Detection Of Donor-specific Antibodies Research Articles

Immunosuppression with mTOR inhibitors prevents the development of donor-specific antibodies after liver transplant.

Donor-specific antibodies (DSAs) are an important cause of complications after solid organ transplant. Risk factors and, thus, strategies for preventing DSA development are not well defined. The DSA status of 400 patients who underwent liver transplant (LT) at the outpatient clinic of the University Hospital Essen was determined. Human leukocyte antigen (HLA) antibodies were detected by single-antigen bead technology. The strength of DSAs was reported as mean fluorescence intensity. Detectable DSAs were found in 74 (18.5%) patients and significantly more often in patients who underwent LT for autoimmune liver disease than for all other indications (29.3%; P=.022), but significantly less often found in patients who underwent LT for hepatocellular carcinoma (7.6%, P=.005). The incidence of DSAs increased with time after LT, and the risk was generally higher for female patients. The frequency of DSA detection was significantly lower (10.6%) for patients receiving immunosuppressive treatment with mammalian target of rapamycin (mTOR) inhibitors than for those receiving other regimens (20.5%; P=.025). Autoimmune liver diseases, female sex, and time of more than 8 years since LT predispose patients to the development of DSAs. Immunosuppression with the mTOR inhibitor everolimus protects against DSA development after liver transplant.

Donor-specific antibodies' C1q binding: improvement in kidney graft management?

The diagnosis of acute antibody mediated rejection (ABMR) after kidney transplantation is based on three parameters: 1) decreased renal function, 2) histopathological signs of rejection, 3) presence of Donor Specific Antibody (DSA) [1]. Patient survival, as well as graft survival have improved since 5 to 10 years [2], thanks to several factors, including better management of patients, better immunosuppressive regimens, detection and avoidance of DSA. Although anti-HLA antibodies follow-up have improved ABMR management, its treatment remains challenging, and outcome may be worsened because of a delay in the diagnosis. This article is protected by copyright. All rights reserved.

Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection.

Consistent with Dr. Paul Terasaki's “humoral theory of rejection” numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR) and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production (no rejection, subclinical rejection, and clinical dysfunction with AMR).

P099 The impact of lymphocyte purity on flow cytometry crossmatch (FCXM) assay. It’s not purely theoretical

Aim Lymphocyte flow cytometric crossmatch (FCXM) is used in pre-transplant risk assessment to detect donor specific antibodies (DSA). Many labs use density medium centrifugation to isolate donor cells for FCXM, which yields variable lymphocyte purity (15–90%) depending on the original concentration of lymphocytes and other leukocytes in donor blood, as well as sample quality and age. However, it is unclear whether variation in lymphocyte purity can effect FCXM assay. The goal of this study was to assess the impact of donor lymphocyte purity on FCXM results. Methods Enriched lymphocytes (Ly; >97%), neutrophils (Nu; >98%) and monocytes (Mo; >92%) were isolated from 5 volunteer donors using subset specific EasySep Direct kits (STEMCELL). Whole leukocyte (WL) cell preps were obtained by mixing enriched Ly, Nu and Mo in equal proportion (approx. 33% each) to mimic poor quality cell preps. Ly and WL cell preps were treated with pronase (2.35 kunitz units/ml) or control (PBS) and used as targets in FCXM against negative control (NC) and several dilutions of positive control (PC) sera. Median channel fluorescence (MCF) shifts from NC obtained with Ly vs WL target cell preps were compared. Results T and B cell FCXM NC reactivity was similar with Ly and WL cells. Pronase treatment was equally effective at reducing B cell background in Ly and WL cell preps (191 vs 198 MCF shift). Interestingly, both T and B cells FCXM performed with WL targets showed significantly reduced PC reactivity compared to Ly cells (Fig. 1). This was true at all PC concentrations (1/50–1/400) and was seen with both pronase treated and untreated cells. Average MCF decrease in pronase treated Ly vs WL preps was 49 MCF for T and 68 MCF for B cell FCXM, and in untreated preps it was 41 MCF for T and 61 MCF for B cell FCXM. Conclusion Lymphocyte purity has significant impact on FCXM results. Weak DSA could be missed if low purity lymphocyte preps are used in FCXM assay. Use of highly enriched lymphocytes will improve detection of DSA and reduce variability of FCXM results. Download : Download high-res image (220KB) Download : Download full-size image

P028 Uncovering the invisible; pre-treatment of sera with EDTA relieves prozone effect

Aim High titer HLA antibodies in patient sera can be falsely assessed due to interfering factors, also known as the prozone phenomenon, causing the masking of such antibodies. In this study we assessed the elimination of prozone by treatment of sera with DTT, EDTA and dilution. Methods Sera from 10 patients were treated with DTT, 6% EDTA, and 1:10 dilution. Antibody screening was performed by Luminex single antigen bead (SAB) assay. Effect of each treatment on the negative and positive controls, false-positive, and false-negative beads were compared to one another as well as to the untreated serum. Results Patient sera treated with DTT, EDTA and diluted at 1:10, all showed elimination of interfering factors and resolved the prozone effect. Comparison of the three treatment approaches showed that, DTT resolved the prozone, however it appeared to cause a slight increase in the mean fluorescent intensity (MFI) of the Luminex negative control (NC) bead. EDTA treatment eliminates prozone effect and has no effect on the NC bead. The 1:10 dilution also can resolve prozone, however the sensitivity can be compromised especially for the lesser strength antibodies. Conclusion In circumstances where prozone phenomenon is suspected, such as in the pre-transplant evaluation of highly sensitized patients, or during post-transplant monitoring for detection of donor specific antibodies, DTT, EDTA, and dilution are all effective strategies in elimination of interfering factors. However in addition to a higher NC, DTT-treatment, unlike EDTA, requires an additional incubation step with the sera (45 min) which affects the overall turnaround time. Dilution of serum can affect the sensitivity of the assay and in addition, often one dilution might not be sufficient for an accurate assessment of prozone, and performing multiple dilutions can prove to be costly. Routine treatment of sera with EDTA can accurately assess antibody specificities without affecting the sensitivity, turn around time and test cost.

P014 A simple and fast method for enrichment of lymphocyte subsets for complement dependent cytotoxicity assays

Aim Serological crossmatch analysis (i.e. complement-dependent cytotoxicity (CDC)) is commonly done before solid organ transplantation to detect donor specific antibodies that may lead to graft rejection or dysfunction. Routine analysis is often performed on isolated donor lymphocyte subpopulations together with recipient serum. Enrichment of cells for CDC can be exceedingly time consuming or results in co-enrichment of non-target or dead cells. We aimed to develop a fast and convenient method for enrichment of donor lymphocyte subsets directly from whole blood. Methods Using MACSxpress technology, untouched human leukocyte subsets can be isolated within only 20 min from up to 30 ml of anticoagulated whole blood. For the enrichment of B, T or a mixture of BT cells whole blood or cell suspensions obtained from rinsed donor spleens were incubated with the respective bead cocktails for 5 min and the tube placed for 15 min in the magnetic field of a separator. With the tube inside the magnetic field, the supernatant, containing the enriched target cells, was collected. Magnetically labeled non-target cells as well as aggregated erythrocytes are retained in the tube. Purity and viability of the enriched cell populations were determined by flow cytometry and functionality was shown in CDC assays. Results MACSxpress-enriched lymphocyte subsets (B/T/BT cells) from whole blood had average purities of 87/96/98% with yields of 60/54/38% ( n = 27/29/23). Cell enrichment from spleen single cell suspensions resulted in purities of 97/96/98% and recoveries of 27/32/29% ( n = 5) (B/T/BT cells). CDC analysis of positive and negative control sera with enriched cells showed no influence of fluorescent readout by the enrichment process. Conclusion Using MACSprep HLA Isolation Kits, lymphocytes for crossmatch analysis can be enriched within 20 min from whole blood without the need of expensive lab equipment or preparation of PBMC by density gradient centrifugation. G. Winkels: Employee; Company/Organization; Miltenyi Biotec.

P146 The use of surrogate crossmatching allows for the detection of a possible donor specific antibody to an HLA-DQ heterodimer

Aim The development of de novo donor-specific antibodies (DSA) post-transplant is an area of interest for the transplant community. There are several challenges in detecting DSA, including the use of recombinant HLA antigens vs. affinity-purified antigens from a phenotype, and the lack of donor typing at many loci. Herein, we describe a case where surrogate flow-cytometric crossmatch (FCXM) was used in conjunction with solid-phase immunoassays for the detection of DSA in a heart transplant recipient. Methods Post-transplant serum was tested by solid-phase immunoassays for the detection of DSA to the following donor antigens: A2,23; B7,8; Bw6; Cw7; DR15,17; DR52; DQ2,6. Confirmation of antibody specificity was obtained using FCXM using three surrogate donor cells. Results Initial testing by One Lambda LABScreen® PRA and Single Antigen assays provided negative results for HLA class I, and inconclusive results for HLA class II, suggesting possible reactivity to HLA-DQ2, HLA-DQA1∗05:01/DQB1∗02:01, or false-positive reactivity. In order to confirm the possible specificities obtained, surrogate crossmatching was performed utilizing pronased cells from donors with the following HLA types: Donor 1- DQB1∗02:XX, 03:XX (DQ9); DQA1∗02:01; Donor 2- DQB1∗02:01; DQA1∗05:01; Donor 3- DQB1∗02:01, 03:01 (DQ7); DQA1∗05:01, with recipient serum at dilutions of Neat, 1:2, 1:4, and 1:16. T cell crossmatch results at all dilutions were negative for each donor cell tested. Donor 1 tested B cell negative at all dilutions tested, while donor 2 tested positive at serum dilutions of Neat, 1:2, and 1:4, and donor 3 tested positive at serum dilutions of Neat and 1:2. This evidence allowed for us to assign a strong antibody, defined by our laboratory as predicted to yield a positive FCXM, to the DQA1∗05:01/DQB1∗02:01 heterodimer. Since donor HLA-DQA1 typing was not available, it is inconclusive whether the antibody detected should be considered donor-specific. Conclusion We conclude that while solid-phase immunoassays are useful tools for the detection of HLA antibodies, there are several challenges present which could require the use of additional assays for confirmation of test results. In addition, the lack of complete donor typing often makes it difficult for the laboratory to accurately interpret the findings of such results.

Donor-Specific Anti-HLA Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) is a curative treatment for a wide variety of hematological diseases. In 30% of the cases, a geno-identical donor is available. Any other situation displays some level of human leukocyte antigen (HLA) incompatibility between donor and recipient. Deleterious effects of anti-HLA immunization have long been recognized in solid organ transplant recipients. More recently, anti-HLA immunization was shown to increase the risk of primary graft failure (PGF), a severe complication of AHSCT that occurs in 3–4% of matched unrelated donor transplantation and up to 15% in cord blood transplantation and T-cell depleted haplo-identical stem cell transplantation. Rates of PGF in patients with DSA were reported to be between 24 and 83% with the highest rates in haplo-identical and cord blood transplantation recipients. This led to the recommendation of anti-HLA antibody screening to detect donor-specific antibodies (DSA) in recipients prior to AHSCT. In this review, we highlight the role of anti-HLA antibodies in AHSCT and the mechanisms that may lead to PGF in patients with DSA, and discuss current issues in the field.

Pretransplant angiotensin II type 1-receptor antibodies are a risk factor for earlier detection of de novo HLA donor-specific antibodies.

Angiotensin II type 1 receptor antibodies (AT1Rabs) have been associated with significantly reduced graft survival. Earlier graft loss has been observed in patients who had pretransplant AT1Rabs and posttransplant donor-specific antibodies (DSA). The main goal of this retrospective cohort study was to examine the association between AT1Rabs and the time period to detection of de novo human leukocyte antigen (HLA-DSA) posttransplantation in living donor kidney transplant recipients (KTR). The analysis included 141 KTRs. Pretransplant frozen serum samples were tested for AT1Rabs by ELISA and HLA-DSA by SAB (Luminex) at both the pre- and post-KT time points. The median AT1Rab level was 9.13 U (interquartile range 5.22-14.33). After a mean follow-up period of 3.55 years, 48 patients were found to harbour de novo HLA-DSAs. The presence of AT1Rabs [hazard ratio (HR) 1.009, 95% confidence interval (CI) 1.002-1.01, P = 0.010], male-to-male transplantation (HR 2.57, 95% CI 1.42-4.67, P = 0.002) and antecedent borderline changes or acute cellular rejection (ACR) (HR 2.47, 95% CI 1.29-4.75, P = 0.006) were significantly associated with de novo DSA detection. A dose-dependent association between AT1Rab levels (<10 U, 10.1-16.9 U, 17-29.9 U and >30 U) and de novo DSA detection was observed (log-rank P = 0.0031). After multivariate analysis of AT1Rab levels (continuous variable), AT1Rabs >30 U, male-to-male transplantation, donor age, higher class I percentage of Panel Reactive Antibody and antecedent borderline changes or ACR remained as independent significant risk factors for the detection of de novo DSAs. The findings suggest that higher levels of pretransplant circulating antibodies against AT1R (>30 U) in kidney graft recipients constitute an independent risk factor for earlier de novo HLA-DSA detection during the posttransplant period.

(644) - Intravenous Immunoglobulin in Sensitized Lung Transplant Recipients and Early Outcomes

The presence of donor specific antibodies (DSA) and/or non-DSA anti-human leukocyte antigen (HLA) antibodies post-transplant have been associated with worse outcomes. Pretransplant HLA antibiotic detection (sensitization) is a strong predictor for post-transplant HLA antibodies and DSA. Therefore, our program instituted an intensive intravenous immunoglobulin (IVIG) peri-operative protocol for all sensitized patients after transplant. In July 2014, we made a systematic change to a more conservative approach where only recipients with pretransplant panel reactive antibody (PRA) ≥ 25% would receive the IVIG protocol. This study was to assess early outcomes of primary graft dysfunction (PGD), detection of DSA, need for ECMO and 30-day readmissions under a more conservative use IVIG protocol. All patients transplanted between 7/1/2014 and 6/30/2015 were eligible for analysis. Exclusion criteria included retransplant and multi-organ transplants. Recipients with pretransplant HLA antibodies were divided into those receiving and those not receiving IVIG based on PRA level. Recipients receiving IVIG off protocol were excluded. Descriptive statistics and nonparametric tests used for comparison of groups. 104 recipients during the study period were eligible for analysis. 28 recipients had any pretransplant PRA. Of these, 11 had PRA 25% or less and did not receive IVIG, 15 had PRA≥ 25% and received IVIG and 2 patients were excluded due to receiving IVIG off protocol (PRA less than 25%). Results are showed in the table. The groups did not differ by PGD grade 3, ECMO, DSA or readmissions by nonparametric tests. A conservative IVIG protocol where only recipients with pretransplant PRA≥ 25% receive IVIG appears to have no impact on early outcome measures in this initial group. A larger sample size and longer follow will confirm if this conservative IVIG approach is appropriate.

Immunological risk assessment: The key to individualized immunosuppression after kidney transplantation.

The wide range of immunosuppressive therapies and protocols permits tailored planning of the initial regimen according to the immunological risk status of individual patients. Pre-transplant risk assessment can include many factors, but there is no clear consensus on which parameters to take into account, and their relative importance. In general younger patients are known to be at higher risk for acute rejection, compounded by higher rates of non-adherence in adolescents. Donor age and recipient gender do not appear to exert a meaningful effect on risk of rejection per se, but black recipient ethnicity remains a well-established risk factor even under modern immunosuppression regimens. Little difference in risk is now observed between deceased- and living-donor recipients. Immunological risk assessment has developed substantially in recent years. Cross-match testing with cytotoxic analysis has long been supplemented by flow cytometry, but development of solid-phase single-bead antigen testing of solubilized human leukocyte antigens (HLA) to detect donor-specific antibodies (DSA) permits a far more nuanced stratification of immunological risk status, including the different classes and intensities of HLA antibodies Class I and/or II, including HLA-DSA. Immunologic risk evaluation is now often based on a combination of these tests, but other assessments are becoming more widely introduced, such as measurement of non-HLA antibodies against angiotensin type 1 (AT1) receptors or T-cell ELISPOT assay of alloantigen-specific donor. Targeted densensitization protocols can improve immunological risk, notably for DSA-positive patients with negative cytotoxicity and flow cross-match. HLA mismatch remains an important and undisputed risk factor for rejection. Delayed graft function also increases the risk of subsequent acute rejection, and the early regimen can be modified in such cases. Overall, there is a shift towards planning the immunosuppressive regimen based on pre-transplant immunology testing although certain conventional risk factors retain their importance.

‘;Unacceptable’ Human Leukocyte Antigens in Lung Re-transplantation

The role of human leukocyte antigen (HLA)-sensitization in the absence of detectable donor-specific antibodies in lung re-transplantation is unclear. Antigens of the second donor matching the HLA typing of the first donor are considered ‘unacceptable’ by some tissue typing laboratories, especially in kidney re-transplantation. Thus, we performed a retrospective analysis of all lung re-transplantations at our center focusing on the impact of HLA-homologies between the first and the second donor (‘unacceptable’ antigens) on patient and graft survival.

The importance of donor-specific anti-HLA antibodies (DSA) identification in renal transplant patients with C4d-negative biopsies

Two index cases of living-related donor renal allografts patients developed C4d-negative rejection. Both cases had negative cytotoxic crossmatch and negative flow crossmatch before transplantation. The serum creatinine levels were tabulated. Both cases experienced augmented anti-T cell therapy (intravenous methyl prednisolone) at the time of rejection, which failed to improve renal function. Meantime, our HLA lab identified circulating anti-class I and/or II HLA antibodies towards donor mismatched antigens by Luminex multiplex bead array. Additional therapy included high-dose IVIg and plasma exchange. The renal function improved significantly. Furthermore, the donor-specific antibody strength decreased after combined plasmapheresis and IVIG therapy. These cases highlight the importance of donor-specific antibody detection by sensitive solid phase assays in the context of C4d-negative ABMR.

Analysis of Panel Reactive Antibodies in Renal Transplant Recipients Detected by Luminex: A Single-Center Experience.

The role of panel reactive antibody has gained universal acceptance in solid-organ transplant. This parameter is used to gauge the level of sensitization of prospective solid-organ recipients. More than one-third of patients on wait lists for kidney transplant are sensitized. Most have previously formed donor-specific and non-donor-specific serum antibodies and/or positive crossmatch by complement-dependent cytotoxicity and/or flow cytometry. We present the rate of positivity at our institution for human leukocyte antigen antibodies and describe the condensation of antibodies in human leukocyte antigens for renal pretransplant recipients. Between January 2011 and December 2012, six hundred twenty consecutive renal transplant recipients on the wait list at the Baskent University were evaluated for this retrospective study. Panel reactive antibody screening and definition tests were studied with Luminex assays for the combination of class I (A, B, C) and class II antigens (DR, DQ). We found a panel reactive antibody screening positivity in 20.4% of our patients on renal transplant waiting list. Panel reactive antibody defining tests were meaningful in 12.2% of the whole list. We observed that only panel reactive antibody class I positivity was seen in 2.2%, only panel reactive antibody class II positivity was seen in 2.7%, and both panel reactive antibody class I and class II positivities were seen in 7.2% of the defining tests. The estimated risk of sensitization for patients with a living donor is determined from the combined results of the crossmatch with the donor and those of the recipient's panel reactive and donor-specific antibodies. Compared with complement-dependent cytotoxicity crossmatch, Luminex assays provide greater sensitivity and specificity in detection of donor-specific antibodies.

The use of circulating donor specific antibody to predict biopsy diagnosis of antibody-mediated rejection and to provide prognostic value after heart transplantation in children

Antibody-mediated rejection (AMR) is a significant cause of mortality after heart transplantation (HT). Although the presence of donor specific antibody (DSA) is a risk factor for developing AMR, serial DSA testing is not widely performed. We aimed to investigate the predictive values and prognostic implications of circulating DSA using endomyocardial biopsy as the gold standard for AMR diagnosis in pediatric recipients of HT. We performed a retrospective study in pediatric recipients of HT followed during the period 2009-2013 with at least 1 biopsy paired with DSA testing. Positive DSA was defined at mean fluorescent intensity (MFI) ≥2,000 using single antigen bead testing. Statistical analyses included 2 × 2 contingency tables, receiver operating characteristic analysis for optimal MFI cutoffs, Spearman correlation of MFI strength to AMR grade, and Kaplan-Meier analysis of event-free survival. Of 66 children included, 27 (41%) had ≥1 DSA positive test. DSA testing had a sensitivity of 92.6%, specificity of 62.2%, positive predictive value of 24.0%, and negative predictive value of 98.5% for biopsy diagnosis of AMR at our institution. There was a statistically significant correlation between higher MFI and higher AMR grade. Patients with positive DSA and AMR had similar survival early after DSA detection but trended toward lower cardiovascular event-free survival later compared with patients without DSA and a negative biopsy. The results of DSA testing in this cohort showed excellent sensitivity and negative predictive value for biopsy-diagnosed AMR, suggesting that DSA testing may aid in the non-invasive prediction of AMR absence in HT. The correlation of DSA MFI strength with higher AMR biopsy grade and the trend toward differences in longer term cardiovascular outcomes provide evidence for routine DSA monitoring after pediatric HT.

Luminex crossmatch for pretransplant evaluation of renal transplant recipients

Aim To evaluate the usefulness of Luminex crossmatch for detection of donor specific antibodies. Methods Four hundred and eighty-nine (434 patients/450 donors) consecutive patient samples including 52 (10.6%) retransplants were tested. Complement Dependent Cytotoxicity (CDC) B and T cell crossmatch by Extended NIH method and Luminex crossmatch was done for all patients of which thirty-six samples were tested multiple times for treatment monitoring. Panel reactive antibody (PRA) screen was done on 243 samples (49.3%). Results CDC crossmatch was positive in 21 patients. HLA class II IgG was detected in 162 samples (31%), class I in 41 samples (8.1%) and both classes of IgG were present in 68 samples (14.1%). Correlation with clinical and/or laboratory parameters was observed in 386/489 (78.9%) samples. One hundred and three samples (21.1%) were discordant including 29 unsensitized patients and 74 with negative antibodies on further testing. Table 1 shows the correlation of LXM with other parameters. Conclusion Luminex crossmatch must be combined with antibody screen for pretransplant evaluation as false positive results were detected for nearly one-fifth of samples. Download : Download full-size image

Prospective Monitoring of Donor-specific Anti-HLA Antibodies After Intestine/Multivisceral Transplantation: Significance of De Novo Antibodies.

Presence of circulating donor-specific antibodies (DSA) may be associated with worse clinical outcomes after intestine/multivisceral transplantation. In 79 intestine/multivisceral recipients, sera were prospectively screened for DSA by Luminex Single antigen test at 1, 3, 6, 9, 12, 18, 24, and 36 months after transplantation. Standard immunosuppression included thymoglobulin-rituximab induction and tacrolimus-prednisone maintenance. C4d staining was performed retrospectively on biopsies in patients that developed acute rejection (AR). Twenty-two (28%) patients developed de novo DSA at a median posttransplant period of 3 (1-36) months. De novo DSA were observed in 10 of 40 liver-including and 12 of 39 liver-excluding transplants (P = 0.57). Occurrence of AR was slightly higher in patients with de novo DSA (45% vs 33%, respectively; P = 0.41). Similarly, chronic rejection (14% vs 5%; P = 0.21) and graft loss due to AR (18% vs 7%; P = 0.14) were numerically higher in patients with de novo DSA. Only 35% patients experiencing AR had circulating de novo DSA at the time of AR. Antibody-mediated rejection was diagnosed in 6 patients based on C4d staining, of these 2 patients had circulating de novo DSA at the time of biopsy. De novo DSA formation, particularly early in the posttransplant course may be associated with trends toward worse outcomes. However, its significance in the pathophysiology of AR remains uncertain. Studies focusing mechanisms of DSA-related graft injury and intragraft DSA detection might provide further insight into this issue.

Clinical Implications of Angiotensin II Type 1 Receptor Antibodies in Antibody-mediated Rejection Without Detectable Donor-specific HLA Antibodies After Renal Transplantation

BackgroundSolid-phase immunoassays have improved detection sensitivity for donor-specific HLA antibody (DSHA) and permitted the accurate diagnosis of antibody-mediated rejection (AMR). However, DSHA is not always sufficient to explain the cause of AMR. Consequently, a means of assessing non-HLA antibodies is required to determine the cause of AMR. The aim of the present study was to evaluate the clinical implications of antibodies (Abs) targeting angiotensin II type I receptor (AT1R) in recipients with AMR but without serum DSHA. MethodsNon-HLA AMR cases diagnosed between January 2011 and June 2014 were included. Levels of anti-AT1R Abs (U/mL) were quantified by using AT1R assay kits (One Lambda, Calif, United States) with collected sera pretransplantation and at biopsy (cut-off value: 15 U/mL). ResultsSeventy-two patients were diagnosed with AMR during the above-mentioned period. Of them, 12 recipients (16.7%) had no DSHA. The sera of these 12 patients were tested (2 patients were only checked at time of biopsy). Nine patients (9/10) were presensitized for anti-AT1R Abs (median, 25.0 U/mL; range, 12.9 to 50.0 U/mL). Ten patients (10/12) were anti-AT1R− positive at time of biopsy (median, 23.2 U/mL; range, 11.4 to 50.0 U/mL). The mean time from transplantation to biopsy was 73 months. Eight patients experienced acute AMR, and 4 developed chronic AMR. Four patients showed negative C4d staining in peritubular capillaries (4/12). Patients were treated with plasmapheresis, low-dose intravenous immunoglobulin, and/or rituximab. ConclusionsAT1R Abs may play a significant role in AMR without detectable DSHA. Pretransplantation detection of AT1R Abs may be helpful for assessing the risk for non-HLA AMR.

P016 : PRONASE TREATMENT OF T LYMPHOCYTES DECREASES CDC CROSSMATCH TESTING TIME BY 50% AND MAINTAINS DSA COMPLEMENT ACTIVATING SPECIFICITY

Crossmatching (XM) via AHG-CDC and Flow cytometry (FC-XM) has enhanced sensitivity for detecting donor specific antibodies (DSA) but limits IgG functional characterization. We propose Pronase treated lymphocytes enhance Amos-CDC XM sensitivity by decreasing CD55 complement (C ′) inhibition without loss of functional specificity. Five well characterized sets of T-cells/sera combinations were chosen to give borderline (≈60–120 MCS) and strong (>300 MCS) FC-XM results. Antibody specificities were determined using LabScreen® SAB. T-cells were isolated using HLA Cell Prep I Dynabeads® and pronase treated (optimized for CDC testing, 0.5 mg/mL for 10 min at 37 °C). Pronase treated Amos-CDC (PT-Amos-CDC) XM was modified for C′ optimization (reduced incubation to 20 min). Standard Amos-CDC, AHG-CDC and FC-XM were performed in parallel. A total 15 XM were performed. Cumulative Class I DSA strength ranged from 5451 to 35,162 normalized MFI. Of those, 9 were positive with all methods, 1 was positive with all methods except Amos-CDC, 3 were only positive by FC-XM, 1 was positive by PT-Amos-CDC and FC-XM, and 1 was only positive by PT-Amos-CDC. PT-Amos-CDC T-cell XM markedly reduces testing time by 50% and maintains DSA specific C′ activation that is not assessable via AHG-CDC and FC-XM methods. This provides valuable reduction in ischemia time. Pronase and C′ steps require optimization to enhance antibody mediated C′ activation without inducing non-specific C′ sensitivity. Although PT-Amos-CDC did not appear as sensitive as AHG-CDC and FC-XM, some cases are better potentiated than others and appears to be more sensitive than the standard Amos-CDC. Further studies will be useful in characterizing these antibodies (e.g., C1q-SAB & IgG subclassing) and the potential use of human C’ in the assay. PT-Amos-CDC provides a rapid cell based in vitro assay that we believe will correlate with in vivo DSA C′ fixing potential. This makes the assay valuable not only to reduce ischemia time but also in assessing risk of hyperacute and accelerated acute rejection. Additional studies will be needed to determine if the assay can be adapted for B-cell XM as well.

OR52: DE-NOVO DEVELOPMENT OF DONOR MISMATCHED HLA IS SIGNIFICANTLY REDUCED IN ABO-INCOMPATIBLE RENAL TRANSPLANT RECIPIENTS: IMPLICATION FOR LONG TERM ALLOGRAFT FUNCTION

Aim To determine whether the abrogation of development of donor specific antibodies (DSA) following ABO incompatible (ABOi) transplants is due to “accommodation” resulting in better graft outcome of ABOi renal transplants in comparison to ABO compatible (ABOc) renal transplants (RTx). Method RTx recipients with ABOi (n = 18) and ABOc (n = 47) with similar demographics were selected for this study [Table 1]. Pre and post-transplant sera were analyzed for DSA to Class I and II human leukocyte antigen (HLA) using LabScreen Single Antigen assay. Both groups had no detectable HLA and were crossmatch compatible. ABOi patients underwent plasmaheresis, intravenous immunoglobulin and Rituximab to reduce antibodies to blood group antigens, while ABOc patients received intravenous thymoglobulin in the immediate post-transplant period. Both groups were maintained on identical immunosuppressive regimen with triple therapy. Results Allo-immune response in ABOi and ABOc RTx patients was analyzed by measuring de novo development of DSA. None of the ABOi group developed DSA either to HLA Class I or Class II. In contrast, ABOc recipients developed DSA, 11 of 47 (23%). It is of significance that ABOi recipients with similar HLA mismatches to ABOc recipients failed to develop any detectable DSA which may account for lower incidence of acute rejection in ABOi group. In the ABOi only 2 of 18 (11%) developed acute rejection episodes which were reversible compared to 9 of 47 (20%) in the ABOc group. At the end of one year, glomerular filtration rate was also significantly better (p Conclusion Our study demonstrates that desensitization using Rituximab, intravenous immunoglobulin and plasmapheresis for depleting antibodies in ABOi RTx patients prior to transplantation abrogates de novo development of DSA, likely due to accomodation, may account for the better outcome of the ABOi renal transplants.