analysis could be an effective method to estimate immunizing eplets, which may provide ancillary information for better understanding of the SAB assay. To perform epitope analysis appropriately, it is necessary to understand the basic principles related to histocompatibility testing and the characteristics of the SAB assay. Therefore, knowledge of the properties and limitations of the SAB assay is critical. In this review, we aim to describe the fundamental concepts regarding immunobiological assessment, including HLA, anti-HLA antibodies, and SAB assay, and explain epitope analysis using examples.

Clinical significance of low pre-transplant donor specific antibodies (DSA) in living donor kidney recipients with negative complement-dependent cytotoxicity crossmatches (CDCXM), and negative flow cytometry crossmatches (FLXM) – A single-center experience

BackgroundIt is controversial whether all donor-specific antibodies (DSA) detected by the solid-phase single antigen bead (SAB) assay negatively affect kidney transplantation outcomes. The study aimed to evaluate the possible clinical significance of low pre-transplant DSA in living donor kidney recipients. We analyzed a group of patients with HLA-A, B, and -DR DSA reactivities below a virtual crossmatch (VXM) value of 5000 MFI but with all VXM DSA reactivities at HLA-DQ, -DP, and -Cw, which were not typed routinely for donors prior to transplantation. We also investigated the incidence of persistent and de novo DSAs in available posttransplant SAB assays. MethodsFrom the historical cohort of living donor recipients transplanted between 2014 and 2018 at our center (n = 82), 55 patients met the inclusion criteria, namely: these patients were > 18 years old with non-HLA identical sibling donors, who were not desensitized, who had available pre-transplant SAB results, and who had negative both complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FLXM) results. An additional donor HLA typing, performed for all 55 recipients, identified donor additional HLA-DQ, -DP, and -Cw DSA reactivities. These patients were then divided by SAB reactivity into three groups: 1) those with DSA-positive reactivities; 2) those with non-donor-specific anti-HLA reactivities (NDSA); and, 3) those who were anti-HLA-negative. All these recipients were followed for three years and checked for their de novo or persistent DSA. ResultsIn the studied cohort, DSA-positive, NDSA reactive, and anti-HLA negative recipients constituted 33%, 36%, and 31% of 55 patients, respectively. Non-routinely considered pre-transplant HLA-DQ, -DP, and -Cw DSA-positive reactivities were shown in as many as 78% of DSA-positive cases (group 1) with the lowest MFI value of 319 to DP4 and the highest MFI of 5767 to DQ2. Of the pre-transplant HLA-A, B, and -DR DSA reactivities, only -DR52 DSA reactivity reached the highest MFI value of 2191. These detected DSAs did not reduce the mean estimated glomerular filtration rate (eGFR) values and did not increase the incidence of proteinuria in recipients. While the 3-year graft survival was lower in the DSA-positive group (94.4%) with one recipient who lost kidney transplant, the difference was not significantly different (p = 0.7) from the NDSA (100%) and negative (100%) groups. In terms of the incidence of de novo acute antibody-mediated rejection (AMR) at three years after transplantation, no case has been reported in the cohort. This may suggest that low DSA-positive recipients do not experience higher rejection rate. However, DSA-positive recipients had a tendency for a higher frequency of C4d deposits in peritubular capillaries (PTC) and de novo DSA. ConclusionOur 3-year follow-up of patients with low pre-transplant DSA found no association with a deterioration in graft function and worse graft survival. Furthermore, we did not observe an increase in AMR in our patients with low DSA. A larger cohort and a longer follow-up period may be needed to evaluate the tendency of low DSA-positive recipients towards the higher incidence of C4d deposits in PTC and/or de novo DSA.

The utility of second single antigen bead assay: Clearing the water or stirring up mud?

Though solid-phase single antigen bead (SAB) testing has provided major advances to the HLA community and organ allocation, it has not been without limitations. In particular, false-positive reactions lead to interpretative challenges and the potential to preclude a transplant if the corresponding antigens are deemed unacceptable. Two different vendor platforms are commercially available for SAB testing, one more recent than the other. The aim herein was to assess the benefit of using the newer SAB platform in situations where the primary platform yielded suspicious (specifically, false positive) reactions. Therefore, 42 serum samples with commonly encountered false-positive patterns observed in our laboratory were tested with the newer platform. Cases were classified as resolved, equivalent, or divergent based on whether the second platform produced no reactivity, the same pattern, or a distinctly different pattern compared to the primary platform, respectively. Approximately 33% of cases were resolved, 46% were equivalent, and 21% were divergent. The project revealed advantages of adding a second SAB platform to the laboratory’s test menu including resolving challenging samples and including broader coverage of different alleles and unique class II alpha/beta subunit combinations. However, the challenges of validating, maintaining, and billing for another test method in the laboratory may be barriers to routine use.

Evidence for the alloimmune basis and prognostic significance of Borderline T cell-mediated rejection.

Prognostic biomarkers of T cell–mediated rejection (TCMR) have not been adequately studied in the modern era. We evaluated 803 renal transplant recipients and correlated HLA‐DR/DQ molecular mismatch alloimmune risk categories (low, intermediate, high) with the severity, frequency, and persistence of TCMR. Allograft survival was reduced in recipients with Banff Borderline (hazard ratio [HR] 2.4, P = .003) and Banff ≥ IA TCMR (HR 4.3, P < .0001) including a subset who never developed de novo donor‐specific antibodies (P = .002). HLA‐DR/DQ molecular mismatch alloimmune risk categories were multivariate correlates of Banff Borderline and Banff ≥ IA TCMR and correlated with the severity and frequency of rejection episodes. Recipient age, HLA‐DR/DQ molecular mismatch category, and cyclosporin vs tacrolimus immunosuppression were independent correlates of Banff Borderline and Banff ≥ IA TCMR. In the subset treated with tacrolimus (720/803) recipient age, HLA‐DR/DQ molecular mismatch category, and tacrolimus coefficient of variation were independent correlates of TCMR. The correlation of HLA‐DR/DQ molecular mismatch category with TCMR, including Borderline, provides evidence for their alloimmune basis. HLA‐DR/DQ molecular mismatch may represent a precise prognostic biomarker that can be applied to tailor immunosuppression or design clinical trials based on individual patient risk.

P093 Daily quality control can prevent misanalysis of solid phase single antigen bead assay and prevent technical issues

P093 Daily quality control can prevent misanalysis of solid phase single antigen bead assay and prevent technical issues

Crossing low-level donor-specific antibodies in heart transplantation.

Donor-specific antibodies (DSA) detected by solid-phase single-antigen bead (SAB) immunoassays have been associated with antibody-mediated rejection (AMR), cardiac allograft vasculopathy (CAV) and decreased survival after heart transplantation. The clinical relevance of low-level DSA is equivocal. This review examines the techniques used to define low-level DSA, the limitations of these techniques and recent clinical experience crossing low-level DSA. Solid-phase multiplex bead immunoassays were introduced to solid-organ transplantation over 15 years ago. These technologies have a much greater sensitivity and specificity than older cell-based immunoassays. It was hoped that this increased resolution would lead to better outcomes by avoiding donors with antigens that transplant candidates produced antibodies against. Although some transplant patients with DSA show increased risk of AMR and decreased survival, a subset of patients with DSA at the time of transplant have outcomes comparable with patients with no DSA. Recent studies have demonstrated that DSA delineated according to titration studies and C1q assays better define low-level DSA that are well tolerated to cross. Early experience with crossing low-level DSA shows promise in kidney and heart transplantation. Preliminary findings from heart and kidney transplant patients show acceptable outcomes after crossing low-level DSA. The policy of crossing low-level DSA increases the donor pool for sensitized heart transplant candidates.

The Road to HLA Antibody Evaluation: Do Not Rely on MFI.

Technological advances in HLA laboratory testing undoubtedly improved the sensitivity and specificity of HLA antibody assessment but not without introducing a set of challenges regarding data interpretation. In particular, the introduction of solid-phase single-antigen bead (SAB) antibody assessment brought the belief that mean fluorescence intensity (MFI) was a quantifiable value. As such, MFI levels heavily influenced HLA antibody reporting, monitoring, and clinical practice. However, given that SAB testing was neither intended for nor approved to be quantifiable, is the use of MFI in current clinical and laboratory practice valid? What, if anything, does this numerical value actually reveal about the pathogenic potential of the antibody? What are the pitfalls and caveats associated with reporting MFI? Herein, we travel the road to HLA antibody assessment and explore the reliability of MFI values to make clinical decisions.

P157 Accelerated graft loss due to non-complement fixing donor-specific antibody against a single DP epitope

Aim The clinical significance of donor-specific antibody (DSA) solely to DP antigens remains controversial. Here we report a case of accelerated graft loss attributed to DSA against a single DPB epitope which was exacerbated by the “peanut butter” phenomenon observed with the solid-phase single antigen bead (SAB) assays. Methods A 48-yo white female with history of ESRD due to hypertension received her 3rd renal transplant (TX) from a deceased donor with 5/6 ABDR mismatch and a 100% cPRA. Her other sensitizations included a pregnancy and multiple transfusions. Results Despite the presence of DSA to DPB1 ∗ 02:01 at 7100 MFI, TX was proceeded in light of clearly negative flow crossmatches (FCXM). Post-TX urine output initially improved but trended down on post-operative day (POD) 2 with elevated serum creatinine ⩾ 6 mg/dL. While SAB on POD3 indicated a decline of DP2 DSA to 4200 MFI, biopsy revealed a Banff IA rejection, with peritubular capillaritis and endothelial cell and vascular injury, suggestive of humoral components. Subsequent SAB on POD13 showed the addition of de novo DSA to B37 at 4300 MFI. Despite immediate intervention with ATG/IVIG/Rituxan/plasmapheresis, the patient developed a profound septic syndrome and a nephrectomy was performed on POD24. Allograft biopsy showed strong evidence of AMR with diffuse cortical necrosis and hemorrhage, arterial thromboses, intimal arteritis, with C4d+ in the peritubular capillaries. Epitope analysis suggested the preexisting DP DSA was in fact directed to a single epitope, 56EE, shared amongst 9 beads comprising 6 DPB1 alleles (02:01/04:02/10:01/18:01/28:01), thereby inadvertently diluting the actual DSA titer as shown on individual beads (“the peanut butter effect”). While it was not being detected by FCXM, the cumulative pre-TX MFI of the DP DSA was indeed > 65,000. Retrospective testing indicated that this DP DSA was never complement fixing, suggesting an involvement of antibody dependent cell-mediated cytoxicity. An intermediate level of anti-AT1R antibody was detected post-TX, which may have further accelerated the graft rejection process. Conclusions This case illustrates the deleterious effect of DSA solely to DP antigens. Epitope analysis may facilitate a more accurate assessment of the actual DSA titer, especially for the highly sensitized patients.

Many de novo donor-specific antibodies recognize β2 -microglobulin-free, but not intact HLA heterodimers.

Solid‐phase single antigen bead (SAB) assays are standard of care for detection and identification of donor‐specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β2‐microglobulin‐free human leukocyte antigen (β2‐m‐fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post‐transplant sera from 55 patients who were positive for de novo donor‐specific antibodies on a SAB solid‐phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β2‐m‐fHLA or native HLA (nHLA). Antibodies to β2‐m‐fHLA were present in nearly half of patients being monitored in the post‐transplant period. The frequency of antibodies to β2‐m‐fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non‐DSA). Among the seven patients with clinical or pathologic antibody‐mediated rejection (AMR), none had antibodies to β2‐m‐fHLA exclusively; thus, the clinical relevance of β2‐m‐fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β2‐m‐fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post‐transplant period.

Importance of routine comparisons between Luminex instruments within a single laboratory

Aim The development of solid phase single antigen bead (SAB) assays for HLA testing has allowed for better identification and monitoring of HLA antibodies. However, standardization of testing and interpretation has not occurred and several variables can contribute to variation within and between laboratories. Instrument standardization, when multiple machines are used in the same laboratory, is of critical importance especially when following patients longitudinally pre- or post-transplant. The purpose of this report is to highlight the value of routine instrument comparisons. Methods To compare fluorescence MFI values between Luminex instruments, 6 informative samples were tested using Class I SABs (One Lambda, Inc). Testing was performed in one tray and immediately prior to running; samples were split to two trays. This allowed the same test sample to be run simultaneously on 2 different machines. Data were collected and differences in MFI values for the same bead were compared between instruments. Results Initial comparison of individual bead MFI values between Lum-1 and Lum-2 showed significant variance of up to 20% (acceptable value 10%)(Fig. 1A). Subsequently, service was performed on both instruments to align, standardize and calibrate. Lum-1 was found to have a failing laser even though it passed daily QC. Testing was then repeated. Post instrument service now showed a marked decrease in the variance between the 2 instruments ( 6%).(Fig. 1B) Conclusion Our data clearly show the value of performing sample comparisons between Luminex instruments within the same laboratory. Luminex controls do not assure instrument comparability. Instruments not meeting predetermined standards should be serviced and acceptable values reestablished. Data should be collected on a semi-annual basis to reconfirm that the results between instruments are comparable. This practice ensures that MFI values are similar between instruments and allows labs to confidently evaluate and compare MFI values from multiple instruments. Download : Download full-size image

OR27: Bw4/Bw6 ON HLA-A AND HLA-C: THE FORGOTTEN SEROLOGICAL PROPERTIES OF HLA CLASS I ANTIGENS

Aim Bw4 and Bw6 are mutually exclusive epitopes associated with all HLA-B antigens. However, certain HLA-A (23, 24, 25, 32) and HLA-C (1, 3, 7, 8, 9, 10, 12, 14, 16:01) antigens also bear the Bw4 and Bw6 epitopes, respectively. Current OPTN policies dictate that the assignment of Bw4/Bw6 should be based exclusively on HLA-B antigens. We sought to determine the extent of Bw4/Bw6 misrepresentation in the deceased donor populations. Methods HLA typing of 5250 deceased donors from two organ procurement organizations (OPO) were analyzed. Molecular typing was performed by PCR-SSP, PCR-rSSOP or the real-time PCR method. Results Of our deceased donor cohort, 47% were reported as positive for both Bw4 and Bw6, 15% as positive for Bw4 only, and 38% as positive for Bw6 only. Of the Bw4-only donors, 62% were found to have at least one of the Bw6-positive HLA-C antigens (9% overall); and of the Bw6-only donors, 32% were found to have at least one of the Bw4-positive HLA-A antigens (12% overall). These data indicate that the actual Bw4/Bw6 assignment is misrepresented in 21% of deceased donors. The antigenicity of the Bw4-positive HLA-A and the Bw6-positive HLA-C antigens was confirmed by: (1) Solid phase single antigen bead analysis using Bw4- and Bw6-specific monoclonal antibodies; and (2) Flow cytometry crossmatches using patient sera with well-defined Bw4 or Bw6 antibody specificity. Conclusions With the serological assays being phased out in histocompatibility testing, the actual serological properties of HLA antigens and their interaction with allo-antibodies are increasingly being neglected. This study suggests that the Bw4/Bw6 serological properties of the HLA class I antigens are misrepresented in 1 of every 5 deceased donors. This is mainly due to the limitations of the current molecular typing methodologies exacerbated by the lack of appropriate regulatory guidance. Potential recipients with unacceptable antigens to Bw4 or Bw6 are not being excluded from match runs with donors carrying the Bw4/Bw6-positive HLA-A and -C antigens. These undeclared, yet highly antigenic and immunogenic epitopes may contribute to unexpected positive crossmatches resulting in delayed organ allocation and organ discards, as well as post-transplant immunologic complications resulting in recipient morbidities.

P011 : MAKING THE MOST OF HLA SINGLE ANTIGEN ANTIBODY TESTING

Aim Solid-phase single antigen (SA) HLA antibody (Ab) testing is utilized to determine virtual cross matches (VXM) and to inform the degree of donor/recipient (in) compatibility, thus assisting in choosing induction, maintenance, and therapeutic approaches post transplant. Serum samples from highly sensitized patients (pts) are prone to inhibition “Prozone” effects, which complicate accurate assessment of antibody strength. EDTA pre-treatment or C1q-binding Ab (C1q) assays, as well as titration studies, were proposed to alleviate this problem. Methods Serum from 55 highly sensitized pts (77 SA bead assays; 7600 data points) were tested. MFI values of SA-IgG and C1q assays, as well as titration data, were compared. SA-IgG data was stratified into Strong, Strong Moderate, Weak Moderate, Weak and Negative (>10,000; 5001–10,000; 2501–5000; 1000–2500, and 10,000; 5001–10,000; 1001–5000; 200–1000; and Results “Prozone” effect, as revealed by titration studies, was observed in 47/55 pts (56/77 tests; 73%), with most samples reaching their highest MFI values at a titer of 1:16. Importantly, in many cases, discordant antibody strengths were observed between neat MFI values and titrations. Specifically, 34/55 pts (40/77 tests) exhibited Weak–Weak Moderate MFI values while antibody titers were high (1:256–>1:1024). Conversely, 14/55 pts (18/77 tests) exhibited Moderate Strong–Strong MFI values while antibody strengths by titration were low (1:1–1:8; 17 additional tests had antibody titers of 1:16). Positive C1q test results correlated with SA-IgG assays with titers of 1:32–64 and higher. Conclusion “Prozone” effect is frequently observed in SA-IgG testing for highly sensitized pts. While EDTA pretreatment of sera or C1q testing can alleviate this issue, titration studies provide additional granular information regarding the strength (affinity/avidity) of the antibody which can assist in planning for desensitization or treatment of AMR. In our hands, use of titration studies lowered the assay-to-assay variability known to affect luminex based multiplex assays and, as we have previously reported, increase the accuracy of VXM prediction.

Pronase Treatment Reduces Denatured HLA Antigen On Single Antigen Beads (SABs).

PURPOSE: The development of the solid-phase single antigen bead (SAB) assays has increased the sensitivity of HLA antibody detection, but the specificity is sometimes questionable. False positive results have been reported in patient sera due to denatured antigen present on the beads, likely as a result of the manufacturing process. In laboratories using SABs as the sole method of detection these denatured specificities can be inappropriately designated as unacceptable antigens, thereby unnecessarily restricting patient access to transplantation. Limited studies have suggested that transplant across antibodies to denatured antigen does not result in inferior outcomes, at least in the short term. In this study, we sought to determine if it was feasible to reduce the level of denatured antigen on the SABs. METHODS: Pronase, a combination of proteolytic enzymes, reduces nonspecific reactivity in cell-based crossmatches. Therefore, we investigated the efficacy of pronase treatment to reduce the level of denatured antigen on SABs. Briefly, SABs were treated with pronase at a working dilution of 2.35 U/mL in RPMI media at 37°C for 15min. Selected, well characterized sera (N=10) known to demonstrate reactivity to denatured antigens and/or true HLA reactivity were tested using SABs that had been treated with pronase. RESULTS: Pronase treatment of SABs eliminated reactivity of patient sera documented to be reactive with denatured HLA antigens. By contrast, pronase treatment had no effect on antibodies reactive to native HLA antigens on the same beads. Additionally, testing with monoclonal antibodies to Class I and Class II revealed that pronase did not reduce the level of native HLA antigen on any of the SABs. CONCLUSIONS: These results demonstrate that pronase treatment of SABs may be used to enhance the specificity of antibody reactivity in patient sera. Pronase treatment reduces reactivity to denatured antigens on SABs without effecting the expression of native HLA proteins. Whether the denatured antigen on SABs represents truly unfolded HLA molecules or co-purified contaminants is under investigation. Nonetheless, pronase treatment may be used to better define HLA antibody reactivity with SABs.

Abstract 18156: Donor Specific Alloantibodies to HLA II Antigens Result in Accelerated Cardiac Allograft Vasculopathy Assessed by 3d Volumetric Ivus

BACKGROUND: The immunologic risk associated with preformed donor-specific allo-antibodies (DSA) against human leukocyte antigens (HLA) in heart transplant (HTX) recipients is unclear. The goal of this study was to evaluate potential role of DSA on the progression of cardiac allograft vasculopathy (CAV) in HTX recipients. METHODS: The presence of DSA against HLA was evaluated using solid phase Single Antigen Bead assay (One Lambda, Inc., Canoga Park, CA; Luminex platform) before transplant in 35 HTX recipients. Using alloantibody specification analysis, 11% (4/35) and 20% (7/35) had DSA against donor Class I and II HLA respectively including the one patient who had both (DSA I &amp; II+) . 71% (25/35) of patients had no demonstrable DSA (DSA-). All patients had negative cytotoxicity cross match. Baseline three-dimensional intravascular ultrasound studies (3D IVUS) were performed at 1.0 [0.18, 1.4] year after transplant. Follow up 3D IVUS exams were performed 0.92 [0.85, 1.04] years after the baseline exam. RESULTS: The increase in mean plaque (media and intima) volume (PV) was accelerated in the DSA II+ group as compared to the DSA- group (0.87±1.9 mm (3)/mm vs. 0.16±1.8 mm (3)/mm) but the vessel volume index (follow up vessel volume/ baseline vessel volume) was similar between groups (1.08±0.5 vs. 1.09± 0.4; p=0.9). Plaque index (PI) (PV/vessel volume x 100 [%]) increased significantly in the DSA II+ group (17.9±6.3%) but not in the DSA- group (-13.6±13.2%, P=0.02). None of these values were significant in the DSA I+ only group. Treatment with Sirolimus did not affect PI progression between the DSA II+ and DSA- groups (PI progression: 15.9±8.7% vs. -11.7±7.1%, P=0.03). Age, gender, lipid and uric acid levels, prevalence of diabetes, ischemic time, time between IVUS examinations, the use of steroids, mycophenolate vs. azathioprine or calcineurin inhibitors vs. sirolimus were not different between the groups (P&gt;0.4 for all). CONCLUSIONS: Heart transplant recipients with clearly defined anti-Class II DSA are at risk for accelerated cardiac allograft vasculopathy. Whether interventions to minimize the risk of DSA such as virtual crossmatch, desensitization and complement inhibition will retard CAV remains to be proven.