Positive XM Research Articles

Impact of a positive crossmatch on pediatric heart transplant outcomes

BackgroundPediatric heart transplant (HT) candidates experience high waitlist mortality due to a limited donor pool that is constrained in part by anti-HLA sensitization. We evaluated the impact of CDC and Flow donor-specific crossmatch (XM) results on pediatric HT outcomes. MethodsAll pediatric HT between 1999 and 2019 in the OPTN database were included. Donor-specific XM results were sub-categorized based on CDC and Flow results. Primary outcomes were treated rejection in the first year and time to death or allograft loss. Propensity scores were utilized to adjust for differences in baseline characteristics. ResultsA total of 4695 pediatric HT patients with T-cell XM data were included. After propensity score adjustment, a positive T-cell CDC-XM was associated with two times higher odds of treated rejection (OR 2.29 (1.56,3.37)) and shorter time to death/allograft loss (HR 1.50 (1.19,1.88)) compared to a negative Flow-XM. HT recipients who were Flow-XM positive with negative/unknown CDC-XM did not have higher odds of rejection or shorter time to death/allograft loss. An isolated positive B-cell XM was also not associated with worse outcomes. Over the study period XM testing shifted from CDC- to Flow-based assays. ConclusionsA positive donor-specific T-cell CDC-XM was associated with rejection and death/allograft loss following pediatric HT. This association was not observed with a positive T-cell Flow-XM or B-cell XM result alone. The shift away from performing the CDC-XM may result in loss of important prognostic information unless the clinical relevance of quantitative Flow-XM results on heart transplant outcomes is systematically studied.

A Positive CDC T-cell Crossmatch is Strongly Associated with Allograft Loss and Early Rejection in Pediatric Heart Transplant Recipients

<h3>Purpose</h3> A recent multi-center study (CTOT-04) reported similar first year outcomes in children with CDC T-cell crossmatch (XM) + and XM- heart transplant (HT) but was underpowered and had only 11 children in the CDC+ HT group. We assessed the association of donor specific T-cell CDC and Flow XM HT with the risk of rejection and allograft loss in US pediatric recipients over 20 years. <h3>Methods</h3> We queried the OPTN database for all pediatric HT performed between 1999 and 2019. Donor specific T-cell XM results were categorized as CDC+, Flow+/CDC Unknown, Flow+/CDC-, and Flow-. Covariate adjustment using a propensity score balanced risk factors between the XM+ and XM- groups. <h3>Results</h3> Of 4695 pediatric HT, there were 165 CDC+, 288 Flow+/CDC Unknown, 94 Flow+/CDC-, and 4148 Flow- recipients. The median age at HT was 7 years, 44% female, 55% white, and 44% had a pre-HT diagnosis of CHD. Pre-HT diagnosis, hemodynamic support, and year of heart transplant differed between the XM+ and XM- groups prior to propensity score adjustment. After adjusting for propensity score, children with a CDC+ HT were significantly more likely to lose their allograft or develop rejection requiring treatment in the first post-HT year (<b>Table</b>). Propensity score matching and stratification by propensity score quintile demonstrated similar results. Over the period of the study, there was a significant shift in the type of XM assays performed (1999: 90% of HT with CDC XM, 32% Flow XM; 2019: 28% of HT with CDC XM, 87% Flow XM). <h3>Conclusion</h3> Children receiving a CDC+ XM HT are at higher risk of graft loss and rejection during the first post-transplant year, suggesting higher anti-HLA antibody burden in such recipients. The lack of differences in allograft loss and rejection with a positive Flow XM alone may be due to current treatment strategies. The shift away from performing a CDC XM results in a loss of important prognostic information. Prospective clinical trials of donor acceptance and antibody treatment strategies are needed.

Stability of Anti-HLA Sensitization Profiles in Highly Sensitized Kidney Transplantation Candidates: Toward a Rational Serological Testing Strategy.

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

Causes of Positive Pretransplant Crossmatches in the Absence of Donor-Specific Anti-Human Leukocyte Antigen Antibodies: A Single-Center Experience.

Pretransplant crossmatch (XM) testing is widely used for detecting preformed donor-specific antibodies (DSAs) against human leukocyte antigen (HLA). However, in some cases, there is a positive XM result in the absence of HLA-DSAs, the cause of which was rarely identified. We reviewed the causes of sequential positive XM results at a single center and analyzed the presence of non-HLA antibodies in patients with an unexplained positive pretransplant XM result. Among 251 patients with T-cell/B-cell complement-dependent cytotoxicity (CDC) or flow cytometric crossmatch (FCXM) positivity, HLA-DSAs were confirmed in 88 (35.1%) by a single antigen bead (SAB) assay, 150 (59.8%) used rituximab (anti-CD20), and 13 (5.2%) had neither HLA-DSAs nor a desensitization history. Anti-angiotensin II type 1 receptor IgG and 33 non-HLA antibodies were tested in the 13 patients with an unexplained positive pretransplant XM result, and more than one non-HLA antibody were revealed in all these patients; 11 patients had non-HLA antibodies reported to be associated with graft rejection, and two patients experienced rejection episode after kidney transplantation. Our study suggests considering non-HLA antibodies testing when a CDC or FCXM test is positive without a definite cause. Assessing non-HLA antibodies might be useful for interpreting XM results and evaluating immunologic risk in transplant recipients.

Correlation analysis between virtual and Complement-Dependent-Cytotoxicity crossmatch in a monocenter retrospective series of 118 allografted patients

Purpose of the StudyThe detection of patients' anti-HLA antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) may affect post-transplant outcome, due to a potential detrimental impact on engraftment or toxicity-related issues. Crossmatch (XM) techniques provide support to physicians during the pre-transplant phase but the role of Complement-Dependent Cytotoxicity XM (CDC-XM) is not well-defined when performed routinely and in parallel with the virtual XM. Patients and MethodsWe report here our experience with both virtual and CDC-XM tests on n = 118 patients undergoing search for a donor other than HLA-identical sibling from July 2013 to June 2018 at our Institution. When anti-HLA antibodies (Abs) were present, they were classified as donor-specific Abs (DSA) or non-DSA. ResultsOn the n = 118 patients, n = 35 (29.7 %) had a positive virtual XM test (of which one of more DSA were found in n = 8; 6.8 %) and n = 5 had a positive CDC-XM test. These latter, positive for HLA class II only, were interpreted as false-positive results due to prior administration of anti-CD20 to the patients, all affected by lymphoma; none of them had a positive virtual XM for anti-HLA Abs of class II. Importantly, all these patients successfully engrafted, further supporting the lack of significant impact of CDC-XM positive results in this series. ConclusionsAccording to our data on more than a hundred patients, routinely performed CDC-XM does not seem to add significant information with respect to virtual XM. We cannot exclude the usefulness of CDC-XM in specific situations, although a positive CDC-XM result was an unfrequent event.

High Immunologic Risk Lung Transplantation Using Risk Stratified Induction

<h3>Purpose</h3> Donor specific antibodies (DSA) are associated with antibody-mediated rejection, chronic lung allograft dysfunction (CLAD) and increased mortality, making lung transplant in the presence of DSA high risk. Our center transplants these patients using risk-stratified induction. We sought was to assess survival and time to CLAD onset in these patients compared to low risk recipients. <h3>Methods</h3> We reviewed lung transplant recipients transplanted from January 2010 to December 2017. Recipients were assigned to three risk categories: low [no DSA, negative crossmatch (XM)], moderate [positive DSA, negative XM] and high [positive DSA, positive XM]. Patients with DSA were treated with anti-thymocyte globulin and intravenous immune globulin, with five cycles of pheresis for positive XM. Low risk patients received basiliximab. We used Cox regression to test the association between risk category and time to death or retransplant as well as time to CLAD onset, and logistic regression to test the relationship between risk category and severe primary graft dysfunction (PGD). <h3>Results</h3> 313 patients met inclusion criteria and 76 died during follow up. 256 (81%) were low risk, 27 (9%) moderate and 30 (10%) high. Patients with pre-transplant DSA were younger and more likely to be bridged to transplant. There was no effect of risk status on survival either unadjusted (Figure 1, p=0.83) or adjusted for age, diagnosis, bridging status and transplant type (p=0.65). There was no relationship to time to CLAD onset (p=0.61). Patients with DSA were at increased risk of severe PGD, both unadjusted (p=0.002) and adjusted for age, diagnosis and bridging status (p=0.01), mainly high vs. low risk (adjusted odds ratio 0.29, [95% CI 0.12-0.70]). <h3>Conclusion</h3> Using a risk stratified induction strategy, DSA at time of transplant was not associated with increased risk of death or CLAD onset in lung transplant recipients, suggesting this is a safe way to improve access for sensitized recipients. The novel PGD association warrants further investigation.

Effect of simultaneous presence of anti-blood group A/B and -HLA antibodies on clinical outcomes in kidney transplantation across positive crossmatch: a nationwide cohort study

ABO-incompatible (ABOi) and positive crossmatch (XM) kidney transplantation (KT) have been considered immunologically challenging. The present study analyzed the clinical outcomes in XM positive KT based on ABO incompatibility. We used data from the Korea Organ Transplantation Registry, a nationwide database, and a single-center registry. A total of 263 patients with positive XM were divided into an ABO compatible (ABOc) & XM positive (ABOc/XM+, n = 176) group and an ABOi & XM positive (ABOi/XM+, n = 87) group. The overall rejection rate one year after KT was significantly higher in the ABOi/XM+ group than in the ABOc/XM+ group (P < 0.01). A total of four mortalities occurred, all in the ABOi/XM+ patients (P < 0.01). There were no differences in surgical complications or the occurrence of infection-related complications, including BK virus nephropathy. Multivariate analysis indicated that female vs. male (odds ratio (OR), 2.27; P = 0.03), DSA class I (MFI/1000) (OR, 1.10; P = 0.03), DSA class II (MFI/1000) (OR, 1.10; P < 0.01), and ABOi & XM+ status (OR, 2.38; P < 0.01) were significant risk factors for acute rejection during the year after transplantation. Overall graft survival was inferior in ABOi/XM+ patients than in ABOc/XM+ patients (P = 0.02). ABO incompatibility in XM-positive KT patients was found to be a significant risk factor for the development of rejection within one year after transplantation as well as for long-term graft survival. The anti-blood group A, B and anti-HLA antibodies may show synergistic activity.

OR17 Epitope load does not predict development of de novo antibodies to HLA in patients with BK virus nephropathy

Aim Emerging data suggest that DSA development is a frequent complication post BKV replication, and most DSA are directed against donor HLA-DQ. In the current investigation, we quantified DQ epitope mismatches between the recipient and donor in individuals with BKVN diagnosis and investigated the association between DQ epitope load and development of DSA. Methods In this pilot study, using the most probable 4-digit HLA typings, we investigated epitope mismatches in individuals who developed DSA post BKVN diagnosis compared to those who did not and correlated them to DSA development and graft dysfunction defined as an increase in serum creatinine by > 0.5 mg/dL at 12 months post-BKVN diagnosis. We had SSOP based intermediate HLA typing available for recipients and donors in 13 recipients who developed BKVN confirmed by renal allograft biopsy. Of the 13 recipients, 2 were excluded: 1 for early graft loss and 1 who had zero DQ mismatches. None of the 11 recipients with BKVN diagnosis had a positive XM or DSA at time of transplantation. Results Time from kidney transplantation to BKVN diagnosis was 8.5 ± 5.1 months. Serum creatinine at time of diagnosis was 1.78 ± 0.56. Of the 11 with BKVN, 36% developed de novo DSA. All DSAs were directed against DQ mismatches. Time from BKVN diagnosis to DSA development was 5.5 ± 2.3 months. Table below lists the epitope mismatch number at each locus. Of the 4 patients with de novo DSA, 75% experienced graft dysfunction during the 12 months post BKVN and of the 7 patients without de novo DSA, 29% experienced graft dysfunction by 12 months. Our data demonstrated that the most frequent DQ epitope mismatches in those with de novo DSA were 52PL3, 45EV, 52PQ2, and 52PR. Conclusions In our pilot study, the epitope load was not different between BKVN patients who developed or did not develop de novo DSA. The most frequent target of de novo antibodies were DQ locus mismatched epitopes. Knowledge of epitope targets of de novo DSA in patients with BKVN may facilitate targeted therapies and improve kidney allograft outcomes. Download : Download high-res image (148KB) Download : Download full-size image

P026Timing is everything – Even for DP antibodies

Introduction Donor Specific Antibodies (DSA) can be a contraindication for kidney transplants. The clinical relevance of DSA to DP antigens and correlation with crossmatch (XM) remain unclear. We report the conversion of a negative XM to positive due to development of DSA to DP6, most likely caused by an unreported sensitizing event. Methods A 38 yo female received a deceased donor (DD) kidney transplant (Tx) after negative T/B cell flow XMs using sera drawn 35 and 44 days pre Tx (day −35 and −44). Delta channel shifts (DCS) in these XMs were: T = 0 for both sera; B = 37 and 31 for days −35 and −44. These sera were negative for DSA. We received a serum drawn at admission for Tx (day −1). The patient received blood transfusions within the previous week (day −6). The day-1 serum had high level DSA to DP6 (MFI = 13,723), compared to day −44 (1,321) and day-35 (1,653). By flow XM, the day −1 serum was T negative, B positive (DCS = 228), likely due to the increase in DSA to DP6. In addition to standard immunosuppression, this recipient also received thymoglobulin and basiliximab once the program was notified of high level DSA and positive flow XM. Post Tx monitoring showed an immediate decline in DSA to DP6: post Tx day 6 (MFI = 7,380), day 12 (5,669), day 21 (4,042), day 63 (485). The DSA to DP6 remained low (MFI 400) for five years. However, the recipient had BK viremia and a kidney biopsy five years post Tx showed “arteriolar hyalinosis and arteriosclerosis related to possible CNI toxicity”. The recipient is re-listed in UNET for another kidney Tx, the primary reason for graft failure deemed transplant glomerulopathy. Discussion This case emphasizes the importance of timely antibody testing in relation to XM testing, awareness of sensitizing events, and updated HLA typing. Our lab now employs stricter guidelines to ensure more frequent receipt of patient sera, especially following potential sensitizing events. At the time of this patient’s Tx, DP typing was not performed on DD. After discovering the increase in antibodies at pre Tx day −1, but absence of DSA, we performed DP typing. Finding DP6 as a DD antigen helped explain the change to positive XM, as the level of DSA to DP6 significantly increased following the sensitizing event pre-Tx. This early peri and post Tx antibody response may have contributed to reduced graft survival and transplant glomerulopathy.

P134 Antibodies to inter-locus shared DR11 and DP epitope: Characterization and analysis consideration

Aim Antibodies (AB) to shared HLA epitopes can result in a single sensitizing event conferring incompatibility with a large percentage of donors. In addition, apparent AB strength on Luminex single antigen bead (SAB) assays can be artificially lowered, impacting virtual crossmatch (XM) assessment. ”DEE” and ”DED” epitopes at position 55–57 on DPB1 are known to be immunogenic. Our goal was to further characterize these AB in our patient population, with an interest in the ”DEE” epitope present at position 57–59 on DRB1. Methods Patients with DP AB to either DPB1∗04:02 (DEE) or DP14 (DED) were queried, and SAB results reviewed for reactivity to known immunogenic DP epitopes (DED - DP3, 6, 9, 14, 17, 20; DEE - DP2, 04:02, 10, 18, 28). AB to DR antigens were also noted, as well as patient HLA typing. To confirm AB cross-reactivity to shared sequence, a serum with AB to multiple DP epitopes (DPA1∗02/04, DED/DEE, and DEAV) was adsorbed using lymphocytes containing just the DEE epitope. Results Patients who lacked DED and DEE on their cells formed an AB of combined specificity: 35 to DED/DEE, and another 25 to DED/DEE plus other DP antigens. Of 8 patients with isolated DEE DP AB, typing identified DED epitope in 5 (no typing available for 2); of 7 patients with isolated DED DP AB, typing identified DEE epitope in 3 (no typing available for remaining 4). DR11 AB were co-present in 60 of 68 patients containing DEE DP AB (with or without other specificities); of the patients without DR11 AB for whom typing was available, all 6 typed as DR11. None of the 7 patients with isolated DED AB had DR11 AB, only 2 of whom typed as DR11. Eluate from the adsorption study reacted exclusively with DED and DEE DP antigens as well as DR11. Conclusions DED and DEE are immunogenic epitopes on HLA-DP, although specificity can be to either or both depending on self-antigens. DEE and combined DED/DEE antibodies (but not DED) cross-react with DR11 due to a shared epitope. However, if DR11 is a self-antigen it’s possible to form DP-exclusive DEE AB, confirming that DR-specific sequences outside the ”DEE” epitope are important. AB to shared epitopes are important to recognize because they can cause positive flow XM despite low MFI on SAB.

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

P136 Successful adeptness of donor exchange program in renal transplantation at King Faisal Specialist Hospital Jeddah

Aim Different methodologies were established to overcome HLA immunological barriers to match the highly increasing number of patients with end-stage renal disease in Saudi Arabia, which is currently ranged between 120 and 150 patients per million per annum. At our hospital, King Faisal Specialist Hospital Jeddah, we therefore successfully established the desensitization and ABOi renal transplantation. Recently we have started to use a donor exchange program. We hereby report about our experience to overcome the immunological incompatibilities by using this program. Methods We used an IT program with high grade on flexibilities by performing different options including simple two-pair exchanges, more complicated domino exchanges and chain donations. All recipients with willing donors and with high titer HLA antibodies and/or strong positive cross match were included in the program. HLA typing was performed by using One Lambda SSOr and SSP. HLA Antibody Identification, Single Antigen Class I/II and C1q test, was performed by One Lambda. T and B cell IgG XM is performed by FACSCanto II flow cytometer, where the cut-off for positive XM is determined based on normal human studies. Results 20 patients were transplanted successfully by using the donor exchange program within a period of 12 months. Considering a total number of 220 transplanted kidneys during this time, which makes our hospital as the largest center for renal transplantation in Saudi Arabia, we were able to increase the number of transplanted organs significantly by 9% and we continue to extend and optimize our histocompatibility services at the hospital. Conclusions Our experience demonstrates the high efficiency of the donor exchange program in overcoming immunological barriers in renal transplantation. This program provides in some cases also an alternative to the invasive and costly desensitization protocols. Further efforts and thoughts are going on to include deseased donors as well in this successful donor exchange program.

The Clinical Significance of Pretransplant C1q Assay Status in Positive Crossmatch Kidney Transplantation

Background Patients with a pretransplant donor-specific antibodies (DSA) are at higher risk for antibody-mediated rejection (AMR). However, not all DSA have the same pathogenetic potential. The novel assay for complement binding capacity may help to stratify the risk for AMR. The aim of this study was to investigate the association between complement binding ability of DSA and clinical outcomes in patients with a positive crossmatch (XM). Methods We analyzed 59 patients with a positive XM who underwent living donor kidney transplantation between 2011 and 2016 with desensitization therapy (12 complement-dependent cytotoxic [CDC] XM and 47 flow cytometric XM). We analyzed clinical outcomes according to XM and C1q status.Results Biopsy-proven AMR developed in 18 patients (30.5%). CDC XM patients suffered more AMR than flow cytometric XM patients (P=0.004). Although the incidence of AMR was higher in C1q-positive DSA than in C1q-negative DSA, the difference was not statistically significant (P=0.067). The XM techniques and C1q positivity did no correlated with graft loss. C1q positivity was associated with higher IgG titer (11181 ± 3997 vs. 4823 ± 6132, P=0.002).Conclusion C1q-positive DSA in patients with positive crossmatch was unable to predict AMR, but CDC XM compared to flow cytometric XM did. The C1q binding activity in patients with positive XM largely reflects differences in antibody strength.

P112 Impact of allele-specific anti-HLA class I antibodies on organ allocation

Aim Accurate classification of serologically-distinct antigens (Ag) is critical in organ allocation, and nowadays it is possible to define anti-HLA antibody specificity to the allelic level. We examined class I antibodies (Ab) for allelic specificities that may be serologically distinct and evaluated its impact on crossmatch (XM) results and organ allocation. Methods 6726 sera were tested for anti-HLA Ab using LABScreen Single Ag assay (One Lambda), in which 17 class I HLA Ag are represented by more than one allele. Discordance in MFI values between the two allelic beads was examined as a categorical variable to mimic clinical practice, and as a continuous variable (MFI value) to better reflect biological plausibility. To evaluate the significance of an allele-specific Ab on XM results, we examined an unbiased cohort of deceased donor-candidate testing (125,828 CDC XMs between 2014 and 2017). In our lab, all candidates within a blood group have a CDC XM performed, regardless of whether they have donor-specific antibody (DSA). This uniquely allows us to examine whether a candidate with an allele-specific DSA, who has already been excluded from the match run, would indeed have a positive XM against a donor who has a different allele. Statistical analyses were performed using JMP12Pro (SAS Institute). Results 1. The incidence of discordance between the two allelic beads varied depending on whether MFI values were considered as continuous vs categorical variables. 2. The presence of allele-specific Ab for rare alleles was out of proportion to their frequencies in the population. 3. The presence of Ab against only the rare allele was a poor predictor of a positive CDC XM, with a positive predictive value of 0–7% compared with 52.5% if the candidate had DSA against an A or B locus Ag. Conclusions 9 out of 17 Class I HLA antigens had potential allele-specific reactivity, meaning that candidates with antibody selectivity against rare alleles may be unnecessarily excluded from many organ offers based on definition of unacceptable antigens by serological equivalence. Conversely, for centers relying on virtual-XM, prematurely defining alleles as new serological splits could unfortunately allow transplants for which the recipient has DSA. Further testing is needed to rigorously confirm these allele-specific reactivity patterns.

P009 Profiling for patients with positive autologous or false positive allogenic flow cytometry cross match

To determine the native disease profile for patients who have positive autologous or false positive allogeneic flow cytometry crosshatches (FCXM). We reviewed Allo and auto FCXM performed in the past 3 years. FCXMs were performed with conventional methods using Ficoll-purified cells (old method). After May 2015, FCXMs were performed with the modified Halifax protocol using RoboSep-purified cells (new method). 530 patients waiting for heart, kidney or any kidney-combined organ transplant were studied. 44 (8.3%) had either positive (n = 35) or borderline positive (n = 9) auto FCXMs. There are significantly more positive auto FCXMs with the old method 25/161 (14.6%) vs. the new method (19/359, 5.35%). Patients whose end stage renal diseases were due to autoimmunity (vasculitis, lupus), IgA nephropathy, or toxicity (calcineurin inhibitor, lithium) had more positive auto FCXMs (RR = 3, P = 0.0003) vs. patients with other diseases. The risk is higher (RR = 4.52) with patients tested using the new method. These 3 diseases are named as disease susceptible for false positive XMs (DisFPX). Analysis of patients waiting for kidney-related transplants with native diseases other than DisFPX and tested with the new FCXM method (n = 280) showed that peritoneal dialysis (PD) gives a higher risk for positive auto XM vs. either hemodialysis or pre-emptive (RR = 3.24, p = 0.04). High PRA (>80%) is a risk for positive auto FCXMs when tested with the old method (RR = 2.54, P = 0.01) but not with the new method (RR = 1.21, P = 0.8). Combining DisFPX and PD can explain 56.1% (23/41) of positive auto XM in all renal patients and 70.1% (12/17) of those tested with the new method. Allogeneic FCXM (n = 750) with deceased donors performed in the same period were reviewed. 30 patients were found to have false positive allogeneic FCXMs (N = 12 with old method, n = 18 for new method). Most patients with false positive allo FCXM have either DisFPX/PD (19/30, 66.3%), or DisFPX/PD/PRA > 80% (25/30, 83%). This is the 1st report that other than well-known autoimmune diseases, patients with IgA nephropathy, nephrotoxicity or those on peritoneal dialysis are also prone to positive auto FCXMs or false positive allo FCXMs. This is helpful when interpreting an unexpected positive FCXM after a negative virtual XM.

P243 Successful renal transplantation in elderly patient with multiple disorders across positive cross match

A 74 old Male patient admitted to the hospital for living related (son) renal transplant with a history of Diabetes mellitus due to underlying condition with diabetic chronic kidney disease, Essential hypertension, and End-stage kidney disease for 1.5 year with high blood pressure for more than 20 years. His laboratory workup results showed multiple viral infections, IgG antibodies in serum for HSV1/2, EBV (VCA) and VZ. Creatinine level was at 1075 μmol/L. Pre-transplant laboratory workup showed the following: HLA typing, performed by one lambda SSO for low/intermediate resolution, for the patient HLA-A∗03, 31, HLA-B∗18, 51, HLA-C12, 15, HLA-DRB1∗11:04, 13:01, HLA-DRB3∗01, 02, DQB1∗03:01, 06:03. Donor typing HLA-A∗02, 03, HLA-B∗14, 18, HLA-C∗08, 12, HLA-DRB1∗11:04, 15:03, HLA-DRB3∗02, DRB5∗01, DQB1∗03:01, 06:03. 6/14 antigens mismatch with his son, DR53 as circulating antibody reported by one lambda single antigen beads with cut off of 1000 MFI, no DSA and Negative virtual crossmatch (XM). His initial Flow XM showed positivity in T and B cell IgG +80 and +79 Median Chanel Shift respectively. XM repeated after three months prior to OR result showed positivity in XM of T and B cell IgG as well as positive auto XM for T and B cells IgG, 50+ and 81 + Median Chanel Shift respectively. The patient was induced with basiliximab prior to transplant and without any desensitization procedure. Post-transplant the kidney works immediately, his Creatinine level dropped fast to 92 μmol/L and no de novo antibodies were detected. Conclusion: Positive T and B cell IgG flow XM should not automatically consider as a conclusive immunological risk for renal transplant, especially in light of virtually negative XM and positive auto XM. That might be caused by non-HLA antibodies binding or previous viral infection, as we assumed in this case.

P187 The challenge of moving to the virtual crossmatch (VXM) for deceased organ donor kidney allocation in Australia

Aim At the forefront of Australia’s kidney allocation protocols is the National Interstate change (NIE) designed primarily to facilitate allocation to recipients that are very well HLA matched with the donor. Currently 976 patients (25% cPRA > 90%) are wait listed for a kidney. NIE still relies heavily on a pre-transplant CDCXM as the final determinant prior to the offer of the organ, shipment of the kidney to the interstate transplant units. To facilitate this system, a process of shipping recipient sera to five state HLA labs occurs each month. This is costly and labour intensive. Methods Australia’s population distribution, geographical isolation, HLA diversity were factors considered in the development of the NIE program over 30 years, however with the increase demands on lab resources, after hours services, organ donors and greater complexity of recipient HLA Ab profile, the current system needs to change. Recipient work up involves defining unacceptable antigens on the basis antibodies with >8000 MFI annual SAB test. In March 2016, the allocation system was changed to include cPRA in the matching algorithm. This change has resulted in an increase of highly sensitised patients being transplanted compared to previous years. Results From 1 st March 2016 to 1 st March 2017, 814 kidneys were donated 56 allocated and transplanted via NIE with 658 transplanted locally. Over 650 recipients were XMed, with only 13 CDC positive XMs which resulted in offers not progressed or declined. These positive CDCXMs were the result of IgG or IgM HLA Abs not detected during the routine screening. Conclusions Although the incidence of positive CDC XM is low, transition to a VXM, we will need to improve patient antibody profiling. Increasing the frequency of recipient screening will improve the definition of unacceptable donor antigens. Assessment of the titre of the HLA antibody not based purely on MFI, is also required in order to have a better understanding of the recipients true level of sensitisation. This is required, in order to gain clinical confidence in accepting a kidney offer without a prospective CDCXM between recipient and donor. Re-evaluation of the current system is required however gaining national acceptance of the change maybe an even larger challenge.

P163 Antibody identification and detection assays {Lab Screen Mixed (LSM), flow PRA screen, and Single Antigen Beads (SAB)}: Which one to believe?

Aim Negative SAB results in the context of positive LSM prevent accurate prediction of virtual crossmatch (XM) in the absence of sensitization history. The issue is particularly problematic in the event of unpredictable positive XM during a deceased donor workup. Unmissed HLA-antibody has to be excluded with certainty by other test. With different assays it may be difficult to choose the best combination. Here we describe the results of correlation between two antibody screening; LAB Screen Mixed using Luminex & FlowPRA Screen on conventional flowcytometry & compare their performance with LAB Screen SAB & XM. Methods 60 samples were tested by the two screening methods according to manufacturer instructions (One Lambda) & tested by SAB for Class I/Class II simultaneously. Sera demonstrated reactivity were crosmatched with surrogate cells bearing the corresponding antigens. Discrepant result was verified by flow specific beads. Results Table 1 summarized the correlation of the results for the three assays. The overall concordance for both screening methods was 72.5% (61.7 for Class I & 83.3 for Class II). In the discrepant results the SAB correlate better with the flow PRA screening. False positive screening was reported only by LSM. Failure of identification of antibody by SAB in presence of positive both screening methods was found in one case (figure1), interestingly the flow PRA of this case was suspicious, DR7 reactivity was confirmed by flow specific bead & weakly positive B-FCXM. In the Four positive cases detected by LSM but not with flow PRA, SAB identify weak antibodies likely to be false positive. Conclusions Flow PRA gives extra evidences about doubtful antibody & provides a rapid risk assessment on the same crossmatch platform.

P002 Patient outcomes following liver-kidney transplantation with donor specific antibodies

Aim Simultaneous liver and kidney transplantation (SLKT) remains as the gold standard for patients with chronic renal and liver diseases. Pre-transplant donor-specific antibody (DSA) to class I antigens have been shown to decrease following SLKT; whereas, DSA to class II antigens may remain detrimental to long term graft survival. Methods We analyzed a cohort of SLKT performed at our institution from 2011 to 2017. We ran both flow and cytotoxic crossmatches pre-transplant and crossmatch positive samples were rerun in the post transplant period. We monitored for DSA both pre and post transplant using a single antigen bead assay. (One Lambda). Results There were 30 SLKT performed, of which 11 had pre-formed DSA (four class I and II DSA, five class I DSA only, two class II only). Strong donor specific anti-HLA antibodies were eliminated following SLKT for class I and II specificities while preserving anti-HLA antibodies against antigens that were not expressed on the donor. The presence of DSA lead to six positive flow cytometry crossmatches of which three were cytotoxic crossmatch positive. Repeat crossmatching with recipient serum post SLKT was found to be negative which correlated with the sharp decrease in DSA MFI detectable in recipient serum. Whether DSA in recipient serum was against class I, II or both antigens there was no significant difference in one-year patient or graft survival. Conclusions The liver absorbed the impact of DSA in our cohort of SLKT recipients. The SLKT recipient did well following transplant in the presence of DSA to class I, II or both. Given the high priority SLK patients receive, having strong DSA no matter the class with a positive XM result should not be a contraindication to transplant.

P274 Discrepancy between DSA and HLA flow cross match associated with high frequency of prozone phenomenon in solid phase assay in organ transplantation

Aim Human Leukocyte Antigen (HLA) antibody identification solid phase assays play a key role in defining Donor Specific Antibody (DSA) which can determine the compatibility between the recipient and donor in cross match results. Luminex bead array test becomes high acceptance and wide reach in HLA antibody detection. However, HLA antibodies with high titer lead to activation of complement and might remove C1 complex on the bead. Subsequently, Interference of HLA antibodies and the secondary IgG antibodies might be occurred. This may lead to false-negative or weak antibody results, which is known as prozone phenomena. Technical methods, like treatment with EDTA, dithiothreitol (DTT), heat inactivation and dilution with normal saline can detect this phenomenon. Methods HLA Typing was performed for all recipients and donors before renal transplantation using One Lambda Sequence specific oligonucleotide revers and sequence specific primers (SSOr/SSP). HLA Antibody Identification, Single Antigen Class I/II and C1q test, was performed by One Lambda. T and B cell IgG XM is performed by FACSCanto II flow cytometer, where the cut-off for positive XM is determined based on normal human studies. Native serum samples of 11 recipients were diluted 1:10 with normal saline when clear discrepancy was found between DSA and HLA Flow XM (DSA 300 MCS). Results All cases (N = 11) showed strong elevating of DSA after 1:10 dilution, often >15000 MFI, which was correlated with strong positive HLA flow cross match results. Conclusions: Our results demonstrate the large incidence of prozone effect in solid phase assay. It is therefore strong recommended to treat the serum -in our cases dilution- for prozone effect always and whenever there is no correlation between strong positive FXM and weak or negative DSA.