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T-cell mediated rejection (TCMR), de novo anti-HLA donor-specific antibodies (dnDSAs) and ensuing antibody-mediated rejection (ABMR) reduce kidney transplantation (KT) survival. The immunomodulatory effects of 25-hydroxyvitamin D [25(OH)D] could be beneficial for KT outcomes. We aimed to evaluating the association between 25(OH)D levels, the development of dnDSAs, clinical TCMR and ABMR, and graft survival. This single center retrospective study included 253 KT recipients (KTRs) transplanted without preformed DSA between 2010 and 2013. We measured 25(OH)D in successive serum samples: at KT (M0) and M12 for the entire cohort, and additionally at M24 and/or M36 when sera were available. We assessed graft outcomes up to 5 years post-KT. The proportion of KTRs having sufficient 25(OH)D at KT (M0) was high (81.4%) and then dropped at M12 (71.1%). KTRs with sufficient 25(OH)D at M0 experienced less clinical TCMR (HR, 0.41; 95% CI, 0.19-0.88 in multivariate analysis). A sufficient 25(OH)D at M12 was independently associated with a longer dnDSA-free survival (HR, 0.34; 95% CI, 0.17-0.69). There was no association between 25(OH)D and clinical AMBR. Studying the KTRs with 25(OH)D measurements at M12, M24 and M36 (n = 203), we showed that 25(OH)D sufficiency over the 3 first-years post-KT was associated with a longer graft survival in multivariate analyses (HR, 0.39; 95% CI, 0.22-0.70). To our knowledge, this study is the first showing an association between 25(OH)D sufficiency post-KT and dnDSA occurrence in KTRs. Moreover, we reinforce previously published data showing an association between 25(OH)D, TCMR and graft survival in KT., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Highly sensitized (HS) anti-HLA patients awaiting kidney transplantation benefit from specific allocation programs. Serological monitoring at 3-mo intervals is recommended to prevent unexpected positive crossmatch (XM), but this strategy is not evidence-based. Therefore, we assessed its relevance when using single-antigen flow bead (SAFB) and screening flow bead (SFB) assays., Methods: We included 166 HS patients awaiting a transplant and assessed their SAFB profile during the year preceding their inclusion. Anti-HLA antibodies were evaluated by SAFB assay and compared within patients as serum pairs at 3, 6, and 9 mo. We assessed the performance of SFB for detecting changes in SAFB profiles with 35 serum pairs., Results: On comparing 354, 218, and 107 serum pairs at 3, 6, and 9 mo, respectively, only 0.6%, 0.7%, and 1% of all antigens tested exceeded for the first time the unacceptable antigen threshold (mean fluorescence intensity ≥2000) in the most recent sample. Irrespective of the follow-up period, the calculated panel-reactive antibodies increased by a mean of 1%, and there was no significant increase in the proportion of donors at risk for positivity of flow- or complement-dependent cytotoxicity XM. The SFB did not accurately detect the variations of SAFB profiles., Conclusions: Changes in HS patient profiles are anecdotal and show little association with transplant access or risk for positive XM. Less-frequent monitoring in HS patients should be considered to improve cost-effectiveness without affecting transplant safety., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Rituximab has been proposed as induction therapy in kidney transplant recipients (KTRs) with preformed donor-specific antibodies (DSA) or a positive flow cross-match. We here evaluated whether adding rituximab was associated with a higher incidence of post-transplant malignancies (PTM) due to greater immunosuppression., Patients and Methods: Forty-eight HLA-sensitized KTRs received induction therapy with anti-thymocyte globulin (ATG) and rituximab because of preformed DSA or a positive flow cross-match (RTX group). They were compared with a control group of 154 patients receiving ATG alone., Results: Thirty-nine of 202 (19.3%) patients developed PTM; the rate was similar in the RTX and no-RTX groups (14.6% vs. 20.8%, respectively, P = .3). The distributions of the types of cancer were similar between the two groups, with the majority being non-melanoma skin cancer (NMSC, n = 24). The risk factors for PTM were male gender, age, history of cancer, and azathioprine., Conclusion: Our data do not indicate a higher rate of post-transplantation de novo malignancies after kidney transplantation in high-immunological risk patients who received induction therapy based on ATG and rituximab., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Anti-denatured HLA-Cw antibodies are highly prevalent, whereas anti-native HLA-Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti-HLA-Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti-denatured and anti-native HLA-Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA-Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty-three (31.6%) were anti-denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ = 0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ = 0.289). We studied 52 kidney recipients with preformed anti-HLA-Cw donor-specific antibodies. Those with anti-native HLA antibodies experienced more acute and chronic antibody-mediated rejections (P = .006 and .03, respectively), and displayed a lower graft survival (P = .04). Patients with anti-native HLA-Cw antibodies more frequently had previous sensitizing events (P < .000001) or plausibility of their antibody profile according to known anti-native HLA-Cw eplets (P = .0001). Anti-native but not anti-denatured HLA-Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Pathogenicity of donor-specific antibodies (DSAs) can be assessed using the single-antigen flow beads (SAFB) assays through mean fluorescence intensity (MFI) with or without serum ethylenediaminetetraacetic acid (EDTA) treatment, measurement of C1q or C3d binding and/or their intragraft detection [graft-bound donor-specific antibody (gDSA)]. We aimed to investigate which of these markers best associates with antibody-mediated rejection (ABMR) and kidney allograft loss at the time of a for-cause biopsy., Methods: This retrospective, single-centre study included 77 kidney transplant recipients who underwent a for-cause biopsy between December 2004 and July 2013. All displayed serum DSAs were identified on the same day as the biopsy. Sera were tested in parallel with the classical SAFB assay with or without serum EDTA treatment, C1q- and C3d-binding assays. gDSAs were eluted from biopsy fragments and identified with SAFB., Results: The median time between transplantation and biopsy was 25 months (range 0.5-251). The median follow-up was 36 months (range 0-140). ABMR was histologically proven in 40% of recipients. The sensitivity and specificity of C1q, C3d and gDSA assays for predicting ABMR were 68% and 61%, 52% and 70% and 64.5% and 56.5%, respectively. At the time of biopsy, only the DSA MFI after EDTA treatment and C3d positivity were associated with graft loss. In multivariate analyses, glomerular filtration rate, transplant glomerulopathy and C4d positivity were the only factors associated with graft loss., Conclusions: Our findings weaken the rationale for systematically implementing C1q, C3d or gDSA assays in this situation, because they do not independently predict ABMR and graft loss.

Background: The differential pathogenicity of anti-HLA donor-specific antibodies (DSAs) is not fully understood. The presence of complement-binding DSAs helps in better defining the prognosis of acute antibody-mediated rejection (ABMR). The evolution of these antibodies after the treatment of ABMR is unknown., Methods: We included patients from the French multicenter RITUX ERAH study diagnosed with acute ABMR within the first year of renal transplantation, with circulating anti-HLA DSAs and treated randomly by rituximab or placebo (and intravenous immunoglobulins, plasma exchange). We centrally analyzed serum samples at the time of ABMR, 3 and 6 months after ABMR, with anti-HLA DSAs specificities and C1q-binding capacity assessment., Results: Twenty-five patients were included: 68% had C1q-binding DSAs at the time of ABMR. The presence of C1q-binding DSAs was associated with a poorer evolution of chronic glomerulopathy at 6 months (P = 0.036). The persistence of C1q-binding DSAs at 3 and/or 6 months after ABMR was associated with more severe chronic glomerulopathy (P = 0.006), greater C4d score deposition score at 6 months after ABMR (P = 0.008), and graft loss 5 years after ABMR (P = 0.029). C1q-binding capacity was associated with the DSA MFI but 5 C1q-binding DSAs in 4 patients had low MFI values without a prozone effect., Conclusion: The presence and persistence of anti-HLA C1q-binding DSAs after ABMR is a detrimental marker, leading to transplant glomerulopathy and graft loss. Assessment of the complement-binding capacities of DSAs could help decide treatment intensification.

Background: In virtual crossmatch (XM) strategies, a correct anticipation of XM results is required for appropriately allocating organs. We reassessed the ability to predict T lymphocyte flow cytometry and complement dependent cytotoxicity XM results with the mean fluorescence intensity (MFI) in Luminex class I single antigen flow beads (SAFB) assay, after correction of complement interference and exclusion of antidenatured HLA antibodies., Methods: Among 432 XM with T lymphocytes (T-XM), 407 were analyzed after exclusion of antidenatured HLA antibodies. Only ethylenediaminetetraacetic acid-treated serum MFI was considered. Only 1 cellular target HLA antigen for the serum was expressed in 238 cases, 209 and 29 being heterozygous and homozygous for this antigen, respectively. For the remaining 169 cases, at least 2 antigens were recognized. Single antigen flow bead MFI thresholds allowing XM positivity to be predicted were calculated with receiver operating characteristic curves., Results: T-XM results were tightly associated with SAFB MFI values. Anti-HLA-A and anti-HLA-B antibodies behaved similarly. Prediction and sensitivity SAFB MFI thresholds were determined, respectively, assessing the highest sensitivity/specificity ratio and at least 95% sensitivity for predicting T-XM positivity. Both were slightly lower for HLA-B than for HLA-A, whereas anti-HLA-Cw antibodies induced random XM results. Both thresholds were only slightly diminished for homozygous and for multiple HLA targets, considering the immunodominant, but not the sum, of antibodies MFI., Conclusions: Antibodies against HLA-A, -B, or -Cw behave differently. A homozygous HLA target does not trigger a twice higher XM positivity. Multiple antibodies are better evaluated through the immunodominant DSA MFI than through the sum of DSA MFI.

C1q-binding ability may indicate the clinical relevance of de novo donor-specific anti-HLA antibodies (DSA). This study investigated the incidence and risk factors for the appearance of C1q-binding de novo DSA and their long-term impact. Using Luminex Single Antigen Flow Bead assays, 346 pretransplant nonsensitized kidney recipients were screened at 2 and 5 years after transplantation for de novo DSA, which was followed when positive by a C1q Luminex assay. At 2 and 5 years, 12 (3.5%) and eight (2.5%) patients, respectively, had C1q-binding de novo DSA. De novo DSA mean fluorescence intensity >6237 and >10,000 at 2 and 5 years, respectively, predicted C1q binding. HLA mismatches and cyclosporine A were independently associated with increased risk of C1q-binding de novo DSA. When de novo DSA were analyzed at 2 years, the 5-year death-censored graft survival was similar between patients with C1q-nonbinding de novo DSA and those without de novo DSA, but was lower for patients with C1q-binding de novo DSA (P=0.003). When de novo DSA were analyzed at 2 and 5 years, the 10-year death-censored graft survival was lower for patients with C1q-nonbinding de novo DSA detected at both 2 and 5 years (P<0.001) and for patients with C1q-binding de novo DSA (P=0.002) than for patients without de novo DSA. These results were partially confirmed in two validation cohorts. In conclusion, C1q-binding de novo DSA are associated with graft loss occurring quickly after their appearance. However, the long-term persistence of C1q-nonbinding de novo DSA could lead to lower graft survival., (Copyright © 2016 by the American Society of Nephrology.)

Background: It is widely accepted that HLA donor-specific antibodies (DSA) are associated with antibody-mediated rejection and graft loss. However, in many transplant programs, preformed anti-HLA-Cw and anti-HLA-DP DSA are not considered in organ allocation policies because their clinical relevance is still uncertain., Methods: We analyzed the clinical impact of Cw/DP DSA through a retrospective study, comparing 48 patients transplanted with isolated preformed Cw/DP DSA (Cw/DP DSA group) with (i) 104 matched HLA-sensitized kidney transplant recipients with No DSA at D0 (No DSA group) and (ii) 47 kidney transplant recipients with preformed A, -B, -DR, -DQ DSA (A/B/DR/DQ DSA group)., Results: A positive flow cytometry crossmatch in the Cw/DP DSA group was more frequent than in the No DSA group and as frequent as in the A/B/DR/DQDSA group. Two years after transplantation, the biopsy-proven acute rejection-free survival was worse in the Cw/DP and A/B/DR/DQ DSA groups than in the No DSA group (65%, 84%, 93%, P = 0.001 and P = 0.05, respectively). Accordingly, graft survival was lower in the Cw/DP and the A/B/DR/DQ DSA groups than in the No DSA group (87%, 89%, 95%, P = 0.02 and P = 0.1, respectively)., Conclusions: These results suggest that preformed anti-HLA-Cw and anti-HLA-DP DSA are as deleterious as anti-HLA A/B/DR/DQ DSA. It justifies their inclusion in kidney allocation programs and in immunological risk stratification algorithms.

Outcome of patients with transplant glomerulopathy (TG) is poor. Using B-cell targeting molecules represent a rational strategy to treat TG during chronic antibody-mediated rejection. In this pilot study, 21 patients with this diagnosis received four doses of intravenous immunoglobulins and two doses of rituximab (IVIG/RTX group). They were retrospectively compared with a untreated control group of 10 patients. At 24 months post-biopsy, graft survival was similar and poor between the treated and the untreated group, 47% vs. 40%, respectively, p = 0.69. This absence of response of IVIG/RTX treatment was observed, regardless the phenotype of TG. Baseline estimated glomerular filtration rate (eGFR) and decline in eGFR during the first six months after the treatment were risk factors associated with 24-month graft survival. The IVIG/RTX therapy had a modest effect on the kinetics of donor-specific alloantibodies at M24, compared to the untreated group, not associated with an improvement in graft survival. The mean number of adverse events per patient was higher in the IVIG/RTX group than in the control group (p = 0.03). Taken together, IVIG/RTX treatment for severe TG during chronic antibody-mediated rejection does not seem to change the natural history of TG and is associated with a high incidence of adverse events., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Single antigen flow beads assays may overestimate sensitization because of the detection of supposedly irrelevant antibodies recognizing denatured class I human leukocyte antigens (HLAs)., Methods: Sera of 323 HLA-sensitized kidney transplant candidates positive with a class I HLA single antigen flow beads assay were retested after acid treatment of the beads. Denatured HLA antibodies were identified according to ratio between the measured fluorescence intensity for treated and nontreated beads. T-lymphocyte flow cytometry crossmatches were performed to characterize the ability of these antibodies to recognize HLA on normal cells as a surrogate of their potential clinical relevance. Their impact on organ allocation was evaluated through a calculated panel reactive antibody. The utility of single antigen flow beads largely devoid of denatured HLA (iBeads) was also evaluated., Results: Denatured HLA antibodies were detected in 39% of the patients. They provided much less positive flow cytometry crossmatches than anti-native HLA antibodies (16% vs. 83%, P<0.0001). Removing the HLA-A and HLA-B antigens targeted by denatured HLA antibodies from unacceptable antigens lowered the calculated panel reactive antibody for 90 patients, sometimes dramatically. The iBeads assay demonstrated nearly the same ability to predict crossmatch results than the acid treatment assay., Conclusion: Denatured class I HLA antibodies are common, but the antigens they target should not be considered as unacceptable in most cases, because they negatively impact access to a transplant while predominantly providing negative sensitive crossmatches. The iBeads assay seems to be a valuable alternative to better define unacceptable antigens.

The single antigen flow bead (SAFB) assay greatly improves the identification of antigenic specificity of anti-HLA alloantibodies. However, it may underestimate or miss high titer antibodies due to the prozone phenomenon caused by a competition between the fluorescent anti-IgG conjugate and serum complement, for the alloantibody. We explored this effect in our cohort of transplant candidates and transplanted recipients. Among a total of 292 and 269 patients with at least three different sera tested with class I and/or II SAFB assays respectively, we identified 9 patients (6 in class I and 3 in class II) who displayed a profound drop (≥ 75%) followed by a subsequent rise (≥ 100%), in strong (mean fluorescence intensity >8000) antibody levels, across an 18-month period. We postulated that such abrupt fluctuations were not explainable by naturally occurring transient desensitization. Sera were analysed with the SAFB assay using EDTA-treated serum and direct complement C1q staining, and with complement-dependent cytotoxicity and flow cytometry crossmatches (CDCXM and FCXM respectively). The prozone phenomenon was involved in all cases. Because it relies on complement activation, the CDCXM was not sensitive to this phenomenon, but the FCMXM was not either, although it resembles in its principle to the SAFB assay. Four additional anti-human conjugates targeting the IgG Fc fragment or the light chains did not circumvent the SAFB drawback. Therefore, a quick decrease in antibody strength must alert against a potential risk for recipients at the time of the transplant, using virtual crossmatch strategies. A prospective pre-transplant crossmatch still remains an ultimate safeguard., (© 2013.)

Allograft pathology, antibody-tissue interaction as demonstrated by C4d deposition and serological evidence of donor-specific antibodies (DSA) are the cardinal diagnostic features of antibody-mediated lesions (AML) in kidney transplantation. However, discrepancy between histological and serological findings is common, and more reliable diagnostic tools are called for. Here, we asked whether the in situ detection of DSA could serve as marker for AML. To that end, we applied the anti-HLA single antigen flow bead assay to eluates from 51 needle core graft biopsies performed for cause. Intragraft antibody profiles were correlated to serum DSA (sDSA), histological data and transplant outcome. The prevalence and the mean number of intragraft DSA (gDSA) were lower than that of sDSA (15/51 gDSA+ vs. 37/51 sDSA+ patients; 1.64 gDSA vs. 2.24 sDSA per patient). DSA were detected in all anti-HLA antibody-positive biopsies (15/15). The presence of gDSA was significantly associated with (1) microcirculation lesions taken individually (g, cg) and analyzed in functional clusters (ptc + g + cg > 0, cg + mm > 0), (2) C4d positivity and (3) a worse short-term transplant outcome (p = 0.05). These associations were not found for patients presenting only sDSA. Taken together, these results indicate that gDSA is a severity marker of antibody-mediated pathogenic process., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Prognosis of renal transplants with positive flow cytometric crossmatch (FCXM) remains controversial., Methods: We analyzed the outcome of these kidney transplant recipients by human leukocyte antigen (HLA) donor-specific antibodies (HLA-DSA) using single-antigen bead (SAB) assays in major histocompatibility complex classes I and II. We compared them with controls with a negative FCXM., Results: Forty-five patients consecutively transplanted with a positive FCXM had significantly more acute rejection episodes than the control patients (33.3% vs. 8.9%, P=0.002). Risk of acute rejection was increased with day 0 (D0) positive T-cell FCXM (odds ratio [OR]=9.04, P=0.002), D0 positive B-cell FCXM (OR=7.43, P=0.02), and D0 HLA-DSA identified by SAB assay (OR=6.5, P=0.03). The 21 patients with D0 positive FCXM and D0 HLA-DSA had more acute rejection (62%, P=0.0001) and a lower estimated glomerular filtration rate 1-year posttransplantation (P=0.0001), when compared with controls. Mainly anti-Cw and anti-DP HLA-DSA were found in patients displaying acute rejection. The remaining FCXM-positive patients displayed short-term outcomes similar to controls. The presence of HLA-DSA detected only by the SAB assay in the context of a negative FCXM crossmatch was not associated with increased risk of acute rejection., Conclusion: Identification of HLA-DSA in D0 sera by the two sensitive techniques FCXM and SAB assay indicates which patients are at highest risk of subsequent acute allograft rejection and chronic allograft dysfunction.

HLA antibody detection with single antigen flow beads (SAFB) assays is impaired by complement interference whose frequency, predictability and distribution among HLA antigens have not been analyzed in large cohorts. We compared in two patients' cohorts the routine follow‐up SAFB profiles obtained in class I (n = 129) and class II (n = 85) with and without ethylenediaminetetraacetic acid (EDTA)‐treatment. The presence of complement interference was defined according to the reproducibility of the SAFB assays evaluated with our class I and II routine positive control sera. Interference occurred in 29.5% and 45.9% of patients in class I and II, respectively. In the untreated condition, at serum level, neither the number of positive beads, the highest bead mean fluorescence intensity (MFI) nor MFI at bead level, satisfactorily predicted interference. HLA‐C were the least affected among class I beads. HLA‐DQ beads were the most affected in class II. At least one antibody specificity was falsely negative without EDTA for about 3% of sera in class I and 9% in class II. EDTA‐treatment did not significantly modify the low‐MFI strengths (500‐3000 range). This study emphasizes the high frequency of complement interference and the importance and advantages of systematically pretreating sera with EDTA before performing SAFB assays. [ABSTRACT FROM AUTHOR]