Influence of Test Technique on Sensitization Status of Patients on the Kidney Transplant Waiting List

The exquisitely sensitive single antigen bead (SAB) technique was shown to detect human leukocyte antigen (HLA) antibodies in sera of healthy male blood donors. Such false reactions can have an impact on critical decisions, especially with respect to the determination of unacceptable HLA‐antigen mismatches in patients awaiting a kidney transplant. We tested pretransplant sera of 534 patients on the kidney waiting list using complement‐dependent cytotoxicity (CDC), enzyme‐linked immunosorbent assay (ELISA) and SAB in parallel. Evidence of HLA antibodies was obtained in 5% of patients using CDC, 14% using ELISA, and 81% using SAB. Among patients without history of an immunizing event, 77% showed evidence of HLA antibodies in SAB. In contrast 98% of these patients were negative in ELISA and CDC. In patients without an immunizing event, SAB‐detected antibodies reacted not always weakly but with mean fluorescence intensity (MFI) values as high as 14 440. High‐MFI‐value antibodies were found in some of these patients with HLA specificities that are rather common in general population, consideration of which would lead to unjustified exclusion of potential kidney donors. False SAB reactions can be unveiled by testing with additional antibody assays. Denial of donor kidneys to recipients based on HLA‐antibody specificities detected exclusively in the SAB assay is not advisable.     

Interpretation of HLA single antigen bead assays

The introduction of single antigen bead (SAB) assays for detection and quantitation of HLA antibodies has improved our ability to identify and manage allosensitized transplant candidates and recipients and to improve organ allocation, and was critical to the creation of national paired kidney exchanges. The principal limitations of the technology have been detailed in the literature and include artifacts resulting in non-specific background, variability, lack of standardization, and interpretive challenges. Accurate interpretation of SAB assays requires consideration of a number of factors, including identification of epitope reactivity patterns, mean fluorescence intensity (MFI) values, patient history, and appreciation of individual bead and assay nuances. The MFI value provides an estimate of relative HLA antibody levels although limited by saturation and epitope distribution effects. A better understanding of SAB assays and MFI values will be necessary to ensure appropriate application of these assays clinically and a higher quality of antibody data used in support of published clinical studies.     

Determining donor‐specific antibody C1q‐binding ability improves the prediction of antibody‐mediated rejection in human leucocyte antigen‐incompatible kidney transplantation

Detrimental impact of preformed donor‐specific antibodies (DSAs) against human leucocyte antigens on outcomes after kidney transplantation are well documented, however, the value of their capacity to bind complement for predicting antibody‐mediated rejection (AMR) and graft survival still needs to be confirmed. We aimed to study DSA characteristics (strength and C1q binding) that might distinguish harmful DSA from clinically irrelevant ones. We retrospectively studied 60 kidney‐transplanted patients with preformed DSA detected by single antigen bead (SAB) assays (IgG and C1q kits), from a cohort of 517 kidney graft recipients (124 with detectable anti‐HLA antibodies). Patients were divided into DSA strength (MFI < vs. ≥ 15 000) and C1q‐binding ability. AMR frequency was high (30%) and it increased with DSA strength (P = 0.002) and C1q+ DSA (P < 0.001). The performance of DSA C1q‐binding ability as a predictor of AMR was better than DSA strength (diagnostic odds ratio 16.3 vs. 6.4, respectively). Furthermore, a multivariable logistic regression showed that C1q+ DSA was a risk factor for AMR (OR = 16.80, P = 0.001), while high MFI DSAs were not. Graft survival was lower in high MFI C1q+ DSA in comparison with patients with C1q? high or low MFI DSA (at 6 years, 38%, 83% and 80%, respectively; P = 0.001). Both DSA strength and C1q‐binding ability assessment seem valuable for improving pretransplant risk assessment. Since DSA C1q‐binding ability was a better predictor of AMR and correlated with graft survival, C1q‐SAB may be a particularly useful tool.     

The correlation of results of panel reactive antibody,identification, and single antigen beads in detection of anti-HLA antibodies: Istanbul Faculty of Medicine,tissue typing laboratory experience

BackgroundWe have performed a retrospective analysis of anti-HLA class I MHC and class II MHC antibodies measured using a single antigen bead (SAB) assay and a panel reactive antibody (PRA) assay.Material and methodsA group of 256 patients with end-stage renal disease (ESRD) was tested for anti-HLA antibodies in the tissue typing laboratory between 2017 and 2020. In the cohort, the serum samples of patients waiting for transplantation were tested. Both the PRA and SAB tests of these patients were analyzed using the Luminex (Immucor) method. The threshold of positivity was accepted as median fluorescence intensities (MFI) ≥1000 for PRA screening and MFI ≥750 for SAB screening.ResultsOverall, antibodies to HLA antigens were detected in 202 (78.9%) out of 256 patients in the PRA study. Antibodies against both class I/II antigens were detected only in 15.6% of these patients, whereas antibodies against only against class I HLA in 31.3% and only against class II HLA in 32.0%. By comparison, the SAB study found that 66.8% of patients were positive for HLA antigens. Furthermore, donor-specific antibodies (DSA) were detected in 52.0% of PRA-positive patients and 52.6% of SAB-positive patients. It was shown that 168 patients (83.2%) out of 202 PRA-positive patients were found to be SAB-positive. In addition, 51 patients negative in the SAB assay (94.4%) were also negative in the PRA assay. Statistical analysis established a significant correlation between the PRA and SAB positivity (p > 0.001).It was also shown that MFI ≥3000 PRA positivity for class I HLA antigens (p = 0.049) and MFI ≥5000 PRA positivity for class II antigens (p < 0.001) correlated with the SAB positivity in patients.ConclusionOur results showed the importance of both PRA and SAB assays to define the status of sensitization in patients.     

Frequency and Determinants of Pregnancy‐Induced Child‐Specific Sensitization

The aim of this study was to define the frequency and determinants of pregnancy‐induced child‐specific sensitization shortly after full‐term delivery using sensitive single HLA‐antigen beads (SAB) and high resolution HLA‐typing of the mothers and their children (n = 301). A positive SAB result was defined by a background normalized ratio >1 or a mean fluorescence intensity (MFI) >300, >500 and >1000, respectively. The overall frequency of pregnancy‐induced sensitization determined by SAB shortly after full‐term delivery was between 45% (MFI > 1000 cut‐off) and 76% (ratio cut‐off). The rate of child‐specific sensitization at the HLA‐A/B/C/DRB1 loci was between 28% (MFI > 1000 cut‐off) and 38% (ratio cut‐off). The number of live birth was associated with a higher frequency of sensitization, which was driven by child‐specific, but not third party HLA‐antibodies. There was a clear hierarchy of sensitization among the investigated loci (B‐locus: 31%; A‐locus: 26%; DRB1‐locus: 20%; C‐locus: 15%; p < 0.0001). Some mismatched paternal HLA‐antigens led to a significantly higher rate of sensitization than the average (e.g. HLA‐A2, HLA‐B49, HLA‐B51, HLA‐C*15). Furthermore, the mother's own HLA‐phenotype—especially HLA‐A/B homozygosity—was associated with a higher rate and broadness of sensitization. The number of mismatched HLA‐A/B/C eplets strongly correlated with the rate of child‐specific class I sensitization.     

C3d Assay in Correlation With Single Antigen Bead Assay for Human Leukocyte Antigen Antibodies

Background

Single antigen bead assay (SAB) is a sensitive method for detecting HLA antibodies, but it does not specifically identify clinically relevant subsets. Recently, a new assay has been developed for detection of C3d bound to HLA antibody-antigen complex. We evaluated the C3d assay regarding its correlation with SAB in renal patients.

The shared epitope phenomenon—A potential impediment to virtual crossmatch accuracy

With the implementation of the new kidney allocation system (KAS), there is increased reliance on a virtual crossmatch/histocompatibility risk assessment (vXM) for evaluating potential presence, as well as strength, of HLA antibodies against a potential donor. The accuracy of such an assessment depends on the precision in the identification of the recipient's antibody profile and the potential donor's HLA typing. While the development of the single antigen bead (SAB) multiplex assay has improved the sensitivity and specificity of HLA antibody detection, several limitations of the assay (specific to certain sensitized patients) can complicate accurate interpretation of results. In this report, we focus on the “shared-epitope” phenomenon, a condition in which antibody strength can be underrepresented, or its presence completely missed, due to binding of the antibody to competing targets on multiple antigens (beads), effectively “diluting” the resulting MFI readout. Here, we provide a relevant background to understand this phenomenon and present a couple of case studies illustrating how it can be investigated, leading to a more accurate histocompatibility consultation.     

Comprehensive Assessment and Standardization of Solid Phase Multiplex‐Bead Arrays for the Detection of Antibodies to HLA

Solid phase multiplex‐bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte‐based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R²), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.     

A case of successful desensitization using therapeutic plasma exchange in high-risk HLA incompatible kidney transplantation

Advances in immune suppression therapies and desensitization have made possible kidney transplantation regardless of HLA incompatibility. Single antigen bead assay (SAB) is a semi-quantitative estimation of the amount of human leukocyte antigen (HLA) antibodies present in the recipient plasma, and mean fluorescence intensity (MFI) generated gives this rough estimation of the antibodies present in the recipient. Here we present a case of successful kidney transplantation in a patient who expressed DSA with high MFI. A 33-yr-old male, diagnosed with chronic kidney disease (CKD) on regular maintenance hemodialysis, opted for second kidney transplant with his sibling as prospective donor and was referred to the department of Transplant Immunology for histocompatibility testing. Patient had HLA incompatibility with multiple DSA identified by SAB. Patient undergone 20 sessions of plasma exchange till discharge and finally till 6 months graft was functioning well. The authors thus conclude that the option of a high-risk HLA incompatible kidney transplant can be offered to recipients with high MFI DSA, who wish to undergo transplantation for end stage renal disease.     

Antibodies to cryptic epitopes on HLA class I and class II heavy chains bound to single antigen beads: Clinically relevant?

Cell surface HLA class I consists of trimers, i.e., alpha - heavy chain, beta – 2 - microglobulin, and a peptide, termed closed conformers (CC) on non-activated lymphocytes. HLA class I and class II may also exist, respectively, as alpha-chain only or alpha and beta - chain only on activated cells termed open conformers (OC). We extend previous studies using an OC-specific monoclonal antibody that demonstrate LABScreen HLA class I and II single antigen beads (SABs) contain a mixture of open and closed conformers. LIFECODES SABs have bound CC only. More HLA class I and class II LABScreen SABs were reactive than LIFECODES SABs due to the presence of OC on LABScreen SABs. We hypothesized that antibody against OC on HLA B antigens would not be detected in cell based cross matches (XMs) with typical lymphocyte targets since anti-HLA OC antibodies would not react with native HLA CC on the cell surface. To test this hypothesis, we performed flow cytometry XM (FCXM) assays with sera of sufficient strength that most laboratories would likely predict positive FCXMs. Sera that reacted strongly with LABScreen SABs (>13,000 MFI) but weakly or not at all with LIFECODES SABs (<1000 MFI) gave negative T and B cell FCXMs. In contrast, sera that reacted with LIFECODES SABs (>13,000 MFI) but weakly with LABScreen SABs (<2100 MFI) exhibited positive FCXMs. Detection of antibodies directed against OC in SAB assays, may lead to inappropriate listing of unacceptable antigens, a decision not to XM or pre-or post – transplant desensitization procedures.     

The effectiveness of the combination of rituximab and high‐dose immunoglobulin in the immunomodulation of sensitized kidney transplant candidates

Kidney transplantation faces many challenges not the least of which is the presence of pre‐formed HLA antibodies. At our institution, we have used a combination of methods to immunomodulate sensitized patients. Most recently, this has been attempted with a combination of immunoglobulin (IVIG) and rituximab (Rituxan; Genetech, CA, USA). A total of 31 patients were followed for up to one yr following treatment with IVIG (2 gm/kg on day 1 and day 30) and rituximab (1 g – day 15). Antibody levels were followed serially at designated time points via solid‐phase single‐antigen beads (SAB) method (One Lambda, Inc., Canoga Park, CA, USA). Concentration of antibodies was based on median fluorescence intensity (MFI). The majority of patients had both class I and class II antibodies (79%). Our results showed that this protocol appeared to be patient and antibody specific. The most pronounced MFI reduction in antibodies occurred within the 30‐ to 100‐d period post‐treatment. Calculated panel‐reactive antibodies decreased but rebound tended to occur by 104 d after antibody MFI nadir. Because of this rebound, it can be inferred that the patients did not show a durable increase in their potential for transplantation. The search for a more effective method to immunomodulate patients continues.     

Pretransplant human leukocyte antigen antibodies detected by single‐antigen bead assay are a risk factor for long‐term kidney graft loss even in the absence of donor‐specific antibodies

Clinical relevance of ELISA‐ and single‐antigen bead assay (SAB)‐detected pretransplant HLA antibodies (SAB‐HLA‐Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death‐censored graft loss was found in patients with pretransplant SAB‐HLA‐Ab [HR: 4.46; 95% confidence interval (CI): 1.47–13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB‐HLA‐Ab but without SAB‐detected donor‐specific Ab (SAB‐DSA) (HR: 4.91; 95% CI of 1.43–16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA‐DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long‐term graft loss in patients with pretransplant SAB‐HLA, even in the absence of DSA. SAB‐HLA‐Ab‐positive patients, being negative in ELISA or CDC assay, might profit from a well‐HLA‐DR‐matched graft and intensified immunosuppression.     

Clinical impact of preformed donor‐specific denatured class I HLA antibodies after kidney transplantation

Class I single‐antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA‐sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor‐specific antibodies (DSA) using two different assays: an acid‐treated SAFB assay (anti‐dHLA DSA) and the iBeads assays (SAFB+/iBeads‐ DSA). Eighty‐five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520–13 882]). Anti‐dHLA and SAFB+/iBeads‐ DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500–1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti‐dHLA DSA or only SAFB+/iBeads‐ DSA developed acute clinical antibody‐mediated rejection in the first‐year post‐transplantation, and their five‐yr death‐censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T‐cell flow cytometry cross‐match. Therefore, both anti‐dHLA DSA and SAFB+/iBeads‐ DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.     

Assessing Antibody Strength: Comparison of MFI,C1q,and Titer Information

The presence of donor‐specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen–antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)‐treated samples, as well as titration studies and peak MFI values of over 7000 Luminex‐based single‐antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen–antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.     

Pretransplant Donor‐Specific HLA Class‐I and ‐II Antibodies Are Associated With an Increased Risk for Kidney Graft Failure

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q‐fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG‐DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q‐fixing DSA. Patients with both class‐I plus ‐II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q‐fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan‐IgG SAB assay are generally related to C1q‐fixation. We conclude that the presence of class‐I plus ‐II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q‐fixing IgG‐DSA could not be assessed due to their low prevalence.     

Comparison Between Total IgG,C1q,and C3d Single Antigen Bead Assays in Detecting Class I Complement-Binding Anti-HLA Antibodies

Background

Complement-binding donor-specific antibodies (DSAs) are associated with antibody-mediated rejection and allograft loss. Novel single antigen bead (SAB) assays—that is, complement component 1q (C1q) and complement component 3d (C3d) assays—have been developed to specifically detect complement-binding DSA, but it remains unclear whether these assays have an improved ability to detect complement-binding DSA as compared with using the total IgG SAB assay with a high mean fluorescence intensity (MFI) cutoff. The aim of this study was to compare the ability of the total IgG, C1q, and C3d SAB assays in detecting complement-binding anti-HLA antibodies.

A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect.Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer.     

Donor‐specific antibodies after pediatric liver transplantation: a cross‐sectional study of 50 patients

The role of donor‐specific HLA antibodies (DSAs) after pediatric liver transplantation (LT) is inadequately established. We conducted a cross‐sectional study on the prevalence of DSAs and their association with liver histology and biochemical variables after pediatric LT. Serum samples were drawn for HLA antibody analyses from 50 patients (76% of 66 eligible patients) operated on at age <18 years between 1987 and 2007 with a median of 10.0 (interquartile range 4.0–16.4) years after deceased donor LT. Mixed and single‐antigen beads with Luminex were used for HLA antibody screening and detection. A mean fluorescence intensity (MFI) value of 1000 was used for positive cutoff. Twenty‐six patients (52%; 95% confidence interval (CI) 39% to 65%) had DSAs. In 22 (85%) patients, DSAs were against class II HLA antigens with a mean (standard deviation) MFI of 13 481 (4727). The unadjusted prevalence ratio for portal inflammation in DSA‐positive compared to DSA‐negative patients (n = 47; 9/24 vs. 1/23) was 8.6 (95% CI 1.6 to 50.9). Laboratory values at the time of study were comparable between DSA‐positive and DSA‐negative patients. In conclusion, approximately half of patients studied had DSAs after pediatric LT. Portal inflammation was associated with DSA positivity although the wide confidence interval around the ratio estimate warrants cautious interpretation.     

Discrepant Antibody Testing Results: Which One to Believe?

The impact of solid-phase immunoassay for HLA antibody detection on the field of transplantation has been extremely significant by providing the most sensitive and precise method for characterization of HLA antibodies. However, despite all the benefits, technical limitations and inherent artifacts represent significant challenges, particularly with Luminex-based single-antigen bead (SAB) assay. Discordant results between antibody detection (screening assay) and identification (SAB) is not uncommon. Positive SAB assay in the context of negative screening testing is well documented and attributed to altered tertiary structure of HLA molecules exposing new epitopes or detection of naturally occurring antibodies. However, there are few reports that addressed the opposite scenario when negative SAB appeared in the context of positive screening assay.In such discrepant results, unmissed HLA antibody has to be excluded with certainty by other tests; however, with the availability of variable assays it may be difficult to choose the best combinations that clarify discrepancy without adding more confusion. Here we describe the results of correlation between 2 antibody screening solid-phase immunoassays (LABScreen Mixed using Luminex and FlowPRA Screen) on conventional flow cytometry and compare their outcomes with SAB and crossmatch results.     

Revisiting Traditional Risk Factors for Rejection and Graft Loss After Kidney Transplantation

Single‐antigen bead (SAB) testing permits reassessment of immunologic risk for kidney transplantation. Traditionally, high panel reactive antibody (PRA), retransplant and deceased donor (DD) grafts have been associated with increased risk. We hypothesized that this risk was likely mediated by (unrecognized) donor‐specific antibody (DSA). We grouped 587 kidney transplants using clinical history and single‐antigen bead (SAB) testing of day of transplant serum as (1) unsensitized; PRA = 0 (n = 178), (2) third‐party sensitized; no DSA (n = 363) or (3) donor sensitized; with DSA (n = 46), and studied rejection rates, death‐censored graft survival (DCGS) and risk factors for rejection. Antibody‐mediated rejection (AMR) rates were increased with DSA (p < 0.0001), but not with panel reactive antibody (PRA) in the absence of DSA. Cell‐mediated rejection (CMR) rates were increased with DSA (p < 0.005); with a trend to increased rates when PRA>0 in the absence of DSA (p = 0.08). Multivariate analyses showed risk factors for AMR were DSA, worse HLA matching, and female gender; for CMR: DSA, PRA>0 and worse HLA matching. AMR and CMR were associated with decreased DCGS. The presence of DSA is an important predictor of rejection risk, in contrast to traditional risk factors. Further development of immunosuppressive protocols will be facilitated by stratification of rejection risk by donor sensitization.