Negative Complement-dependent Cytotoxicity Crossmatch Research Articles

#6257 PROGNOSTIC VALUE OF A POSITIVE CROSSMATCH BEFORE DESENSITIZATION IN A COHORT OF HIGHLY-SENSITIZED PATIENTS

Abstract Background and Aims Human Leukocyte antigen (HLA) sensitization in patients is a barrier for Kidney Transplantation (KT) access, and highly-sensitized patients remain on the waiting list longer than other patients which results in a higher morbidity and mortality. Desensitization may be an option to increase their access to KT. All patients transplanted in this context have a negative Complement-Dependent Cytotoxicity crossmatch (CDC-CM) the day of KT. However, the stratification of the long-term risk of graft loss according to flow cytometry crossmatch (FC-CM) and to CDC-CM during the desensitization period remains unknown. Method In this retrospective study, all highly-sensitized patients (Panel Reactive Antigen (PRA) ≥ 85) in our center were included. CDC-CM and FC-CM were performed on samples before and during the desensitization period and clinical follow-up data were retrieved. Pre-KT desensitization strategy consisted in two Rituximab infusions (375mg/m2) and a variable number of apheresis (mostly semi-specific immunoadsorption) associated with a classical immunosuppression-based regimen (tacrolimus, mycophenolate and steroids). Results A total of 183 highly-sensitized recipients received a KT between 2014 and 2022. Of these, 23 were desensitized prior to KT and among them 18 had a positive FC-CM before KT and 5 had no positive FC-CM on all assessed serums. Mean age was 52.9 ± 13 years. Mean follow-up was 5.2±4 and 3±2 years for non-desensitized and desensitized patients respectively. PRA before desensitization was higher in desensitized patients (98%) versus non-desensitized patients (95%) and the mean time on the waiting list was 5.8±4 versus 7.5±4 years respectively. Death-censored graft survival and patient's raw survival, using cox-survival analysis, were not statistically different between the desensitized kidney transplanted patients and the highly-sensitized patients without desensitization (p = 0.77 and p = 0.57 respectively). In sub-group analyses, the positivity of FC and/or CDC-CM did not add an additional risk of graft loss or patient mortality. Conclusion Desensitization allows highly-sensitized patients to access KT with an HLA-incompatible graft (with a positive CDC or FC-CM on the serum collected prior to desensitization), with the same graft survival as similarly sensitized patients transplanted with an HLA-compatible graft without desensitization.

Correlation of Luminex-Based Single Antigen Based Results With Complement-Dependent Cytotoxicity Crossmatch and Flow Cytometry Crossmatch Results: A Single-Center Experience From Istanbul

This study aimed to retrospectively investigate the correlation of mean Class I donor-specific antibody (DSA) intensity values detected in Luminex-based techniques with the results of complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FC-XM) results. A total of 335 patients with kidney failure and their living donors whose CDC-XM, FC-XM, and single antigen based (SAB) tests were studied between 2018 and 2020 for transplant preparation from living donor candidates were included in the study. Patients were divided into 4 groups according to their mean fluorescence intensity (MFI) values of SAB assay. Anti-HLA antibodies (class I and/or class II) were detected using SAB in 91.6% patients included in the study (MFI >1000). Class I DSA was positive in 34.8% of patients with anti-HLA antibodies. When CDC-XM and FC-XM results were evaluated in the 4 groups separated according to MFI values, 3 patients with DSA MFI <1000 had negative CDC-XM and T-B-FC-XM results. Of 32 patients with DSA-MFI between 1000 and 3000, 93.75% (n=30) had T-B-FC-XM or CDC-XM-negative results, and 6.25% (n=2) had B-FC-XM-positive results. The CDC-XM, T, and B-FC-XM were negative in all 17 patients with DSA-MFI between 3000 and 5000. Our results showed that MFI >5834 DSA values were significantly correlated with positive T-FC-XM (P < .001), and MFI >6016 values were significantly correlated with positive CDC-XM (P=.002). In addition, MFI values >5000 were associated with both CDC-XM and FC-XM in our study. The MFI values >5000 correlated with both CDC-XM and FC-XM.

RE: Imlifidase-generated Single-cleaved IgG: Implications for Transplantation.

With great interest, we read the article by Bockermann et al1 on crossmatch assays after the administration of the novel desensitization drug Imlifidase. Proteolysis by Imlifidase generates single-cleaved immunoglobulin G (IgG) in a rapid first reaction and subsequently 2 F(ab’)2 fragments and a fully separated Fc fragment in a longer second reaction.2 Bockermann et al1 demonstrated that Luminex single antigen bead (SAB) testing, and crossmatching with both flowcytometry (FCXM) and the anti-human IgG-amplified complement-dependent cytotoxicity crossmatch (CDCXM) do not fully distinguish single-cleaved IgG from intact IgG. Bockermann et al1 suggest that the nonamplified CDCXM is best capable of demonstrating relevant residual intact IgG. Our center performed the first Imlifidase reimbursed HLA-incompatible kidney transplantation in March 2022. Contrary to Bockermann et al,1 we had difficulties in the interpretation of nonamplified CDCXM after Imlifidase treatment. A 29-y-old female with 6-y dialysis vintage after 2 failed kidney transplants was running out of vascular access. Despite high-urgency status and priority allocation via the Eurotransplant Acceptable Mismatch program, she remained without kidney offers. This was caused by her virtual panel-reactive antibody status of 100%. Her historical SAB mean fluorescence intensity (MFI) levels were high with broad reactivity in B-cell memory, with current MFI levels in the lower ranges. To enable for a timely HLA-incompatible kidney transplant, we delisted all unacceptable antigens with corresponding MFI levels of <3000, provided that CDC screening was negative. By this delisting, we increased her donor frequency of 0.000%–3.95%. Eight days later, this resulted in an HLA 1-2-2 mismatched kidney offer, with 2 “delisted DR-unacceptables.” Imlifidase was administered based on a virtual crossmatch. Serum was sampled 2 and 4 h after Imlifidase administration. Nonamplified CDCXMs with donor spleen cells were performed with preblood simultaneously tested with post 2-h and post 4-h sampling. Pre-CDCXM without dithiothreitol (DTT) treatment (which dissolves the disulfide bridges of IgM tetramers) was positive, indicating autoreactivity. DTT treatment revealed a negative pre-Imlifidase CDCXM. To our surprise, both the 2- and 4-h Imlifidase CDCXMs with DTT were positive (value 35 of 100). We had to decide whether or not to proceed with transplantation based on the nonamplified CDCXMs alone, as additional FCXM and SAB testing were scheduled for the next day. Because our patient had not recently received blood transfusions, was without infectious episodes, and had a negative pre-Imlifidase CDCXM, we expected assay interference by Imlifidase and decided to proceed with transplantation. The retrospectively performed pre-Imlifidase FCXM was negative for T cells and was 1.94 MCS for B cells. Luminex SAB was positive on 2 donor DR loci with 2500 and 2200, and became negative after Imlifidase. We investigated CDCXM on frozen donor spleen cells retrospectively and noticed CDC-positivity 2 and 4 h after Imlifidase, with negative CDCXMs on day 3. The patient did experience a vascular rejection after 1 wk, without signs of AMR. SAB testing revealed HLA antibody rebound from day 4 onward; however, DSA remained negative until current follow-up of 4 mo. This case demonstrates that even in nonamplified CDCXM, an interference by Imlifidase may occur. We advise to combine different assays when interpreting desensitization results after Imlifidase treatment.

Positive Single-Antigen Bead Assay with Negative Flow Crossmatch in a Renal Transplant - A Case Report

The occasion is when there is discordance between the results of the cell-based and virtual crossmatch and requires further workup before considering a patient for renal transplant. A case of the positive single-antigen bead (SAB) with donor-specific antibodies (DSA) against HLA-A*33:01 with negative complement-dependent cytotoxicity crossmatch and flow crossmatch was identified. The acid treatments of the beads were done to denature the antigen to understand the nature of identified DSA. There was variation in the Mean fluorescence intensity (MFI) observed in between patient sera with acid-treated beads (significantly increased) in contrast to regularly untreated SAB. This indicated additional antigens become available by the denaturation process. Hence, before making a final decision about the transplant in cases of ambiguity, SAB results should be interpreted in light of other compatibility results.

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 influence of the antithymocyte globulin dose on clinical outcomes of patients undergoing kidney retransplantation

Optimizing antithymocyte globulin (rATG) dosage is critical for high immunological risk patients undergoing a repeat kidney transplant. This natural retrospective cohort study compared clinical outcomes of two successive cohorts of consecutive recipients of retransplants receiving 5 x 1 mg/kg (rATG-5, n = 100) or a single 3 mg/kg (rATG-3, n = 110) dose of rATG induction therapy. All patients had negative complement-dependent cytotoxicity crossmatch and no anti-HLA A, B, DR donor-specific antibodies (DSA). The primary endpoint was efficacy failure (first biopsy-proven acute rejection, graft loss, or death) at 12 months. There was no difference in the cumulative incidence of efficacy failure (18.0% vs. 21.8%, HR = 1.22, 95% CI 0.66-2.25), respectively. There were no differences in 3-years freedom from biopsy proven acute rejection, and patient, graft, and death-censored graft survivals. There were no differences in the incidence of surgical complications (25.0% vs. 18.2%; p 0.151), early hospital readmission (27.8% vs. 29.5%; p = 0.877) and CMV infections (49% vs. 40%; p = 0.190). There were also no differences in the incidence (59.6% vs. 58.7%, p = 0.897) and duration of delayed graft function but a stable difference in estimate glomerular filtration rate was observed from month 1 (54.7±28.8 vs. 44.1±25.3 ml/min/1.73 m2, p = 0.005) to month 36 (51.1±27.7 vs. 42.5±24.5, p = 0.019). Mean urinary protein concentration (month 36: 0.38±0.81 vs. 0.70±2.40 g/ml, p = 0.008) and mean chronic glomerular Banff score in for cause biopsies (months 4-36: 0.0±0.0 vs. 0.04±0.26, p = 0.044) were higher in the rATG-3 group. This cohort analysis did not detect differences in the incidence of efficacy failure and in safety outcomes at 12 months among recipients of kidney retransplants without A, B, and DR DSA, receiving induction therapy with a single 3 mg/kg rATG dose or the traditional 5 mg/kg rATG.

Tailored immunosuppression after kidney transplantation - a single center real-life experience

BackgroundKidney allograft survival continuously improved with introduction of novel immunosuppressants. However, also immunologically challenging transplants (blood group incompatibility and sensitized recipients) increase. Between 2006 and 2008, a new tailored immunosuppression scheme for kidney transplantation was implemented at the University Hospital in Zurich, together with an ABO-incompatible transplant program and systematic pre- and posttransplant anti-human leukocyte antigen (HLA) antibody screening by Luminex technology. This study retrospectively evaluated the results of this tailored immunosuppression approach with a particular focus on immunologically higher risk transplants.MethodsA total of 204 consecutive kidney transplantations were analyzed, of whom 14 were ABO-incompatible and 35 recipients were donor-specific anti-HLA antibodies (DSA) positive, but complement-dependent cytotoxicity crossmatch (CDC-XM) negative. We analyzed patient and graft survival, acute rejection rates and infectious complications in ABO-compatible versus -incompatible and in DSA positive versus negative patients and compared those with a historical control group.ResultsOverall patient, death-censored allograft survival and non-death-censored allograft survival at 4 years were 92, 91 and 87%, respectively. We found that (1) there were no differences between ABO-compatible and -incompatible and between DSA positive and DSA negative patients concerning acute rejection rate and graft survival; (2) compared with the historical control group there was a significant decrease of acute rejection rates in sensitized patients who received an induction with thymoglobulin; (3) there was no increased rate of infection among the patients who received induction with thymoglobulin compared to no induction therapy.ConclusionsWe observed excellent overall mid-term patient and graft survival rates with our tailored immunosuppression approach. Induction with thymoglobulin was efficient and safe in keeping rejection rates low in DSA positive patients with a negative CDC-XM.

A novel approach reveals that HLA class 1 single antigen bead-signatures provide a means of high-accuracy pre-transplant risk assessment of acute cellular rejection in renal transplantation

BackgroundAcute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach.ResultsThe data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA < 30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity = 76.5%, specificity = 88.9%).ConclusionsThe resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information.Trial registrationClinicalTrials.gov NCT00724022. Retrospectively registered July 2008.

Value of a flow cytometry cross-match in the setting of a negative complement-dependent cytotoxicity cross-match in heart transplant recipients.

Complement-dependent cytotoxicity cross-match (CDCXM) is used for evaluation of preformed HLA-specific antibodies in patients undergoing heart transplantation. Flow cytometry cross-match (FCXM) is a more sensitive assay and used with increasing frequency. To determine the clinical relevance of a positive FCXM in the context of negative CDCXM in heart transplantation, the United Network for Organ Sharing (UNOS) database was analyzed. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess graft survival for three different patient cohorts defined by cross-match results: T-cell and B-cell CDCXM+ ("CDCXM+" cohort), CDCXM- but T-cell and/or B-cell FCXM+ ("FCXM+" cohort), and T-cell/B-cell CDCXM- and FCXM- ("XM-" cohort). During the study period, 2558 patients met inclusion criteria (10.7% CDCXM+, 18.8% FCXM+, 65.5% XM-). CDCXM+ patients had significantly decreased graft survival compared to FCXM+ and XM- cohorts (P=.003 and <.001, respectively). CDCXM- and FCXM+ patients did not have decreased graft survival compared to XM- patients (P=.09). In multivariate analysis, only CDCXM+ was associated with decreased graft survival (HR 1.22, 95% CI 1.01-1.49). In conclusion, positive FCXM in the context of negative CDCXM does not confer increased risk of graft failure. Further study is needed to understand implications of CDCXM and FCXM testing in heart transplant recipients.

Successful Renal Transplantation Across HLA Barrier: Report from India.

Organ donors are sometimes found “unsuitable” due to the presence of donor-specific anti-HLA antibodies in the recipient. In recent years, improved desensitization protocols have successfully helped to overcome HLA incompatibility hurdle. We present three cases where optimum desensitization was achieved in patients with the donor-specific anti-HLA antibody (DSA) leading to successful renal transplantation. All patient–donor pair underwent HLA typing, complement dependent cytotoxicity crossmatch (CDC-XM), flow cytometry XM (FC-XM), and panel reactive antibody. If any of the three tests was positive, single antigen bead assay was performed to determine the specificity of the anti-HLA antibody (s). Patients with DSA were offered organ-swap or anti-HLA antibody desensitization followed by transplantation. Desensitization protocol consisted of single dose rituximab and cascade plasmapheresis (CP) along with standard triple immunosuppression. The target DSA mean fluorescence index (MFI) was <500, along with negative CDC-XM and FC-XM for both T- and B-cells. Three patients with anti-HLA DSA, who did not find a suitable match in organ swap program, consented to anti-HLA antibody desensitization, followed by transplantation. Mean pre-desensitization antibody MFI was 1740 (1422–2280). Mean number of CP required to achieve the target MFI was 2.3 (2–3). All the three patients are on regular follow-up and have normal renal function test at a mean follow-up of 8 months. This report underlines successful application of desensitization protocol leading to successful HLA-antibody incompatible renal transplants and their continued normal renal functions.

Desensitization Protocol in Recipients of Deceased Kidney Donor With Donor-Specific Antibody–Low Titers

BackgroundKidney transplantation is the better option for end-stage renal disease (ESRD), but for patients with human leukocyte antigen (HLA) sensitization, the wait times are significantly longer than for patients without antibodies. Many desensitization protocols have been described involving strong immunosuppression, the use of apheresis, and B-cell–modulating therapies. We have designed a desensitization protocol from day 0 for deceased donor kidney transplantation. Our aim was to present our initial experience with five kidney transplant patients. MethodsAll patients had a negative complement-dependent cytotoxicity cross-match. The desensitization protocol included five to seven doses of thymoglobulin (1.25 mg/kg) and three sessions of plasmapheresis (PP) within the first week after transplantation, with intravenous immunoglobulin (500 mg/kg) after each PP session and one dose of rituximab on day 8. The presence of donor-specific antibodies (DSA) was analyzed by use of Luminex technology; levels between 1000 and 3000 mean fluorescence intensity were considered for desensitization. ResultsThe median age was 44 years and median renal replacement therapy time was 9 years. All recipients presented 1 to 3 DSA specificities. There were no severe side effects related to PP, infusion of intravenous immunoglobulin, or rituximab. The median follow-up period was 19.3 months. Median serum creatinine level at last follow-up was 1.7 mg/dL. A kidney biopsy was performed in all patients. Graft and patient survival was 100%. ConclusionsUntil now, few data are available concerning whether HLA-incompatible kidney transplantation after desensitization would benefit patients with ERSD. The desensitization strategy using the combination of PP, low doses of intravenous immunoglobulin, and rituximab at our center resulted in a satisfactory clinical outcome.

Deceased donor kidney transplantation across donor-specific antibody barriers: predictors of antibody-mediated rejection

Apheresis-based desensitization allows for successful transplantation across major immunological barriers. For donor-specific antibody (DSA)- and/or crossmatch-positive transplantation, however, it has been shown that even intense immunomodulation may not completely prevent antibody-mediated rejection (ABMR). In this study, we evaluated transplant outcomes in 101 DSA+ deceased donor kidney transplant recipients (transplantation between 2009 and 2013; median follow-up: 24 months) who were subjected to immunoadsorption (IA)-based desensitization. Treatment included a single pre-transplant IA session, followed by anti-lymphocyte antibody and serial post-transplant IA. In 27 cases, a positive complement-dependent cytotoxicity crossmatch (CDCXM) was rendered negative immediately before transplantation. Seventy-four of the DSA+ recipients had a negative CDCXM already before IA. Three-year death-censored graft survival in DSA+ patients was significantly worse than in 513 DSA- recipients transplanted during the same period (79 versus 88%, P = 0.008). Thirty-three DSA+ recipients (33%) had ABMR. While a positive baseline CDCXM showed only a trend towards higher ABMR rates (41 versus 30% in CDCXM- recipients, P = 0.2), DSA mean fluorescence intensity (MFI) in single bead assays significantly associated with rejection, showing 20 versus 71% ABMR rates at <5000 versus >15 000 peak DSA MFI. The predictive value of MFI was moderate, with the highest accuracy at a median of 13 300 MFI (after cross-validation: 0.72). Other baseline variables, including CDC assay results, human leukocyte antigen mismatch, prior transplantation or type of induction treatment, did not add independent predictive information. IA-based desensitization failed to prevent ABMR in a considerable number of DSA+ recipients. Assessing DSA MFI may help stratify risk of rejection, supporting its use as a guide to organ allocation and individualized treatment.

P114 : CHANGE OF THE PATIENT’S ANTI HLA ANTIBODY PROFILE BETWEEN INITIAL AND FINAL WORK-UP

Aim Patients anti HLA antibody profile could change overtime in its specificity, strength and/or behavior. Here we are presenting a case for a patient with end stage renal disease (ESRD) workup for living related kidney transplant, in which antibody profile changed between the initial and final work-up (the day before transplant). Methods HLA Class I highly sensitized patient with anti A26 and anti B50 Donor Specific Antibody (DSA) with 2904, 12168 Median fluorescent Intensity (MFI) respectively, underwent desensitization protocol with Rituximab, high dose IVIG and Plasma Apheresis. One day before transplantation, final Complement Dependent Cytotoxicity crossmatch (CDCXM) as well as Flow Cytometry Crossmatch (FCXM) were performed utilizing historical serum “initial work-up sera prior to desensitization” and current serum post desensitization. Results As can be seen in the table below, the characteristics of the patients’ DSA changed between the initial and final work-up. The DSA changed from complement fixing antibody with a positive CDCXM crossmatch to non-complement fixing antibodies with negative CDCXM crossmatch. Conclusion This case highlights the relevant of frequent testing of anti-HLA antibodies in renal transplant candidates. As seen above, the antibody Isotype switched from fixing to none complement fixing. Thereafter, such conversion could modify the treatment modality. Result. Initial work-up Final work-up CDCXM Positive Negative FCXM Positive Positive Undiluted sera A26 (2904 MFI), B50 (12168 MFI) A26 (12421 MFI), B50 (984 MFI) Diluted sera (1:4) A26 (16680 MFI), B50 (6247 MFI) A26 (5936 MFI), B50 (Negative) C1q Positive Negative

Optimizing Transplantation of Sensitized Kidney Recipients through Prioritization of Unacceptable Antigen Assignment.

Background: The calculated PRA (CPRA) was implemented as part of the OPTN/UNOS kidney allocation system on October 1, 2009, based on the frequency of antigens determined to be unacceptable (UAs) in the donor pool for a given patient. It is incumbent on each center to establish criteria for UAs assignment based on their ability to transplant (tx) sensitized recipients (recips). The aim of our study was to establish an algorithm for assignment of UAs such that a complement dependent cytotoxicity crossmatch (CDC-XM) would be negative and a concomitant flow cytometric crossmatch (FXM) would be weakly positive to allow for successful tx of sensitized kidney recips. Methods: Four antibody (Ab) methods were used to determine specificity, function, and strength of the Ab: C1q binding, CDC panel, Luminex single antigen (LSA), and LSA with a 1:8 dilution. UAs were assigned in the following order: 1) C1q/CDC+; 2) LSA 1:8 >7,500 MFI; and LSA >10,000 MFI. Results: Between January 4, 2010 and July 6, 2013, 345 recips received deceased donor (DD) tx, including 194 with no detectable antibodies to HLA (HLA-Abs) and 151 with HLA-Abs. Of the 151, 20 recips (13%) received zero mismatch grafts and were excluded from further analysis. For the remaining 131 recips, only DD with no C1q binding UAs and negative CDC XMs were considered for tx. The 3 categories of FXM were: 1) T neg/B neg = 66 recips (50.4%); 2) T neg/B pos = 32 recips (24.4%); and 3) T pos/B pos = 33 recips (25.2%) (table). Antibody mediated rejection (ABMR) rates for each category were: 1) 7 (10.6%); 2) 6 (18.8%); 3) 8 (24.2%). The overall ABMR rate for HLA-Abs+ recips = 21.5%. Conclusions: Careful assignment of UAs has allowed for 38% of HLA-Ab+ recips to be tx'ed successfully with HLA disparate DD grafts and an overall rejection rate of 21.5%. The C1q binding assay proved to be valuable to identify those antigens that must be avoided to optimize a negative CDC-XM and weak FXM. Strategies for assigning UAs are imperative to assign a CPRA status reflective of the sensitization level for recips and thereby optimize transplantation of HLA-Ab+ recips.Table: No Caption available.DISCLOSURES:Jordan, S.: Grant/Research Support, CSL-BEHRING, GENENTECH-ROCHE.

Deceased Donor HLA Antibody Incompatible Renal Transplantation without Antibody Removal: High Incidence of Acute Rejection Reduced by T-Cell Depleting Induction Therapy

Introduction: Screening for HLA-specific antibodies (Ab) is routine for patients on kidney transplant waiting lists. In the UK screening is performed at least every 3 months, and HLA antigens to which a patient has detectable Ab are listed as ‘unacceptable mismatches’. Many units use a stringent definition of unacceptable, for example any HLA antigen to which Ab can be detected with an MFI >1000 (using the Luminex® platform). Whilst this approach is effective in preventing a positive crossmatch (XM) following allocation, it also precludes the offer of organs to sensitized patients. Here we describe our experience of deceased donor (DD) renal transplantation knowingly performed in the presence of donor-specific anti-HLA Ab (DSA). Methods: 21 patients received HLA Ab-incompatible (HLAi) DD transplants in one of two circumstances: (1) Patients in whom the threshold for ‘unacceptable’ for any HLA specificity was increased to a Luminex MFI >3000 (n=8), and (2) Patients with a positive flow cytometry B-cell crossmatch (FC-BXM) known to have anti-HLA-DP Ab (n=13). Since donors in the UK are not routinely DP genotyped, the presence of DP DSA was assumed, and confirmed retrospectively by donor DP genotyping. Complement-dependent cytotoxicity crossmatch (CDC-XM) using a recent pre-transplant serum sample was negative in all patients. FC-BXM was positive in 15 (71%). The mean DSA MFI was 5268 (range 1086-16356) and mean follow-up length was 20.8 months (range 1-64). All episodes of antibody-mediated rejection (AMR) and T cell-mediated rejection (TCMR) were analyzed. Results: All patients received tacrolimus, MMF and prednisolone. Induction agents used were Basiliximab (n=8), Alemtuzumab (n=9) and Thymoglobulin (n=4). No planned Ab removal was used. Death-censored 1 year graft survival was 92% (12/13 patients with >12 months follow up). Acute rejection (AR) was significantly more common in those receiving basiliximab at induction compared to a T-cell depleting agent (75% (6/8: 3 with TCMR and 3 with AMR) versus 15% (2/13: both AMR) respectively, p=0.018). Patients developing AMR were more likely to have a positive CDC-XM using historic serum (3/5 with AMR versus 1/16 without AMR, p=0.028). Of the 12 patients with isolated DP DSA, 2 experienced AMR and 2 TCMR. All episodes of AMR were reversed with plasma exchange. Mean creatinine for those patients with graft function at baseline (18/21) was 137μmol/L and at 1 year was 133μmol/L (n=12). There were 3 deaths (post-operative haemorrhage at 2 weeks, disseminated aspergillosis at 3 weeks and a cerebral lymphoma at 4 years) and 1 graft loss (graft rupture with Banff 2B AR at 1 week). Conclusions: HLAi DD renal transplantation is associated with a high incidence of AR, with AMR in 24% of patients despite negative CDC-XM. Our limited experience suggests that: (1) T-cell depleting induction is beneficial, (2) A current negative but historic positive CDC-XM could prompt prospective antibody removal and (3) donor HLA-DP genotyping should be routine practice. Despite high rates of AR, transplantation in these high-risk recipients is still achievable with satisfactory allograft function at up to 5 years post-transplant.

Clinical Relevance of Anti-HLA Donor-Specific Antibodies Detected by Luminex Assay in the Development of Rejection After Renal Transplantation

The role of anti-human leukocyte antigen (HLA) donor-specific antibodies (DSA) detected by Luminex in the development of rejection is not fully understood. A study including 369 patients who received transplants from deceased donors with a negative complement-dependent cytotoxicity crossmatch (XM) was performed. From the total of patients, 151 underwent a renal biopsy because of renal dysfunction, whereas the 218 remaining showed a stable renal function, and no rejection was assumed. Diagnosis and type of rejection was based in biopsy data. Patients with a positive virtual XMs showed more rejection episodes of any types when comparing with patients with negative virtual XMs (P<0.0001). Nevertheless, there were no significant differences between patients without anti-HLA antibodies and patients with anti-HLA no DSA. Allograft impairment was caused by a rejection episode in 84% (32/38) of patients with anti-HLA-DSA but only in 30% (34/113) of patients without anti-HLA-DSA. Regarding the type of rejection detected in the biopsy, all the patients with de novo (after transplantation) anti-HLA-DSA were diagnosed as antibody-mediated rejection (AMR) or AMR+T-cell-mediated rejection, whereas most of the patients without anti-HLA-DSA (68%) were diagnosed with T-cell-mediated rejection, and patients with preexistent anti-HLA-DSA showed a more homogeneous distribution of the different types of rejection. According to our results, patients with preformed or de novo anti-HLA-DSA showed the highest likelihood to suffer rejection episodes. Transplantation with preformed anti-HLA-DSA should be avoided, and an early detection of de novo HLA antibodies is important to treat patients before damage occurs in the graft.

Renal transplantation in sensitized recipients with positive luminex and negative CDC (complement-dependent cytotoxicity) crossmatches

Recently, Luminex-crossmatch (LumXm) was introduced. The aim of this study was to evaluate clinical outcomes in sensitized recipients with a positive Luminex-crossmatch (LumXm (+)) and a negative complement-dependent cytotoxicity crossmatch (CDCXm (-)) after renal transplantation. Fifty-five renal transplant recipients with a CDCXm (-) and PRA class I or II ≥20% were enrolled in this study between February 2008 and December 2010 at Severance Hospital. Eighteen patients displayed LumXm (+) defined as LumXm positive class I or II and 37 patients displayed LumXm (-). Mean duration of follow-up was 18.9 ± 8.3 months. During this period, no patient death or graft loss occurred. The incidence of biopsy-proven or clinically presumed rejection was higher in the LumXm (+) group (n = 12, 66.7%) than in the LumXm (-) group (n = 6, 18.2%) (P = 0.001). All biopsy-proven acute rejections (n = 12) were diagnosed as acute cellular rejection. No significant difference in mean serum creatinine level or eGFR was observed between the groups at 18 months post-transplantation. The short-term outcome of renal transplantation in sensitized patients with a LumXm (+) and a CDCXm (-) may be considered to be acceptable. However, patients with a LumXm (+) have a substantially higher immunological risk for the development of acute cellular rejection.

The impact of pretransplant donor-specific antibodies on graft outcome in renal transplantation: a six-year follow-up study

OBJECTIVE:The significance of pretransplant, donor-specific antibodies on long-term patient outcomes is a subject of debate. This study evaluated the impact and the presence or absence of donor-specific antibodies after kidney transplantation on short- and long-term graft outcomes.METHODS:We analyzed the frequency and dynamics of pretransplant donor-specific antibodies following renal transplantation from a randomized trial that was conducted from 2002 to 2004 and correlated these findings with patient outcomes through 2009. Transplants were performed against a complement-dependent T- and B-negative crossmatch. Pre- and posttransplant sera were available from 94 of the 118 patients (80%). Antibodies were detected using a solid-phase (Luminex®), single-bead assay, and all tests were performed simultaneously.RESULTS:Sixteen patients exhibited pretransplant donor-specific antibodies, but only 3 of these patients (19%) developed antibody-mediated rejection and 2 of them experienced early graft losses. Excluding these 2 losses, 6 of 14 patients exhibited donor-specific antibodies at the final follow-up exam, whereas 8 of these patients (57%) exhibited complete clearance of the donor-specific antibodies. Five other patients developed “de novo” posttransplant donor-specific antibodies. Death-censored graft survival was similar in patients with pretransplant donor-specific and non-donor-specific antibodies after a mean follow-up period of 70 months.CONCLUSION:Pretransplant donor-specific antibodies with a negative complement-dependent cytotoxicity crossmatch are associated with a risk for the development of antibody-mediated rejection, although survival rates are similar when patients transpose the first months after receiving the graft. Our data also suggest that early posttransplant donor-specific antibody monitoring should increase knowledge of antibody dynamics and their impact on long-term graft outcome.

Enhanced Kidney Allocation to Highly Sensitized Patients by the Acceptable Mismatch Program

). Highly sensitized patientshave only a small chance to receive a crossmatch negativedonor kidney, because they have developed antibodiesagainstmanydifferentHLAantigensresultingfrompreviouscontacts with allogeneic cells by pregnancy, blood transfu-sions,orprevioustransplants.Ifnospecialmeasurementsaretaken, these patients will accumulate on the transplant wait-ing lists. More and more transplant centers are developingstrategiestoremovecirculatingHLAalloantibodiesinthesepa-tients to be able to transplant the patient with a donor kidneydespite a positive crossmatch (

Clinical relevance of Luminex donor‐specific crossmatches: data from 165 renal transplants

The clinical significance of the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA) prior to renal transplantation detected solely by solid-phase techniques remains unclear. This study was designed to determine the clinical relevance of the recently introduced bead-based Luminex donor-specific crossmatch (LumXm). A group of 165 patients transplanted between 1997 and 2001 were tested. Of 165 recipients transplanted with a negative complement-dependent cytotoxicity crossmatch, 32 proved to have a positive Luminex crossmatch. Sixteen were positive for class I, 15 were positive for class II, 1 was both class I and II positive and 133 recipients were negative. Acute rejection (AR)-free survival for all recipients was 77%, and there was no difference in AR-free survival between LumXm-positive and LumXm-negative recipients. Overall graft survival after a median follow-up time of 8 years was 56%. Recipients with a positive class I LumXm had worse long-term graft survival (P = 0.006). In recipients with a positive class I LumXm, 5-year graft survival was 41% vs 70% in negative patients and 10-year graft survival was 27% vs 56%. Positivity for class II LumXm was not a significant risk factor for graft failure (P = 0.7). In conclusion, pretransplant DSA detected by the LumXm had no impact on AR episodes. Class II LumXm positivity proved no significant risk factor for graft failure, but the value of the class II LumXm is questionable. A positive class I LumXm resulted in worse long-term graft survival compared with a negative one.