HLA Antibody Monitoring Research Articles

#2806 Role of non-HLA antibodies in pre-transplant evaluation of antibody-mediated damage risk

Abstract Background and Aims Donor-specific HLA antibodies (HLA-DSA) contribute to antibody-mediated rejection (ABMR) after kidney transplantation (KT) and worse graft-survival. Non-HLA antibodies may play a role in the development of ABMR in the presence of HLA-DSA or in the development of microvascular inflammation (MVI) in the absence of HLA-DSA. Considering both antibodies in potential recipients before or after KT could enhance the prediction of post-KT ABMR/MVI. Method Retrospective study of a cohort of 169 KT recipients with: a) pre- and post-KT sera; b) follow-up HLA antibody monitoring; and c) follow-up biopsies. We determined the presence of 60 non-HLA antibodies in sera with a multiplex test: the Single Non-HLA Beads kit (LIFECODES®) on a Luminex® platform. We obtained an absolute number of positive antibodies and a sample MFI ratio sum according to which we stratified results into quartiles. Results Pre-KT, we detected a 15.4% of KT recipients with high HLA sensitization (cPRA >50%), being 8.3% HLA-DSA, and non-HLA antibodies in 85% of KT recipients, with a median of 3 (2-5) antibodies and a median MFI ratio sum of 10.1 (2.9-18.5). During a median follow-up of 60 (18.5-89) months 73 patients developed ABMR/MVI. We observed that a high burden of pre-KT non-HLA antibodies was associated with a higher risk of ABMR/MVI, regardless of HLA sensitization and pre-KT HLA-DSA. Notably, 57% of patients with ABMR/MVI had a high non-HLA burden before KT and 24.6% had a HLA cPRA >50%. Altogether, 68.5% of these patients showed either a high HLA sensitization or a high non-HLA burden before KT (Figure). Conclusion A high non-HLA preTR non-HLA antibody burden detected with a multiplex panel allows identification of patients more likely to develop ABMR/MVI, irrespective of the presence of anti-HLA antibodies.

#2239 Identification of non-HLA antibodies associated with the development of antibody-mediated damage after kidney transplantation

Abstract Background and Aims Non-HLA antibodies may play a role in the development of antibody-mediated rejection (ABMR) in the presence of donor-specific anti-HLA antibodies (HLA-DSA) or microvascular inflammation (MVI) without HLA-DSA in kidney transplantation (KT). The development of multiplex panels allowed the analysis of multiple antibodies simultaneously and new antibodies potentially related to ABMR/MVI have been identified. Method Retrospective study of a cohort of 169 KT recipients with: a) pre- and post-KT sera; b) follow-up HLA antibody monitoring; and c) follow-up biopsies. We determined the presence of 60 non-HLA antibodies in sera using a multiplex test: the Single Non-HLA Beads kit (LIFECODES®) suitable for the SAB assay performed using Luminex® technology. Results Pre-KT, we detected non-HLA antibodies in 85% of the recipients, with a median of 3 (2-5) positive antibodies. During follow-up, 73 patients developed ABMR/MVI. Pre-KT, in a univariate analysis including the presence of HLA-DSA (8.3% in the cohort) we detected 7 antibodies individually associated with ABMR/MVI. In multivariate analysis (Figure) GSTT1, NCL, PLA2R1 and Thyroglobulin antibodies together with HLA DSA were significantly associated with the development of ABMR/MVI, although only GSST1 and Thyroglobulin appear in a relevant number of patients (>10 patients) involved in 32 cases of ABMR and 6 cases of MVI. Post-KT, only the detection of anti-GSTT1 antibodies in sera 1- and 3-years post-TR was associated with ABMR/MVI in 3-year biopsies. Conclusion Pre-KT the detection of antibodies against GSST1 and Thyroglobulin using a multiplex non-HLA antibody detection panel is independently associated with the development of ABMR/MVI during follow-up. Post-KT detection of GSST1 is also associated with ABMR/MVI in 3-year biopsies.

Luminex screening first vs. direct single antigen bead assays: Different strategies for HLA antibody monitoring after kidney transplantation

Main problemLuminex panel and single antigen beads (SAB) are used for screening and DSA specificity determination respectively. The cost of SAB may limit its general use, so some labs perform SAB tests only after positive screening. MethodsWe compared both strategies: 1) SAB only if positive screening with kits from manufacturer A, and 2) direct SAB from manufacturer B, and correlate their sensitivity with histological findings. ResultsWe selected 118 kidney transplant recipients with a normal biopsy (n = 19), histological antibody-mediated damage (ABMR, n = 52) or interstitial fibrosis/tubular atrophy (IFTA, n = 47) following Banff 2015 and 2017 classification.Direct SAB detected DSA in 13 patients missed by screening. Strategy 1 detected DSA in 0% normal, 61.5% ABMR and 8.5% IFTA patients; percentages with strategy 2 were 5.2%, 78.8% and 14.8% (p=0.004). Strategy 2 identified DSA allowing full ABMR diagnosis in 17% cases missed by strategy 1. Thereafter, direct SAB from manufacturer A confirmed DSA in 46% DSA-positive cases with strategy 2 (55.5% ABMR cases). ConclusionsLuminex screening failed to identify clinically relevant HLA antibodies, hampering DSA detection in patients with possible ABMR. Direct SAB testing should be the chosen strategy for post-transplantation monitoring, albeit direct SAB from the two existing manufacturers may diverge in as much as 50% of cases.

Relevance of donor-specific antibody monitoring after kidney transplantation: Findings from the Collaborative Transplant Study and the Heidelberg Transplant Center.

Monitoring of donor-specific HLA antibodies (DSA) has become part of the clinical routine in kidney transplantation. This paper gives a brief overview on data from the Collaborative Transplant Study (CTS) and the Heidelberg Transplant Center on the clinical relevance of post-transplant DSA monitoring in patients undergoing renal transplantation. The obtained findings underline the importance of DSA monitoring in the post-operative course in immunologically high-risk patients and patients with deterioration of graft function. Especially in patients with a pre-activated immune system, a gap in the immunosuppressive therapy appear to lead to persistence, reappearance or de novo occurrence of strong, complement-activating DSA, resulting in severe antibody-mediated rejection (AMR) and, without timely intervention, in AMR-related graft loss.

P111 Analysis of hla antibodies after native nephrectomy in renal allograft recipients

Aim Many centers avoid nephrectomy of chronically rejected kidneys because of fear of sensitization to HLA antibodies. It is not clear if the rise in antibodies is from an overall immune sensitizing event or specific to the removal of the donor organ. We analyzed anti-HLA antibodies following native nephrectomy as a control event to the allograft nephrectomy. Methods Twelve kidney allograft recipients who underwent native nephrectomy for cause had serum samples obtained before the nephrectomy and 1–32 weeks after nephrectomy. All patients remained on clinically indicated immunosuppression. The study was approved as an exempt study by the IRB. The serum samples were tested on a Luminex platform with Single Antigen Class I and Class II beads specific for HLA antibodies. Changes in calculated panel reactive antibody (cPRA) and peak mean fluorescence intensity (MFI) were measured by paired t-test. Results Three of twelve patients (25%) showed an increase in HLA antibodies. One of the sensitized and one of the non-sensitized patients required a blood transfusion during the procedure. Nine patients had no class l or class ll antibody before or after native nephrectomy. The 3 patients who had sensitization to HLA prior to the native nephrectomy all had an increase in antibody post-nephrectomy: pre cPRA = 65 and post cPRA 74, p = 0.02. Two of the 3 sensitized patients showed an increase of MFI in their highest antibody specificity class I MFI (7933 pre and 13733 post) and class II MFI (6200 pre and 8067 post), but this was not significant in this small group. One subject showed increased donor specific antibody, with no significant class I before native nephrectomy, but MFI of A3 of 14800 and B44 of 17600 after native nephrectomy; and class II DR1 MFI of 3300 before (no DR4) and DR1 MFI of 18,200 and DR4 MFI of 15500 after native nephrectomy. There was no incidence of allograft loss. Conclusions HLA antibodies may rise following a native nephrectomy in sensitized patients. Monitoring of HLA antibodies after native nephrectomy may be prudent.

OR52 Ides desensitization therapy: Breaking down antibody barriers to transplantation

Despite increased transplant rates for highly sensitized candidates, inequities for those with CPRA >99.7% remain. This case highlights the efficacy of an endopeptidase (IdeS) to cleave IgG HLA-specific antibodies and complications for donor-specific HLA antibody (DSA) monitoring in the presence of therapeutic agents. DSA was evaluated using flow cytometric crossmatch tests (Flow-XM) and Luminex immunoassays (Lifecodes classes I and II single antigen; Immucor-Lifecodes, Stamford, CT and Single Antigen Beads; One Lambda, Canoga Park, CA). Sera were pretreated using Melon Columns™. A 43 year old male with a CPRA of 99.99% was entered into a clinical trial investigating the efficacy of IdeS to cleave IgG molecules and render them ineffective in eliciting complement activation or antibody-dependent cellular cytotoxicity. A deceased donor was identified to whom the patient had HLA-DP DSA yielding a positive prospective Flow-XM. Within 2 h of IdeS administration, immunoassays and a Flow-XM revealed no detectable DSA. Due to concern for DSA rebound, alternate day DSA monitoring and intense post-transplant immunosuppression were employed. Two single antigen panels confirmed DSA reappearance on POD7 prior to high dose immunoglobulin (IVIG) dosing. Administration of IVIG produced an increase in the negative and positive control beads and reactivity with self HLA-antigens (3400 mean fluorescent intensity, MFI). Utilization of multiple bead assays, Flow-XM tests, and HLA pattern analysis rather than MFI was needed to monitor further increases in DSA that would prompt therapeutic plasmapheresis (TPE). Cumulative DSA test analysis revealed a stabilization of DSA accompanied by declining serum creatinine values with no initiation of TPE. New therapeutics hold promise for removing DSA barriers; however, post transplant monitoring can be challenging due to test interference that accompany these biologics. This case illustrates the use of multiple assays and HLA pattern analysis to guide post transplant care.Download : Download high-res image (270KB)Download : Download full-size image A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; Thermo Fisher Scientific.

Late Steroid Withdrawal Following AB0-Incompatible Renal Transplantation

BackgroundCurrent evidence on steroid withdrawal following AB0-incompatible (AB0i) renal transplantation is low. We compared clinical outcomes of patients who agreed to late steroid withdrawal and patients who remained on steroid treatment. MethodsSteroid withdrawal was carried out in 11 patients at ≥12 months after transplantation (group W). For comparison, we analyzed 19 patients who remained on triple immunosuppression including steroids (group M). Minimum follow-up was 24 months following transplantation and 12 months after steroid withdrawal. ResultsBaseline characteristics, including observation times, were not different between groups W and M. Graft survival was 100% in group W compared with 84% (16/19) in group M (P = .15). In group M, 1 patient experienced graft failure because of suspected antibody-mediated rejection (ABMR) following temporary cessation of mycophenolate treatment after a diagnosis of cryptococcal pneumonia. Two patients died with functioning graft because of sepsis. In group W, we observed 1 episode of ABMR following steroid withdrawal. At the end of follow-up, estimated glomerular filtration rates (eGFR) were 54 (19–91) versus 60 (15–85) mL/min/1.73 m2 in group W versus M, respectively (P = .67). ConclusionsLate steroid withdrawal following AB0i transplantation is feasible at a moderate risk of rejection. We recommend close monitoring of renal function and HLA antibodies during and after steroid withdrawal. On the other hand, the occurrence of severe infections causing death and graft loss in patients on triple maintenance immunosuppression including steroids should remind us to consider the overall immunosuppressive burden.

P180 Moving onto plan C: Finding detours when DSA creates a roadblock to bone marrow transplantation

Aim This case study illustrates potential avenues to consider when identifying hematopoietic stem cell donor options for HLA sensitized patients. Methods HLA typing was performed using reverse sequence specific oligonucleotide probe hybridization assays (One Lambda, Inc.) and Sanger sequence base typing assays (local and commercial kits). HLA-specific antibody was assessed using class I and class II phenotypes (Lifecodes®, Immucor) and single antigen panels (One Lambda Inc.). Results The presence of donor specific antibody (DSA) can adversely impact the likelihood of bone marrow engraftment depending on the specificity and strength of the DSA. DSA assessment and monitoring is becoming crucial as centers are utilizing HLA mismatched donors. In the last 5 years, 60–70% of the overall allogeneic bone marrow transplants at our center utilized HLA mismatched donors. In a majority of our sensitized patients, the availability of multiple eligible donors allowed for either the avoidance of DSA or the minimization of DSA to levels that did not warrant desensitization. But, in 6% of the cases evaluated in 2016, DSA caused roadblocks requiring alternate pathways. The case of a 41 year old female with Acute Lymphocytic Leukemia illustrates the “Moving onto Plan C” mantra. Plan A: A related haploidentical transplant was diverted due to crossmatch positive DSA against the patient’s four eligible donors. Plan B: An unrelated matched transplant was deterred due to DRB3*01 allele specific DSA against several 10/10 HLA loci matched donors with the DRB1*13:01/DRB3*01 association instead of patient’s DRB1*13:01/DRB3*02 association. Plan C: An unrelated mismatched donor search was initiated to identify 9/10 HLA loci matched donors with a DRB1 mismatch likely to avoid the patient’s broad specificity and eliminate the potential of a DRB3*01 typed donor. Conclusion Even though there is a low incidence of patients requiring an alternate route due to DSA, the impact of the DSA is life-threatening to the individuals in this cohort. With the increase of HLA mismatched transplants, there is a greater need for initial HLA antibody assessments, continual HLA antibody monitoring, alternative back-up plans and creative solutions in order to offer all patients the best chance of survival.

P085 Lung re-transplant with repeated mismatch HLA antigens

Avoidance of previous donor mismatched HLA antigens is often considered for lung re-transplant due to concerns of potential HLA sensitization and memory responses. However, this approach may result in the exclusion of a large number of potential donors from consideration. Recently, we successfully performed a re-transplant with a history of multiple repeated mismatches. A 30+ year old male with a past medical history of cystic fibrosis underwent two previous bilateral lung transplants in August 2004 and in November 2010. Both allografts failed due to CLAD/BOS that developed after respiratory viral infections. The patient also subsequently developed detectable class I and II HLA antibodies with a cPRA > 88% in May 2016. Among these antibodies, strong anti-A1, B7 and DQ2 were first donor HLA specific. The second donor shared an A3-B18-DR4 haplotype with the first donor but not the recipient. No HLA antibodies specific to this A3-B18-DR4 haplotype antigens or to any other mismatched HLA antigens of the second donor were detected. A third lung transplant became urgently necessary in October 2016. Adding his previous donors’ mismatched HLA antigens to the unacceptable HLA list resulted in a cPRA > 96%. Therefore, a personalized listing strategy was developed based on discussions between the HLA lab and the transplant program. A deceased donor became available in November 2016. The virtual crossmatch revealed that the donor had no unacceptable HLA antigens based on HLA antibody detection. However, this donor carried the A3-B18-DR4 haplotype which was a repeated mismatch haplotype of the previous two donors. Based on a risk and benefit assessment, this donor was accepted as the source of lungs for the third transplant. The patient received 5 plasmapheresis sessions post-transplant to prevent antibody mediated rejection. Frequent post-transplant HLA antibody monitoring has not detected development of donor specific antibodies to the third donor and the new allograft functions well. Lung re-transplant can be performed with a cross of a repeated HLA mismatch provided that the recipient has no detectable HLA antibodies specific to the current donor. Our preliminary findings obtained from our study conducted on UNOS lung transplant data supports this conclusion. An assessment of risk and benefit of crossing the repeated mismatch HLA antigens should be done on a case-by-case basis.

Pretransplantation monitoring of HLA antibodies by complement dependent cytotoxicity and Luminex-based assays

The introduction of more sensitive techniques, such as Luminex® for HLA antibody screening of patients awaiting organ transplantation has resulted in a better understanding of transplantation immunology and improvements in clinical practice. The interpretation of the results obtained only by Luminex® can lead to inaccurate evaluation of a patient's antibody status and unjustified rejection of a potential organ donor. The aim of this study was to demonstrate the benefits of performing HLA antibody screening in the sera of patients on the waiting list for organ transplantation by two different assays, complement dependent cytotoxicity (CDC) and Luminex®. A retrospective analysis was performed on 563 pretransplant serum samples from 141 patients on the kidney transplantation waiting list in Rijeka, tested from March 2012 until March 2015. All samples were tested in parallel by the CDC assay and the Luminex®-based assay. Out of the 563 samples tested 302 (53.7%) tested negative for HLA antibodies and 88 (15.6%) positive by both assays. From 173 (30.7%) samples with discordant results 149 (26.5%) were CDC negative and Luminex® positive, while 24 (4.3%) were CDC positive and Luminex® negative. Among the Luminex positive patients seven did not experience any immunizing events. Evaluation of the HLA antibody status in patients on a waiting list for organ transplantation should be based on the results of the both CDC and Luminex® (or other sensitive) assays in accordance to information about patient's clinical status and exposure to immunizing events.

Value of Donor-Specific Anti-HLA Antibody Monitoring and Characterization for Risk Stratification of Kidney Allograft Loss.

The diagnosis system for allograft loss lacks accurate individual risk stratification on the basis of donor-specific anti-HLA antibody (anti-HLA DSA) characterization. We investigated whether systematic monitoring of DSA with extensive characterization increases performance in predicting kidney allograft loss. This prospective study included 851 kidney recipients transplanted between 2008 and 2010 who were systematically screened for DSA at transplant, 1 and 2 years post-transplant, and the time of post-transplant clinical events. We assessed DSA characteristics and performed systematic allograft biopsies at the time of post-transplant serum evaluation. At transplant, 110 (12.9%) patients had DSAs; post-transplant screening identified 186 (21.9%) DSA-positive patients. Post-transplant DSA monitoring improved the prediction of allograft loss when added to a model that included traditional determinants of allograft loss (increase in c statistic from 0.67; 95% confidence interval [95% CI], 0.62 to 0.73 to 0.72; 95% CI, 0.67 to 0.77). Addition of DSA IgG3 positivity or C1q binding capacity increased discrimination performance of the traditional model at transplant and post-transplant. Compared with DSA mean fluorescence intensity, DSA IgG3 positivity and C1q binding capacity adequately reclassified patients at lower or higher risk for allograft loss at transplant (category-free net reclassification index, 1.30; 95% CI, 0.94 to 1.67; P<0.001 and 0.93; 95% CI, 0.49 to 1.36; P<0.001, respectively) and post-transplant (category-free net reclassification index, 1.33; 95% CI, 1.03 to 1.62; P<0.001 and 0.95; 95% CI, 0.62 to 1.28; P<0.001, respectively). Thus, pre- and post-transplant DSA monitoring and characterization may improve individual risk stratification for kidney allograft loss.

De-novo donor-specific antibody (DSA) against HLA-DQ antigens resulting in acute antibody mediated rejection in a renal transplant patient

Abstract A highly sensitive single antigen bead assay (SAB) by luminex is a very useful tool for post-transplant monitoring of HLA antibodies for diagnosing antibody-mediated rejection ABMR. Donor-specific anti-HLA antibodies that arise de-novo (de-novo DSA) after transplantation, indicate the highest risk for acute antibody-mediated rejection (AMR) and graft loss. The return of pre-existing antibodies after transplantation or the development of new de-novo anti-HLA or non-HLA DSA may also confer an increased risk for graft loss. A case with de-novo donor-specific antibody (DSA) against HLA-DQ antigens resulting in acute antibody-mediated rejection (ABMR) in a renal transplant patient is presented along with review of literature.

Importance of routine comparisons between Luminex instruments within a single laboratory

Aim The development of solid phase single antigen bead (SAB) assays for HLA testing has allowed for better identification and monitoring of HLA antibodies. However, standardization of testing and interpretation has not occurred and several variables can contribute to variation within and between laboratories. Instrument standardization, when multiple machines are used in the same laboratory, is of critical importance especially when following patients longitudinally pre- or post-transplant. The purpose of this report is to highlight the value of routine instrument comparisons. Methods To compare fluorescence MFI values between Luminex instruments, 6 informative samples were tested using Class I SABs (One Lambda, Inc). Testing was performed in one tray and immediately prior to running; samples were split to two trays. This allowed the same test sample to be run simultaneously on 2 different machines. Data were collected and differences in MFI values for the same bead were compared between instruments. Results Initial comparison of individual bead MFI values between Lum-1 and Lum-2 showed significant variance of up to 20% (acceptable value 10%)(Fig. 1A). Subsequently, service was performed on both instruments to align, standardize and calibrate. Lum-1 was found to have a failing laser even though it passed daily QC. Testing was then repeated. Post instrument service now showed a marked decrease in the variance between the 2 instruments ( 6%).(Fig. 1B) Conclusion Our data clearly show the value of performing sample comparisons between Luminex instruments within the same laboratory. Luminex controls do not assure instrument comparability. Instruments not meeting predetermined standards should be serviced and acceptable values reestablished. Data should be collected on a semi-annual basis to reconfirm that the results between instruments are comparable. This practice ensures that MFI values are similar between instruments and allows labs to confidently evaluate and compare MFI values from multiple instruments. Download : Download full-size image

How do we make the best choice in assays used to monitor HLA antibodies in renal recipients awaiting transplant? A comparison of flowpra beads with luminex single-antigen beads

Introduction Solid-phase HLA antibody assays are sensitive and specific to identify antigen and allele-level specificities. Protocol changes to test frequency and the addition of FlowPRA bead (FPB) screening to monitor HLA antibodies in renal patients required post-implementation monitoring. This study correlated Luminex single-antigen bead (SAB) and FPB test results to monitor the effectiveness of these changes. Methods SAB for antibody ID and FPB for screening are performed quarterly on actively-waitlisted renal recipients. This study evaluated agreement based on current SAB positive cutoff MFI values: 3000 for HLA-A and -B, 5000 for HLA-C and 1000 for all Class II specificities. Class II results with cutoff ⩾3000 were included to gauge sensitivity. The current FPB positive cutoff is ⩾5% over the negative control. Results 615 serum samples from 261 patients tested within a 15-month window after FPB implementation were evaluated for agreement by patient number and number of samples tested. Patient Numbers : SAB and FPB Class I results did not agree in 84 patients (32.2%); 63.1% of patients had false positive (FP) and 36.9% had false negative (FN) results. With a MFI ⩾1000 cutoff, Class II results did not agree in 119 patients (45.6%); 0.8% of patients had FP and 99.2% had FN results. With a MFI ⩾3000, Class II results did not agree in 49 patients (18.8%); 18.4% of patients had FP and 81.6% had FN results. Sample Numbers : SAB and FPB Class I results did not agree in 162 serum samples (26.3%); 60.5% of samples had FP and 39.5% had FN results. With a MFI ⩾1000 cutoff, Class II results did not agree in 312 samples (50.7%); 11.9% of samples had FP and 88.1% had FN results. With a MFI ⩾ 3000 cutoff, Class II results did not agree in 128 samples (20.8%); 33.6% of samples had FP and 66.4% had FN results. Conclusion HLA antibody monitoring is important to anticipate donor-specific reactivity in renal transplant recipients. If ID and screening assays are used to monitor these levels, an acceptable degree of correlation between methods is important. The FPB assay is not as sensitive or specific as SAB, especially in detecting weak, clinically-significant specificities. This study identified weakness in our protocol, leading us to evaluate other HLA antibody assay options.

Comparison of the cost associated with an immune monitoring program to treatment of rejection

Following transplantation, patients often receive a standardized regimen of maintenance immunosuppression. Biomarker monitoring could help to individualize immune therapy, giving adequate immunosuppression to high risk patients while possibly avoiding toxicity and infection in low risk patients. Furthermore, early detection and immunosuppression dosage changes before clinical dysfunction maybe a cost effective strategy to minimize rejection episodes. The aim of our study was to define the cost associated with an anti-HLA antibody monitoring program over 5 years compared to treating patients for a single acute rejection episode. We hypothesize having a donor specific HLA antibody monitoring program would help to define early rejection before clinical symptoms arise and therefore be a cost benefit. Our study included 23 heart transplant patients diagnosed with either acute cellular rejection (ACR), antibody mediate rejection (AMR) or both. We calculated the average cost associated with the rejection episode including hospital days and treatment for an ACR, AMR or both. For comparison we calculated the cost for an donor specific anti-HLA antibody monitoring program using Flow PRA, HLA antibody specificities and specificity titers using One Lambda Reagents over a 5 year follow-up period. We found the average cost to treat AMR was 200 times greater than the cost for ACR. ACR rejections were treated with steroids, whereas AMR required multiple rounds of plasmapheresis, IVIG and Rituxan. The cost for an HLA monitoring program over 5 years including FPRA and HLA antibody specificities was comparable to a round of plasmapheresis or a single endomyocardial biopsy and appears to offer patients at risk for AMR due to strong sensitization history a cost effective early detection method upon which immunosuppression could be adjusted. The data suggest that early prediction of acute rejection may allow for optimization of therapy prior to major graft damage and make earlier cost effective preventative interventions possible. As a cost effective strategy we propose performing dose adjustments of maintenance immunosuppression after detection of DSA, preventing the need for later costly rejection treatment.

The Clinical Spectrum of De Novo Donor-Specific Antibodies in Pediatric Renal Transplant Recipients

The development of donor-specific HLA antibodies (DSA) is associated with worse renal allograft survival in adult patients. This study assessed the natural history of de novo DSA, and its impact on renal function in pediatric renal transplant recipients (RTR). HLA antibodies were measured prospectively using single-antigen-bead assays at 1, 3, 6 and 12 months posttransplant followed by 12-monthly intervals and during episodes of allograft dysfunction. Of 215 patients with HLA antibody monitoring, 75 (35%) developed DSA at median of 0.25 years posttransplant with a high prevalence of Class II (70%) and HLA-DQ (45%) DSA. DSA resolved in 35 (47%) patients and was associated with earlier detection (median, inter-quartile range 0.14, 0.09-0.33 vs. 0.84, 0.15-2.37 years) and lower mean fluorescence intensity (MFI) (2658, 1573-3819 vs. 7820, 5166-11 990). Overall, DSA positive patients had more rapid GFR decline with a 50% reduction in GFR at mean 5.3 (CI: 4.7-5.8) years versus 6.1 (5.7-6.4) years in DSA negative patients (p = 0.02). GFR decreased by a magnitude of 1 mL/min/1.73 m(2) per log10 increase in Class II DSA MFI (p < 0.01). Using Cox regression, independent factors predicting poorer renal allograft outcome were older age at transplant (hazard ratio 1.1, CI: 1.0-1.2 per year), tubulitis (1.5, 1.3-1.8) and microvasculature injury (2.9, 1.4-5.7). In conclusion, pediatric RTR with de novo DSA and microvasculature injury were at risk of allograft failure.

P126 : A CASE STUDY: ACUTE HUMORAL REJECTION DUE TO ANTI-HLA DP ANTIBODIES IN A RENAL TRANSPLANT RECIPIENT

We report the case of a highly sensitized 37 year old male who suffered an acute humoral rejection due to donor specific anti-HLA DP (DP) following a second kidney transplant. A previous living donor kidney graft failed in 2005 due to noncompliance with immunosuppression and he returned to hemodialysis. Past history included two blood transfusions. On evaluation for retransplant in 2010, his cPRA using microarray single antigen beads (One Lambda) was 100% including anti-DP1 (MFI 4221). On 2/17/14, he received a deceased donor kidney (KDPI 17%). T and B cell crossmatches by CDC-AHG/CDC long incubation and by the rosette method (Life Technologies) were compatible. There were no donor specific antibodies (DSA) to HLA A B Cw DR or DQ. DP typing of deceased donors was not performed. Retrospective DP typing indicated that the donor was DP1. The clinical staff was advised. He received Thymoglobulin induction and was placed on Mycophenolate Mofetil (MMF), prednisone and Tacrolimus. By post op day (POD) 10, his creatinine decreased to 1.6 mg/dL. On POD 18, his creatinine increased to 2.5 mg/dL. Biopsy results showed acute glomerulitis and acute tubular necrosis. C4d staining was positive. A biopsy day serum showed anti-DP1 (MFI 7627) by microarray single antigen beads and (MFI 26038) using the C1q binding assay (One Lambda). C1q testing of archived crossmatch serum did not show anti-DP1. After treatment with plasmapheresis and 1 g/kg IVIg, his creatinine stabilized at 1.8–2 mg/dL. He is currently maintained on MMF, Tacrolimus and prednisone. A survey of 1069 renal transplant candidates revealed that 13% had DP antibodies. Two of three transplanted patients with DP antibodies retrospectively determined to be DSA had humoral rejections. Another patient with no pre-transplant DSA received a crossmatch compatible kidney and had a rejection due to de novo anti-DP. These 3 cases were regrafts. Our experience is consistent with recent literature. With increasing evidence that DP antibodies can cause graft rejection and with technology available to perform HLA DP typing and antibody monitoring, the transplant community should take action to recognize the potential risk of DP antibodies in renal transplant, require DP typing of all donors prior to transplant and consider incompatible donor DP antigens unacceptable for regrafts.

De Novo Donor-Specific HLA Antibodies: Biomarkers of Pancreas Transplant Failure

This study assesses the role of posttransplant HLA antibody monitoring in the surveillance of pancreas transplant recipients. Four hundred thirty-three pancreas transplants were performed at the Oxford Transplant Centre 2006-2011 (317 simultaneous pancreas kidney [SPK] and 116 isolated pancreas [IP]). HLA antibody monitoring was performed at 0, 6 and 12 months and annually and during clinical events. There was no association between pancreas graft failure and recipient or donor characteristics. Posttransplant antibody status, available for 354 (81.8%) of recipients, demonstrated that 141 (39.8%) developed de novo HLA antibodies, of which 52 (36.9%) were de novo donor-specific HLA antibodies (DSA) (34 SPK, 18 IP). The development of antibodies to donor HLA, but not to nondonor HLA, was significantly associated with poorer graft outcomes, with 1- and 3-year graft survival inferior in SPK recipients (85.2% vs. 93.5%; 71.8% vs. 90.3%, respectively; log-rank p = 0.002), and particularly in IP recipients (50.0% vs. 82.9%; 16.7 vs. 79.4%, respectively; log-rank p = 0.001). In a multivariate analysis, development of de novo DSA emerged as a strong independent predictor of pancreas graft failure (hazard ratio 4.66, p < 0.001). This is the largest study to examine de novo HLA antibodies following pancreas transplantation and clearly defines a high-risk group in need of specific intervention.

67-P: ROUTINE HLA ANTIBODY MONITORING OF RENAL ALLOGRAFT RECIPIENTS FOR DEVELOPMENT OF DONOR SPECIFIC ANTIBODIES

Aim Recently our renal transplant (Tx) program has implemented routine HLA antibody monitoring of allograft recipients. We determined the incidence of donor specific antibody (DSA) in the 6 months – 1 year post-Tx period. Methods 155 renal allograft recipients (Tx in 2011-2012) were tested for HLA antibody prior to Tx and 6 – 12 months post-Tx. Pre and post-Tx monitoring of antibodies was determined using flow cytometry screening beads and a Luminex-based solid-phase class I and II single antigen bead assay with a positive cutoff of 1000 MFI. Crossmatches (XM) were performed using pronase treated cells and flow cytometry. Correlations between DSA development and serum creatinine were analyzed. Results Overall, 67 of 155 renal Tx recipients were sensitized at time of Tx of which 41 were tested for DSA post-transplant. Of 88 non-sensitized patients, 50 were tested post-Tx. 5 of 41 sensitized patients developed de novo DSA compared to 4 of 50 patients in the non-sensitized group. Of the 9 patients that developed DSA, 7 had DSA to HLA-II antigens while 2 had antibodies against HLA-I antigens. In patients with DSA, serum creatinine level ranged from 1.39 – 5.87. However, no correlation was observed between DSA MFI and serum creatinine level. Conclusions Institution of routine testing for DSA following renal Tx has allowed identification of patients with HLA DSA that may not have been tested in an indication based scheme. A higher percentage of sensitized patients received routine monitoring than non-sensitized patients. More monitoring in sensitized patients is consistent with sensitized patients increased susceptibility to develop DSA. Development of DSA does not correlate with a positive XM but does correlate with sensitization. Consistent with previous reports, the majority of patients developed DSA to HLA-II antigens. Routine testing for DSA will enable a better understanding of possible connections between development of DSA and clinical outcomes in our cohort.

De Novo HLA Antibody Monitoring in Kidney Transplantation: Who Are to Target?

De Novo HLA Antibody Monitoring in Kidney Transplantation: Who Are to Target?