Donor-specific Antibody Strength Research Articles

Cancer Risk Following HLA-Incompatible Living Donor Kidney Transplantation.

To characterize cancer risk in ILDKTr, we evaluated 858 ILDKTr and 12 239 compatible living donor kidney transplant recipients (CLDKTr) from a multicenter cohort with linkage to the US transplant registry and 33 cancer registries (1997-2016). Cancer incidence was compared using weighted Cox regression. Among ILDKTr, the median follow-up time was 6.7 y (maximum 16.1 y) for invasive cancers (ascertained via cancer registry linkage) and 5.0 y (maximum 16.1 y) for basal and squamous cell carcinomas (ascertained via the transplant registry and censored for transplant center loss to follow-up). Invasive cancers occurred in 53 ILDKTr (6.2%) and 811 CLDKTr (6.6%; weighted hazard ratio [wHR] 1.01; 95% confidence interval [CI], 0.76-1.35). Basal and squamous cell carcinomas occurred in 41 ILDKTr (4.8%) and 737 CLDKTr (6.0%) (wHR 0.99; 95% CI, 0.69-1.40). Cancer risk did not vary according to donor-specific antibody strength, and in an exploratory analysis, was similar between CLDKTr and ILDKTr for most cancer types and according to cancer stage, except ILDKTr had a suggestively increased risk of colorectal cancer (wHR 3.27; 95% CI, 1.23-8.71); however, this elevation was not significant after correction for multiple comparisons. These findings indicate that the risk of cancer is not increased for ILDKTr compared with CLDKTr. The possible elevation in colorectal cancer risk is unexplained and might suggest a need for tailored screening or prevention.

423.3: Long-term Outcomes Following HLA-Incompatible Living Donor Kidney Transplantation

Introduction: Incompatible living donor kidney transplantation (ILDKT) offers a survival benefit compared to dialysis for patients with donor specific antibody (DSA) who do not have a compatible living donor. Longer-term outcomes have not been characterized in this population, but must be quantified to inform treatment selection for patients with an incompatible willing donor, and counseling for ILDKT recipients. Method: We compared 1,406 ILDKT to 17,542 compatible LDKT (CLDKT) recipients using a 25-center cohort from the United States 1997-2016, linked to national registry data (SRTR). Participating centers classified ILDKT recipients as positive Luminex, negative flow crossmatch (PLNF, n=376); positive flow, negative cytotoxic crossmatch (PFNC, n=687); or positive cytotoxic crossmatch (PCC, n=343). We used multivariable Cox regression to quantify the risk of mortality and death-censored graft failure (DCGF), and multilevel mixed-effects linear regression to evaluate eGFR decline (CKD-EPI creatinine equation 2021) in ILDKT and CLDKT recipients. Results: The cumulative mortality risk for CLDKT, PLNF, PFNC, and PCC was: 1.6%, 1.9%, 3.2%, and 8.2% at one year; 22.5%, 18.4%, 29.4%, and 36.1% at ten years; and 39.1%, 36.1%, 45.4% and 51.6% at fifteen-years (p<0.001) (Figure 1A). Although this translated to an equivalent mortality risk for PLNF recipients (aHR=0.830.961.12, p=0.6) after adjustment, PFNC and PCC recipients had a 1.48-fold (aHR=1.301.481.68, p<0.001) and 1.66-fold (aHR=1.331.662.08, p<0.001) higher risk compared their CLDKT counterparts, respectively. The cumulative risk of DCGF for CLDKT, PLNF, PFNC, and PCC was: 1.9%, 2.1%, 3.4%, and 11.1% at one year; 16.8%, 20.5%, 33.1%, and 41.0% at ten years; and 25.5%, 33.1%, 45.2% and 52.5% at fifteen-years (p<0.001) (Figure 1B). After adjustment, this translated to higher DCGF across all levels of DSA strength (aHR PLNF=1.141.411.73, PFNC=1.491.711.95, PCC=1.612.082.67; p for all≤0.001). Conversely, ILDKT and CLDKT recipients had comparable average eGFR immediately post-transplant (difference=-4.22-1.511.20 mL/min/1.73m2, p=0.3). However, ILDKT recipients had faster decline in eGFR per year compared to CLDKT recipients (difference in slope=-1.06-0.76-0.47 mL/min/1.73m2 per year, p<0.001; decline over 5 years for ILDKT=-9.01-7.55-6.10 mL/min/1.73m2 vs. CLDKT=-4.19-3.74-3.29 mL/min/1.73m2). Conclusion: Patient and graft survival following ILDKT is good through fifteen years post-transplant, supporting this modality, but survival is worse compared to CLDKT recipients. ILDKT recipients bear close monitoring to maximize graft life.This work was supported by grant numbers K01DK101677 (Massie) and R01DK98431 (Segev) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Dr. Segev is supported by the K24AI144954 (Segev) from the National Institute of Allergy and Infectious Diseases (NIAID).

Delayed graft function and acute rejection following HLA-incompatible living donor kidney transplantation.

Incompatible living donor kidney transplant recipients (ILDKTr) have pre-existing donor-specific antibody (DSA) that, despite desensitization, may persist or reappear with resulting consequences, including delayed graft function (DGF) and acute rejection (AR). To quantify the risk of DGF and AR in ILDKT and downstream effects, we compared 1406 ILDKTr to 17542 compatible LDKT recipients (CLDKTr) using a 25-center cohort with novel SRTR linkage. We characterized DSA strength as positive Luminex, negative flow crossmatch (PLNF); positive flow, negative cytotoxic crossmatch (PFNC); or positive cytotoxic crossmatch (PCC). DGF occurred in 3.1% of CLDKT, 3.5% of PLNF, 5.7% of PFNC, and 7.6% of PCC recipients, which translated to higher DGF for PCC recipients (aOR=1.03 1.682.72 ). However, the impact of DGF on mortality and DCGF risk was no higher for ILDKT than CLDKT (p interaction>.1). AR developed in 8.4% of CLDKT, 18.2% of PLNF, 21.3% of PFNC, and 21.7% of PCC recipients, which translated to higher AR (aOR PLNF=1.45 2.093.02 ; PFNC=1.67 2.403.46 ; PCC=1.48 2.243.37 ). Although the impact of AR on mortality was no higher for ILDKT than CLDKT (p interaction=.1), its impact on DCGF risk was less consequential for ILDKT (aHR=1.34 1.621.95 ) than CLDKT (aHR=1.96 2.292.67 ) (p interaction=.004). Providers should consider these risks during preoperative counseling, and strategies to mitigate them should be considered.

Presence of a survival benefit of HLA-incompatible living donor kidney transplantation compared to waiting or HLA-compatible deceased donor kidney transplantation with a long waiting time

HLA-incompatible living donor kidney transplantation (LDKT) is one of efforts to increase kidney transplantation opportunity for sensitized patients with kidney failure. However, there are conflicting reports for outcomes of HLA-incompatible kidney transplantation compared to patients who wait for HLA-compatible deceased donor kidney transplantation (DDKT) in the United States and United Kingdom. Waiting for an HLA-compatible DDKT is relatively disadvantageous in Korea, because the average waiting time is more than five years. To study this further, we compared outcomes of HLA-incompatible LDKT with those who wait for HLA-compatible DDKT in Korea. One hundred eighty nine patients underwent HLA-incompatible LDKT after desensitization between 2006 and 2018 in two Korean hospitals (42 with a positive complement-dependent cytotoxicity cross-match, 89 with a positive flow cytometric cross-match, and 58 with a positive donor-specific antibody with negative cross-match). The distribution of matched variables was comparable between the HLA-incompatible LDKT group and the matched control groups (waiting-list-only group; and the waiting-list-or-HLA-compatible-DDKT groups; 930 patients each). The HLA-incompatible LDKT group showed a significantly better patient survival rate compared to the waiting-list-only group and the waiting-list-or-HLA-compatible-DDKT groups. Furthermore, the HLA-incompatible LDKT group showed a significant survival benefit as compared with the matched groups at all strength of donor-specific antibodies. Thus, HLA-incompatible LDKT could have a survival benefit as compared with patients who were waitlisted for HLA-compatible DDKT or received HLA-compatible DDKT in Korea. This suggests that HLA-incompatible LDKT as a good option for sensitized patients with kidney failure in countries with prolonged waiting times for DDKT.

P1791HLA DESENSITIZATION- INDIAN EXPERIENCE

Abstract Background and Aims There are many barriers to kidney transplant and one of them is presence of donor specific antibodies (DSAs) in the recipients. Presence of strong DSA is considered a relative contraindication for kidney transplantation, however, if DSAs are of weak to moderate then desensitization is attempted in many centres with good success rate. Desensitizing such patients can be an acceptable approach to increase the donor pool and facilitating transplants. This is a retrospective analysis of patients who underwent desensitization at our centre after availability of luminex single antigen (LSA) assay Method Between April 2014 and December 2018, 825 patients underwent kidney transplantation at our centre. Patients who were CDC negative but positive FCXM were further analysed with LSA to know the presence and strength of DSAs. Our protocol for desensitization consisted of plasmapheresis (PP) 1.5 volume by double filtration on alternate day and low dose IV IG 100 mg/kg after each PP. Whenever MFI was &amp;lt;1000 and/or FCXM was negative, patient was taken for transplant with thymoglobulin induction of 1.5 mg/kg for 2-3 doses. All patients were maintained on triple immunosuppression consisting of tacrolimus, mycophenolate mofetil and corticosteroids. We did not routinely followed DSAs in these patients post-transplant if there was no clinical indication. All adverse events during follow up including new onset diabetes after transplant (NODAT), infections, acute rejections (AR), graft loss and death Results Out of 825 patients, 15 underwent HLA incompatible transplants, of which, 8 were males. All patients were first transplant and 11/13 had history of some sensitizing events in the form of blood transfusion and/or pregnancy. The mean dialysis duration was 8.6 ±14.6 months. FCXM was positive in all the patients with 5 patients had T cell flow positive, 8 had B cell flow positive and 2 had both T &amp; B cell FCXM positivity. Most patients had weak to moderate positive flow cross match. On further evaluation by LSA, all these patients had DSAs, with 3/15 had MFI &amp;lt;1000, 6 had MFI between1000-2000, and remaining 6 had MFI &amp;gt;2000, out of which one patient had MFI of 7195 and six patients had multiple DSAs. These patients underwent desensitization with PP and IVIG and the end point of treatment was either MFI &amp;lt; 1000 or FCXM negative. Post- transplant DSAs were done in patients with high MFI or clinically indicated. Two patients had increase in post-transplant DSA titres requiring post-transplant plasmapheresis. The mean follow up was 29±6 months. On follow up, only 1 patient developed borderline cellular rejection one year after transplant, which responded with pulse steroids. Three patients had biopsy for asymptomatic rise in creatinine but it showed patchy ATN with no evidence of rejection.. One patient developed transient CMV viremia, one patient developed lymph node tuberculosis (TB) and two patients had UTI, all of them responded to treatment. There was no graft or patient loss till last follow up. Conclusion This study shows that HLA desensitisation is feasible and successful in if patients are selected carefully and evaluate thoroughly. HLA incompatible transplant can provide a new lease of life to those patients who would otherwise not get a kidney due to lack of paired exchange and deceased donor program

Beyond 10years, with or without an intestinal graft: Present and future?

Long-term outcomes in children undergoing intestinal transplantation remain unclear. Seventy-one children underwent intestinal transplantation in our center from 1989 to 2007. We report on 10-year posttransplant outcomes with (group 1, n=26) and without (group 2, n=9) a functional graft. Ten-year patient and graft survival rates were 53% and 36%, respectively. Most patients were studying or working, one third having psychiatric disorders. All patients in group 1 were weaned off parenteral nutrition with mostly normal physical growth and subnormal energy absorption. Graft histology from 15 late biopsies showed minimal abnormality. However, micronutrient deficiencies and fat malabsorption were frequent; biliary complications occurred in 4 patients among the 17 who underwent liver transplantation; median renal clearance was 87mL/min/1.73m2 . Four patients in group 1 experienced late acute rejection. Among the 9 patients in group 2, 4 died after 10years and 2 developed significant liver fibrosis. Liver transplantation and the use of a 3-drug regimen including sirolimus or mycophenolate mofetil were associated with improved graft survival. Therefore, intestinal transplantation may enable a satisfactory digestive function in the long term. The prognosis of graft removal without retransplantation is better than expected. Regular monitoring of micronutrients, early psychological assessment, and use of sirolimus are recommended.

The Natural History of Pre-Existing Donor Specific Antibody and Amnestic Responses after Heart Transplantation

Sensitized patients have prolonged wait times for heart transplant and often low-level donor specific antibodies (DSA) will be crossed when accepting a donor heart. The purpose of this study was to determine if positive low-level DSA at the time of transplant increase in strength post transplant and increase the risk of antibody mediated rejection (AMR). We reviewed 72 heart transplant patients between 2010 and 2018 in whom low-level DSA were crossed. DSA strength, as measured by mean fluorescence intensity (MFI) and AMR on biopsy was assessed at 1, 3, 6, 12 months post transplant. Other outcomes assessed: freedom from first-year rejection (acute cellular rejection (ACR), any treated rejection (ATR)), freedom from cardiac allograft vasculopathy (CAV, as defined by stenosis ≥30% by angiography), and freedom from graft dysfunction (LVEF ≤40%). Patients were compared to a contemporaneous control group conditional to 1-year survival without DSA at transplant. Approximately one-third of patients with pre-existing DSA at transplant demonstrated an increase in DSA strength. Patients with pre-existing DSA had decreased freedom from ATR and AMR but no differences in freedom from CAV, graft dysfunction, or ACR compared to the control group. Crossing low-level DSA may increase the risk of AMR, but there is no difference in clinically important sequelae such as CAV or graft dysfunction. Thus, accepting low-level DSA at the time of transplant appears feasible and could broaden the donor pool.

To Evaluate Anti-HLA Antibodies Sensitization in Pre- and Post-renal Transplant Patient’s Serum: A Retrospective Case Series

Introduction: In India, patients of renal failure are dependent on live related or unrelated donor. Because of poor financial condition patients do not go for DSA (Donor Specific Antibody) detection using Luminex. In absence of screening of de-novo production of DSA and don’t get proper management. As a result of which patient undergo acute rejection.&#x0D; Case Series: Here we are presenting 5 acute rejection cases comparing there DSA in pre-transplant and post-transplant sera using solid-phase assays.5 renal transplanted patients undergone acute and hyperacute rejection (Banff's classification) were considered for presented case series. Collected serum (pre and post-transplant on day of rejection) from each patient was subjected to detection of anti-HLA antibody using Luminex-PRA.&#x0D; Conclusion: Presence of donor-specific anti-HLA antibody with their titer was detected in pre and post-transplant serum. It is found that the strength of DSA is directly proportional to graft life.

Deleterious effect of anti-angiotensin II type 1 receptor antibodies detected pretransplant on kidney graft outcomes is both proper and synergistic with donor-specific anti-HLA antibodies.

Both donor-specific antibodies (DSA) and anti-angiotensin II type 1 receptor antibodies (AT1R-abs) have been associated with poor graft outcomes after kidney transplantation (KT). We aimed to understand the impact of pretransplant AT1R-abs with or without concomitant DSA on KT outcomes. Seventy-six patients transplanted in 2009 were studied. DSA (MFI > 1000) and/or AT1R-abs (>10UI) were detected by solid-phase assays in pre-KT sera. Multivariable Cox regression models were used to determine independent predictors of outcomes: acute rejection (AR) and graft failure. At transplant, 48 patients were AT1R-abs (-)/DSA (-), 12 AT1R-abs (+)/DSA (-), 9 AT1R-abs (-)/DSA (+) and 7 AT1R-abs (+)/DSA (+). Incidence of acute rejection at 1-year increased from 6% in AT1R-abs (-)/DSA (-), to 35% in AT1R-abs (+)/DSA (-), 47% in AT1R-abs (-)/DSA (+) and 43% in AT1R-abs (+)/DSA (+) (P < 0.001). No difference in DSA strength and C1q-binding ability was observed between AT1R-abs (-) /DSA (+) and AT1R-abs (+)/DSA (+) patients. Graft survival at 6-years was the lowest in AT1R-abs (+)/DSA (+) (57%), followed by AT1R-abs (+)/DSA (-) (67%), and higher in AT1R-abs (-)/DSA (-) (94%) and AT1R-abs (-)/DSA (+) (89%) patients (P = 0.012). AT1R-abs (+)/DSA (-) (HR = 6.41, 95% CI: 1.43-28.68; P = 0.015) and AT1R-abs (+)/DSA (+) (HR = 7.75, 95% CI: 1.56-38.46; P = 0.012) were independent predictors of graft failure. Acute rejection incidence and graft failure were associated with both DSA and AT1R-abs. These results demonstrate a proper negative effect of AT1R-abs on graft outcomes, besides a synergistic one with DSA. Pretransplant AT1R-abs should be acknowledged to better stratify patients' immunological risk.

It's About Everything: Validation and Optimization of 96-Well Tray Flow Cytometry Crossmatch

Flow cytometric crossmatch (FCXM) is widely used in many centers as part of pretransplant risk assessment to detect donor-specific anti-HLA antibodies. The limited number of crossmatches that can be performed during on-call work-up for deceased donors within reasonable time remains the main obstacle to accommodating the majority of highly sensitized listed patients to be tested by the standard tube FCXM method. This limitation often directs the organs to nonsensitized patients and deprives highly sensitized patients who could be compatible if their sera were included in the crossmatch test.The goal of this study is to optimize a 96-well tray FCXM protocol that allows more sera to be crossmatched without prolonging the overall procedural time while maintaining quality and sensitivity of the assay. The new method was validated against use of the standard tube method and included total of 63 crossmatches performed simultaneously by both methods using 20 donors' cells with patients' sera, pooled positive controls tested on different dilutions, and commercial negative control.In the new protocol we modify various assay parameters including tube platform, incubation time, amount of reagent antibody cocktail, and cell volumes. An overall concordance of 98% was achieved with the protocols with slight improvement in sensitivity (2 negative B-cell reactions converted to positive in presence of weak donor-specific antibodies and mild T-cell reactivity could be picked up at 1:80 diluted positive control by the tray method only). The median channel fluorescence values of the 2 methods were essentially equivalent for both T and B crossmatches (r2 of 0.98 and 0.97, respectively). In conclusion, 96-well tray assay has the potential to increase the probability of highly sensitized patients receiving transplants by allowing increased number of crossmatches to be performed with significant reduction in turnaround time and assay cost. Furthermore, the enhanced sensitivity of the assay will provide more accurate information about sensitization status and strength of donor-specific antibodies to treating physicians, allowing them to choose the best therapeutic option and to provide better patient care.

Microvascular inflammation in renal allograft biopsies assessed by endothelial and leukocyte co-immunostain: a retrospective study on reproducibility and clinical/prognostic correlates.

The most prominent histologic lesion in antibody-mediated rejection is microvascular inflammation (MVI); however, its recognition and scoring can be challenging and poorly reproducible between pathologists. We developed a dual immunohistochemical (IHC)-stain (anti-CD34/anti-CD45 for endothelium/leukocytes) as ancillary tool to improve on the semi-quantitative Banff scores and allow quantification of MVI. We examined the relationship between CD34-CD45 IHC-based quantitative MVI score (the inflamed peritubular capillary ratio, iptcr) and renal-graft failure or donor-specific antibodies (DSA) strength at the time of biopsy. Quantitative iptcr score was significantly associated with renal graft failure (hazard ratio 1.81, per 1 SD-unit [0.13 points] of iptcr-increase; P=0.026) and predicted the presence and strength of DSA (ordinal odds ratio: 2.42; P=0.005; 75 biopsies/60 kidney transplant recipients; 30 HLA- and/or ABO-incompatible). Next, we assessed inter-pathologist agreement for ptc score and ptc extent (focal/diffuse) using CD34-CD45 IHC as compared to conventional stain. Compared to conventional stain, CD34-CD45 IHC significantly increased inter-pathologist agreement on ptc score severity and extent (κ-coefficient from 0.52-0.80 and 0.46-0.68, respectively, P<0.001). Our findings show that CD34-CD45 IHC improves reproducibility of MVI scoring and facilitates MVI quantification and introduction of a dual anti-CD34/CD45 has the potential to improve recognition of MVI ahead of DSA results.

P154 Variability in the sensitivity of the flow cytometric crossmatch

Aim The sensitivity of the flow cytometric crossmatch (FXM) is often variable across labs in the presence of weak donor specific antibodies (DSA). In addition, inter-laboratory variation to the interpretation of Mean Fluorescence Intensity (MFI) values escalates the potential for discordance. The aim of this study is to compare FXM results among laboratories and correlate the results with DSA strength. Methods The study collected and analyzed data reported by participants in the Flow and Virtual Crossmatch Exchange over a period of 4 years. Data collected included antibody specificities, MFI values, T- and B-cell positive cutoffs, mean channel shifts (MCS), and negative control MC. In addition, the coefficient of variations (%CV) were calculated using the center-specific MFI values to analyze the variability in the reporting of antibody strength among labs. Results A total of 24 cells were tested against 48 sera over 12 Exchanges for a total of 96 T/B flow crossmatches. Among the 96 FXM pairs, 84% of T-cell and 88% of B-cell crossmatches were reported with good agreement (>80%). Positive concordant T-cell crossmatches were observed in 92% (57/62) of cases when class I DSA were reported with MFI >3000. Positive concordant B-cell crossmatches were observed in 89% (49/55) of cases when class I and class II DSA were reported with MFI >5000. Conclusions The data shows there is good consistency in T/B Flow crossmatch outcomes among labs in the presence of moderate or strong DSA. However, when DSA strength is weak, the sensitivity of the flow crossmatch is more widely variable. In addition, center practice, such as cutoff values and the reporting of MFI values may influence FXM outcomes. Thus, educational activities should continue to provide standards for quality control for the FXM.

P119 Correlation between the strength of donor specific antibody (DSA) using single antigen bead assay (SAB) and the results of CDC- cytotoxicity and flow cytometric crossmatch

Aim In this study we correlated flow and CDC- crossmatch outcomes with results of Luminex-single antigen bead (SAB). Methods Between 2010–2016 HLA-antibody Sera were tested by screening and SAB class I & II (One lambda) according to manufacturer recommendation. MFI of ⩾1000 were considered positive. These sera were used to perform the following: 1. 80 crossmatches using isolated PB or spleen lymphocytes from healthy deceased or living donors respectively by CDC–crossmatch. Correlation was performed on sera with single antibody or multiple classes I and II antibodies. Titer of CDC was given as the inverse of the highest dilution yielding positive XM. 2.108 FCXM cross using isolated PB donor lymphocytes expressing one HLA- antigen (class I or II) and 78 crossmatches using cells expressing multiple antigens detected by the sera. Analysis performed on FACSCanto II & mean channel shift (MCS) was calculated. The cut off for T &B are 64, 100 respectively. T- Positive, B- negative results in the absence of HLA- antibody were excluded from the study. Results Our results indicate good correlation of FCXM and CDC-XM with LUMINEX -SAB assay. SAB assay has good correlation with T-FCXM (r2 = 0.7) but not with B-FCXM. Fig. 1 A cutoff MFI of 3000 for SAB assay (as single or sum of multiple weak antibodies) had a sensitivity & specificity of 100% in detecting a positive FCXM. The few number of sera with single anti HLA-Cw and DP antibodies did not give positive reaction up to 5000 and 10,000 MFI respectively but were not included in the study. Single anti HLA-A, B, DRB1of ⩾10,000 MFI was associated with positive CDC-XM in most cases and multiple low levels antibody that yield this 10,000 would also give positive reaction. We noticed presence of A2 specificity among multiple anti class I or class II antibodies increased the chance of positive reaction even when sum of MFI is ⩽10000. Conclusions SAB correlates significantly with outcome of both cytotoxic and T-FCXM. Based on the threshold established it is possible to predict correctly results of crossmatch. Download : Download high-res image (164KB) Download : Download full-size image

Correlations between donor-specific antibodies and non-adherence with chronic active antibody-mediated rejection phenotypes and their impact on kidney graft survival

Chronic-active antibody-mediated rejection (CAABMR) is associated with poor kidney graft survival and has no clear effective treatment. Forty-one cases of CAABMR were detected in indication graft biopsies and evaluated according to current Banff classification. We investigated the impact of concurrent donor-specific antibodies (DSA) and their characteristics, together with non-adherence regarding immunosuppression on CAABMR histopathological phenotypes and prognosis. Twenty-four (59%) patients had detectable DSA at biopsy, with 15 of them being considered non-adherent. Graft function at biopsy was similar in DSA (+) and (−) patients. DSA (+) patients had significantly higher tubulointerstitial inflammation (i and ti) and acute humoral (g+ptc+v+C4d) composite score than DSA (−). DSA (+)/non-adherent cases presented additionally with increased microvascular inflammation (ptc and v), besides having a distinctively lower ah score. C1q DSA strength was higher (P = .046) in non-adherent patients and correlated closely with C4d score (P = .002). Lower graft function and ah score, higher proteinuria and ci + ct score, and, separately per each model, DSA (+) (HR = 2.446, P = .034), DSA (+)/non-adherent (HR = 3.657, P = .005) and DSA (+)/C1q (+) (HR = 4.831, P = .003) status were independent predictors of graft failure. CAABMR with concomitant DSA pose a higher risk of graft failure. Adherence should be evaluated, and histopathological phenotyping and DSA characterization may add critical information.

Complement (C1q) Binding De Novo Donor-Specific Antibodies and Cardiac-Allograft Vasculopathy in Pediatric Heart Transplant Recipients.

We hypothesized C1q binding de novo donor-specific antibody (DSA) after heart transplant (HT) is a higher risk for development of coronary artery vasculopathy (CAV) in children. A retrospective analysis of 127 pediatric HT recipients transplanted between January 2005 and December 2014 was used to determine complement (C1q)-binding de novo DSA on the outcomes of HT and the ability of the C1q assay to predict CAV development. Of 127 patients, 59 (46.4%) developed de novo DSA, 37 of those had C1q+ DSA. There was no difference in baseline characteristics except patients who developed C1q+ DSA more often received a donor heart from a female compared with C1q- DSA group (P = 0.034). The DSA median fluorescent intensity (MFI) value of 7000 or greater had 80% sensitivity and 80% specificity (C statistics 0.89, P <0.05) for predicting positive C1q binding. Multivariate analyses identified C1q binding DSA as an independent risk for CAV with a hazard ratio (HR) of 3.25 (95% confidence interval [CI], 1.33-7.93; P = 0.0095). In multivariable Cox proportional hazard models, the covariates associated with graft loss included: C1q+ DSA (HR, 3.2; 95% CI, 1.34-7.86; P < 0.009), pre-HT renal insufficiency (HR, 11.3; 95% CI, 3.71-34.29; P < 0.0001), and pre-HT ventilator support (HR, 3.3; 95% CI, 1.39-7.81; P = 0.007). The DSA strength in MFI correlates with positive C1q-binding activity and hence functional capabilities of DSA. Close monitoring of DSA strength in MFI and function (C1q assay) may be useful for identifying pediatric HT recipient at risk for development of CAV.

369 Therapeutic Plasma Exchange in Small-Bowel Transplant Recipients With Acute Allograft Rejection

Therapeutic plasma exchange (TPE) is used in solid-organ transplant patients to treat antibody-mediated rejection (AMR) by removing donor specific antibodies (DSAs). While ASFA guidelines exist for treating AMR in other solid-organ transplants, no such guidelines exist for small bowel-allograft patients; the benefit of TPE in AMR of small bowel transplant patients has not been studied. Five patients were followed with serial surveillance ileoscopies and allograft biopsies. Tissues were processed using standard protocols (UNMC) for permanent histologic examination. Two H&E stained slides from each biopsy were examined microscopically by gastrointestinal/transplant pathologists for evidence of ACR. To assess for AMR, serial serum DSA levels were obtained via by Luminex LabScreen single antigen bead assay (One Lambda, Canoga Park, CA). Patients were treated with at least five TPE procedures. To determine whether TPE provided benefit, we examined three parameters in the post-apheresis period: DSA presence and strength, histopathologic rejection, and clinical response. All patients developed class I and/or class II DSAs following transplant. No clear change in DSA levels following TPE was seen. Sixty percent of patients (3/5) had histopathologic evidence of acute cellular rejection (ACR) prior to initiation of TPE. Following TPE, biopsies showed no evidence of ACR or did not meet the criteria necessary for evaluation. Management of rejection in small bowel transplant patients, caused by both antibody-mediated and T-cell driven processes, is extremely complex. We examined the impact of performing TPE in patients with histologic and/or clinical evidence of acute rejection. A clear trend in the burden or activity of DSAs, histopathologic improvement of allograft biopsies, or clinical improvement following administration of TPE, was not seen. Thus, in our study, TPE does not appear to have a clear benefit in the treatment of AMR and/or ACR in small bowel rejection. Additional studies will be needed to corroborate our results.

HLA donor-specific antibodies in allogeneic hematopoietic stem cell transplantation: challenges and opportunities.

Allogenic hematopoietic stem cell recipients may have preformed antibodies directed against foreign HLA antigens. The use of partially HLA-mismatched allogeneic hematopoietic stem cell donors allows for the possibility of the presence of circulating HLA donor-specific antibodies (DSAs) in the recipient. The presence of DSAs at the time of stem cell infusion increases the risk of primary graft failure. More recently developed technology using solid phase immunoassays (SPIs) with fluorochrome-conjugated beads has greatly improved the ability to detect and classify DSAs. When used in combination with the classic lymphocytotoxic complement-dependent and flow cytometric crossmatch tests, SPIs help provide DSA strength assessment. Parous females frequently harbor DSAs. DSAs tend to be of higher intensity when directed against haploidentical first-degree relatives. DSA assessment requires frequent monitoring as their relative strength can change over time. Although the criteria that constitutes a prohibitive DSA is unknown, desensitization techniques can result in engraftment rates as experienced in fully HLA-matched allogeneic blood or marrow transplantation recipients.

P033 Cryopreserved donor cell crossmatches: Can we believe the results?

Aim In order to facilitate rapid turnaround, exchange programs such as the NKR require preliminary flow cytometric crossmatching (FCXM) using frozen cells. However, we’ve found that questionable FCXM results occur more often with frozen cells compared to results obtained with fresh donor lymphocytes. In this study, we investigated the frequency of such discrepant results. We compared 17 donor/recipient pairs who were crossmatched using both frozen and fresh donor cells. Methods Fresh lymphocytes were isolated using EasySep™ (StemCell Technologies). Frozen cells were thawed following a validated protocol (ASHI Laboratory Manual). All cells were treated with pronase. FCXM were performed using a rapid protocol (Liwski, et al., H.I. 72, 2011). In all cases, a screening FCXM was performed with frozen cells and a final FCXM was performed with fresh donor cells. For final FCXM, one specimen tested in the initial screening FCXM was included. Donor specific antibodies (DSA) were analyzed using multiple solid phase platforms. Results Six out of the 17 FCXM comparisons were discrepant. Among the discrepant FCXM, 4/6 were T & B cell positive using frozen cells and negative using fresh cells; 1/6 had elevated, but within negative range, MCS using frozen cells and clearly negative MCS ( Conclusions The problem with unreliable frozen cell crossmatches is that false positive results can lead to unnecessary donor denial and false negative results can bring about last minute exchange failure. When a recipient clearly has no DSA, a positive FCXM using frozen cells is not typically problematic and most often a repeat FCXM using fresh cells is negative. On the other hand, for patients with DSA, the intent of an exploratory FCXM with frozen cells is to rapidly evaluate whether or not DSA strength is at an acceptable level. However, in order to be clinically informative, it is important that FCXM results using frozen cells correlate with fresh cells. Download : Download high-res image (279KB) Download : Download full-size image

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.

Prevalence and Clinical Impact of Donor-Specific Alloantibody Among Intestinal Transplant Recipients.

Rejection remains the leading cause of allograft loss, and a major barrier to improving long-term outcomes after intestinal transplantation. Our aim is to define the prevalence and investigate the role of donor-specific antibody (DSA) on intestinal graft outcomes. The study includes 109 transplants performed in 95 recipients at a single center. Patients were screened for DSA pretransplant, monitored regularly posttransplant and when clinically indicated using the single-antigen bead Luminex assay. Standard induction immunosuppression was with interleukin-2 receptor antagonists, and antithymocyte globulin in high-risk recipients. Maintenance regimens were tacrolimus-based. Pretransplant DSA was detected in 12 (11%) recipients with 50% continuing to have circulating antibodies posttransplant. An additional 24 (25%) patients developed de novo DSA, and of these, 71% had persistent antibodies. Recipients with preformed DSA demonstrated elevated risks of early graft failure, whereas those with de novo DSA experienced accelerated graft loss once DSA was detected, reaching a 28% failure rate within 2 years. HLA-DQ mismatch is a significant risk factor for de novo DSA emergence, whereas the persistence of antibodies is predicted by DSA strength and specificity. Although inclusion of the liver in the intestinal allograft imparts an immunological advantage against rejection-related graft loss, this protective effect was lost among recipients with persistent DSA. The presence of DSA is associated with inferior graft outcomes among intestinal transplant recipients. An enhanced understanding of the mechanisms by which DSA causes allograft injury, and effective strategies targeting humoral immune reactivity are needed to improve long-term intestinal graft outcomes.