CDC XM Research Articles

Abstract 12138: Outcomes of Pediatric Cardiac Transplantation With a Positive Retrospective Crossmatch

Introduction: A positive crossmatch (+ XM) has traditionally been associated with adverse outcomes following pediatric heart transplantation. However, more recent studies suggest that favorable intermediate-term outcomes may be achieved despite a + XM. Hypothesis: Children with a + XM have similar long-term survival but higher rate of complications, such as rejection, coronary vasculopathy (CAV), and infection, compared to patients with a negative XM. Methods: The Pediatric Heart Transplant Society Registry database was queried from 2010-2021 for all patients <18 years of age with a known XM. Baseline demographics were compared between + XM and negative XM groups using appropriate parametric and non-parametric group comparisons. Multiphase survival analysis curves were constructed for each outcome to describe both short- and long-term hazard. Similar correlations were performed for different types of + XM (cytotoxic vs Flow). Results: Of 4599 transplants during the study period, XM results were available for 3914, with + XM in 373 (9.5%). There were 70 cases with CDC + XM (39 B-cell, 31 T-cell) and an additional 149 (90 B-cell, 59 T-cell) with flow + XM where CDC XM was either negative, unknown, or not performed. 5-year survival was significantly different in the 2 groups (HR=1.3, p=0.04). Patients with a +XM had shorter time to first rejection (p=.0001) and detection of CAV (p=0.01), while the risk of infection was similar (p=0.11) [Figure]. When comparing CDC+ to Flow+/CDC- patients, there was no difference in 5-year survival or time to first rejection, however CDC+ patients had shorter time to CAV detection (p= 0.01). Conclusions: Pediatric patients transplanted across a + XM experience earlier rejection, earlier development of CAV, and increased graft loss compared to patients with a negative crossmatch. The risk of heart transplantation against a + XM must be balanced with the ongoing risk of waitlist mortality.

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.

HLA Class II Positivity by Lysate Crossmatch in Renal Transplant Scenario-Dangerous if Ignored!!!

The detection of antibodies before transplantation is an important step in assessment of patient immunological risk and exclusion of incompatible donors. Many centers have now implemented donor-specific antibody (DSA) along with complement-dependent cytotoxicity crossmatch (CDC XM) for renal transplant cases. A 34-year-old male with end-stage kidney disease was referred for an ABO-compatible transplant from his mother. The CDC XM done 30 days before transplant was negative. DSA XM was negative for Class I (median fluorescence intensity [MFI] 189) and positive for Class II (MFI 1671). Since CDC and DSA Class I were negative, the nephrologists went ahead with the transplantation. On day 6 posttransplant, serum creatinine showed a rising trend (up to 2.13 mg/dl), and therefore, renal biopsy was done which showed mild acute tubular necrosis with positive C4d staining. DSA XM performed on day 15 posttransplant showed negative Class I (MFI 148) and positive Class II (MFI 9987) confirming antibody-mediated rejection (AMR). The patient was started on steroids, and intravenous immunoglobulin and serial plasma exchanges were performed. Then, DSA Class II levels came down to 1602. DSA levels have been monitored periodically and Class II MFI values have been ranging from 2000 to 4000. The patient is maintained on routine immunosuppression, and a graft is intact with serum creatinine level between 1.7 and 1.8 mg/dl 8 months posttransplant. DSA-isolated Class II positivity in renal transplant recipients correlates strongly with AMR and should be considered clinically significant.

Inactivation of IgM Antibodies as a Crucial Element of Diagnostics in Sensitized Patients Awaiting Kidney Transplant

BackgroundThe positive result of complement-dependent cytotoxicity crossmatch (CDC XM) may not constitute a contraindication to renal transplantation, unless it is mediated by IgM antibodies, particularly of the autologous type. The current work presents the evaluation of the frequency of reactive IgM antibodies in sensitized kidney patients in Poland and their influence on panel-reactive antibodies (PRAs) readout and allocation status. Patients and methodsResults of PRA CDC assay with and without dithiothreitol were elaborated in 53 prospective recipients with historic PRA of ≥50%. Delta PRA (dPRA) was calculated. Retrospective analysis of the results in the context of age, sex, transplant number, and cause of end-stage renal failure was performed. ResultsReactive IgM antibodies were detected in 81% of patients. Panel reactivity completely disappeared in 4% and in 51% of recipients PRA decreased by 2 to 77 percentage points. In 14 patients, PRA increased, and in 10, its level did not change. The allocation was altered in 36% of recipients. Priority status was lost in 8 and gained in 3 cases. Additional points for high sensitization were obtained in 1 and lost in 7 patients. dPRA was significantly greater in patients awaiting the first transplant compared with the second (P = .007) and third (P = .002). Higher dPRA was also symptomatic for autoimmune patients (P = .025). ConclusionsReactive IgM antibodies affected PRA level and allocation in a considerable group of sensitized patients in Poland. Discrimination of IgM from IgG antibodies should be introduced to the recipient qualification algorithm.

SAT-352 THE EFFECT OF IMMUNOLOGICAL RISK ASSESSMENT ON GRAFT AND PATIENT SURVIVAL BEFORE KIDNEY TRANSPLANTATION

Kidney transplantation is the most successful renal replacement therapy for patients with end stage renal disease (ESRD), which offers improving quality of life and survival advantage. In our center, since 1970 complement-dependent cytotoxicity cross match, since 2002 standard panel reactive antibody screening (PRA-class I and II) and since December 2015 flow cytometry crossmatch (FCM-XM) assay and detection of anti-HLA antibodies with more specific methods and measuring mean fluorescence intensity (MFI) titers have been performed pretransplant cadaver and living kidney recipients. The aim of this study was to evaluate the immunological/non-immunological complications, graft and patients survivals and the factors affecting survival in kidney transplant recipients who underwent transplantation between January 1993 and March 2018. 630 patients who underwent kidney transplantation were enrolled. Group I include 148 patients who were just tested with CDC-XM, group II include 96 patients who were tested with CDC XM and PRA screening and group III include 96 patients who were tested with FCM-XM and detailed PRA. Patients’ demographic features, laboratory and transplantation data were compared. Graft and patient survival and factors affecting them were analyzed. Also after determining the differences of these groups patients were matched based on the propensity score match analysis after that analyzes were repeated. Comprasion of patient and transplantation characteristics is shown at Table 1. The mean follow-up period was 113±85 months in group I, 92±45 months in group II, and 25±8 months in group III (p<0.001). The rate of cadaveric donor were similar in each group. The mean number of HLA mismatches was 2±1 in group I, 3±2 in group II, and 3±1 in group III (p<0,001). It has been observed that the number of PRA positive recipients increased in recent periods with known PRA status (p=0,001). The rate of given induction treatment and maintenance immunosuppressive treatment regimens also changed over time. While the average creatinine and glomerular filtration rate (GFR) at discharge was similar in groups (p=0.852), GFR was lower in group I than the other two groups during the follow-up (p<0.001). Acute rejection rate was highest in group I and least in group II (p<0.001). Graft survival was higher in the other period than in the tested only with CDC-XM period (group I) (p=0.019) and patient survival was also higher in group II and III in each years during follow-up (p<0,05). After matching the age, sex, donor type and HLA mismatch number of the patients with propensity score, 48 patients in group I, 55 patients in group II and 51 patients in group III were matched and the analyzes were repeated. After analysis, graft and patient survival were higher in group II and III than in group I. With improvements in immunological risk assessment, the use of related tests before renal transplantation and since 2000s the development of immunosuppressive theraphy have positively affected graft and patient survival in kidney transplant patients in our center, although transplantations were made to higher-risk patients recent years. Detailed assessment of immunological risk and individualization of pretransplant evaulation and induction and maintenance therapies may also have played a role in improving graft and patient survival.

P143 Development and validation of LSAB MFI cutpoints for use in the virtual crossmatch test to predict flow cytometry crossmatch (FCXM) and cdc crossmatch (CDC XM) results

Aim Luminex Single Antigen bead assay (LSAB) derived mean fluorescence intensity (MFI) values of anti-HLA antibodies directed at the potential donor’s HLA are the primary parameter used in the virtual crossmatch to predict physical crossmatch (XM) outcome. We aimed to develop statistically validated LSAB MFI cutpoints for predicting CDC XM and FCXM results. Methods We leveraged the ASHI proficiency testing (PT) 80% consensus results of 7156 T FCXM, 6758 B FCXM, 2917 T CDC XM and 2233 B CDC XM as the reference results to investigate whether LSAB MFI of IgG anti-HLA antibodies predict validated physical XM results. LSAB MFI was determined in our laboratory using One Lambda Single Antigen HLA Class I and Class II beads. The 80% consensus results are from 8 consecutive challenges during 2013 to 2016, and 107 laboratories across USA tested sera and HLA typed cells distributed by ASHI, and reported the results to ASHI for assessment of the laboratory’s proficiency. Data analysis included: summing of MFI of IgG antibodies directed at HLA-A, B and C for investigating their association with T FCXM and T CDC XM results; summing of MFI of antibodies directed at HLA-A, B, C, DR, DQ and DP for investigating their association with B FCXM and B CDC XM results; assigning alternate ASHI challenges to the Discovery set and the Validation set; investigating the association between LSAB MFI and physical XM results by logistic regression analysis corrected for overdispersion; and identification of LSAB MFI cutpoint for maximizing the sum of sensitivity and specificity. LSAB MFI cutpoints derived from the Discovery set to predict FCXM and CDC XM results were investigated in an independent validation set. Results Table 1 demonstrates that LSAB MFI cutpoints from the Discovery set predicts XM outcomes in the Validation set (ROC AUC range from 0.974 to 0.999). Conclusions We have developed and validated LSAB MFI cutpoints for use in the virtual crossmatch to accurately predict physical T FCXM, B FCXM, T CDC XM and B CDC XM results.

P126 Comparison study of CDC and flow crossmatch vs. donor specific antibodies in transplantation

Aim Our approach to transplant highly sensitized patients has been to perform CDC crossmatch (XM) when the results of flow xm are above 200 mean channel shifts (MCS) with donor specific antibodies (DSA). The aim of our study was to determine if the flow crossmatch and solid phase antibody testing can be used as the only measures for decisions to transplant. Methods Results of flow xm, CDC xm and solid phase DSA testing were analyzed for 44 donor and recipient pairs. In all cases, the flow xm were positive with greater than 200 MCS values. Undiluted patient sera were tested by Luminex single antigen (LSA) test for HLA antibodies (ABS). The ABS detected by this method were grouped into weak (MFI 2500–5000) moderate (MFI 5000–7500), and strong (MFI 7500-greater than 17000). The CDC xm was performed with and without DTT treatment using the same sera for flow xm. Results In 70% of this group with positive T and/or B CDC xm at least one DSA detected was in the strong binding range above 17,000 MFI. In 30% of cases multiple DSA detected in weak to moderate range below 7500 MFI values. In 50% of neg T cell and 96% of neg B cell CDC crossmatches, the MFI of DSA detected was in moderate to strong range. In 90% of negative CDC xms the MCS values for flow T was below 300 and flow B below 400 MCS. The range of flow MFI values for positive CDC B cells xm was 156–596 and 238–671 for T cells. Conclusions While the CDC xm test is the classical standard to exclude transplant, it is not always a reliable test. This assay is quite subjective and dependent on quality and strength of complement, technical skills of the laboratory technician and donor cell viability. This assay is also not reliable when patient is under desensitization therapy. There is a need to develop this assay using flow cytometer platform. Use of solid phase testing methods and determination of standards for decision to transplant based on number, type and strength of antibodies can be a better method

OR51 To treat or not to treat: Long term stable kidney allograft function in presence of strong complement-fixing donor-specific antibody (DSA) to HLA-DQ alpha chain

The clinical significance of HLA antibody specific to the DQ alpha chain is controversial. Here we present a case of long term stable kidney allograft function in presence of strong de novo complement-fixing DQA DSA. A 27-year-old Caucasian male (non-sensitized; 0% CPRA) with ESRD to IgA nephropathy received a living donor kidney transplant (TX) 10+ years ago with a negative pre-TX CDC crossmatch (XM). Mismatched donor HLA were A1, A11, B8, DR13, DR17, DR52, DQ2, DQ6. Beginning 6 years post-TX, routine antibody screening by single antigen beads (SAB) revealed the emergence of de novo DQ antibody, with the reactivity to donor DQ2 and DQ6 initially detected at 700–10400 and 100–2400 MFI, respectively. Subsequent SAB profiles indicated that the DSA had progressively converted to be primarily directed against DQA1∗04/05/06, likely a result of sensitization from donor DQA1∗05:01. Epitope analysis suggested that the DSA was directed against a well-defined eplet 40GR3 shared by all DQA1∗04/05/06 alleles and may represent three distinct eplets 40G, 47C, 50V51L53Q, all located in the alpha-1 domain accessible to antibody binding. Testing by phenotype beads confirmed this strong DQA reactivity (5900–14100 MFI) and flow XM with surrogate donors expressing DQA1∗05 were strongly B-cell positive (210–420 MCS), suggesting the DSA was directed against native DQA chain. Functionally, C1q testing demonstrated the DQA DSA’s considerable ability to fix complement (5300–9500 MFI), while CDC XM with DQA1∗05 + surrogate donors were all negative. Despite the prolonged presence of this DQA DSA, other than an episode of acute cellular rejection (Banff II C4d-) diagnosed shortly after transplant which was promptly resolved, the patient’s 10 + years of post-TX course has been unremarkable with a stable kidney function (creatinine 1.5 ± 0.2 mg/dL; BUN 13.4 ± 2.7 mg/dL; GFR 55.0 ± 6.8 mL/min). This case highlights the fact that not all HLA DSA are created equal in terms of their clinical significance, and may also suggest a protective effect of a subset of DSA when directed against only to the DQ alpha chain. Download : Download high-res image (313KB) Download : Download full-size image

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.

Luminex-Based Donor-Specific antibody crossmatching for renal transplant: A 3-Year experience in South India

Introduction: Although CDC has been the gold standard for many years, this assay is certainly not perfect and its use is associated with several problems. The Luminex anti-HLA antibody detection assay is reportedly more sensitive and specific. One of the test is Luminex DSA cross match which is more sensitive than CDC cross match. It is also considered cost effective tool to evaluate pre and post renal transplant cases. Material and Methods: We started Luminex based Donor specific antibody Cross Match tests (DSA Xm) with lysate by the end of 2009 along with CDC after standardization. This study is planned to evaluate the results of DSA Xm of last three years & also to observe antibody medicated rejections after renal transplant. Results: Total 3137 Luminex DSA Cross Match tests were done & 515 tests were positive cross match with negative CDC xm result. Out of 515 positive results 164 were positive for class I, 223 were positive for class II and 128 were positive for both class I & II. In the three years total 1129 renal transplants were done at various centers and total 42 cases of Antibody medicated rejections were reported. Out of 42 patients, 36 patients had pre transplant history of CDC negative & Luminex DSA xm positive. Conclusion: We evaluated our three year results of DSA Xm test and found a cost effective, sensitive and useful supplementary test to evaluate pre and post renal transplant cases.

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.

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.

P122 Virtual crossmatch to assist with timely evaluation of donor compatibility for sensitized national kidney registry recipients

Aim The National Kidney Registry (NKR) has reduced wait time for patients with incompatible donors. Utilizing Virtual crossmatch (vXM) to predict viable NKR donors with sensitized NKR recipients can expedite exploratory crossmatch (EXM) orders and place recipients in a chain swap sooner. Evaluating weak DSA will help clarify the need for an EXM or declining the donor. Methods vXM was done using patient HLA-Ab information and donor allelic level DSA. HLA-Ab assignment was performed by Luminex single antigen beads using EDTA treated patient serum. If clarification was needed, Phenotyping beads and/or Flow Single Antigen beads were tested. A vXM was done to evaluate need for an EXM to prevent swap failures and/or include recipients in a chain swap. EXM were executed using cryo-preserved cells. Due to limited cell yield only flow XM were done for EXM. Final XM were performed using T and B CDC and flow XM. Results At the Cleveland Clinic, cutoff for HLA-Ab reported to the NKR is > 4000 Mean Fluorescence Intensity (MFI). Seventy vXM were performed between January 2016 and March 2017. Twenty-eight vXM had weak (MFI = 1000–3999 MFI) DSA. EXM’s were suggested when weak DSA was observed in the vXM or for highly sensitized patients. Results of twenty-two EXM/screening XM (screening XM were the result of donor preselects) that were ordered showed seventeen cases had single DSA (CI = 9 CII = 8). These were predicted by vXM to be negative. This was 100% concordant to the EXM and final XM (Table 1A). Five cases had multiple DSA (CI = 2, CII = 2, CI & CII = 1), all predicted by vXM to be negative to weak positive. This was 100% concordant to the EXM and final XM (Table 1B). Conclusions vXM based on number and intensity of DSA was 100% concordant with EXM and Final XM. This assists in reliable donor selection for NKR recipients. vXM provides a quick evaluation of NKR donors for highly sensitized patients or patients with weak DSA. Reporting EXM quickly to the NKR reduces wait time and enables NKR recipients to become part of a successful chain swap. Download : Download high-res image (247KB) Download : Download full-size image

P086 MFI, MFI everywhere: Is there a clinically applicable MFI cutpoint anywhere?

Aim Luminex Single Antigen bead assays (LSAB) are robust for detecting and identifying IgG anti-HLA antibodies, and the LSAB mean fluorescence intensity (MFI) is currently used to list unacceptable antigens and to predict physical crossmatch (XM) results. Concerns exist regarding the use of MFI as the criterion because MFI thresholds for accurately predicting physical XM results are far from established. We aimed to address this unmet need. Methods We leveraged the XM results from ASHI Proficiency Testing (PTs) to investigate whether LSAB MFI cutpoints are predictive of physical XM results. ASHI graded (⩾80% consensus) XM results from 8 consecutive ASHI PTs in which up to 30 labs tested 40 sera and 16 cells distributed by ASHI were used to examine whether LSAB MFI data we generated in the same PTs predict physical XM results. Cumulative MFIs of DSA directed at HLA-A, B, and C were tested for their ability to predict T cell Flow XM and T cell AHG XM results; cumulative MFIs of DSA directed at HLA-A, B, C, DRB1, DRB3/4/5, DQB and DP were tested for their ability to predict B cell Flow XM and B cell CDC XM results. Results Our data analysis identified that cumulative MFI of DSA less than 6000 directed at HLA-class I antigens predicted T cell Flow XM results with a 100% negative predictive value (NPV) and MFI > 7000 predicted T cell Flow XM results with a 100% positive predictive value (PPV) (Chi-Square for trend, X 2 = 70, df = 1, P 8000 predicted B Flow XM result with a 100% PPV (X 2 ; =; 67, df = 1, P Download high-res image (343KB) Download full-size image Conclusions Our data demonstrating the feasibility of developing LSAB MFI cutpoints for the accurate prediction of T cell Flow XM, B cell Flow XM, T cell AHG XM, and B cell CDC XM results advance a strategy for the interpretation of virtual crossmatches and the prediction of physical XM results.

P142 Hyperacute rejection in a kidney transplant recipient with no HLA antibodies

A 65 year old, blood group O, African-American male with 0% CPRA received a blood group O deceased donor kidney transplant with a negative crossmatch (XM). During surgery, the transplanted kidney became dusky within minutes of restoring blood flow. There was no suspicion of anti-HLA or anti-ABO antibodies which could mediate hyperacute rejection, so a thrombotic event or anatomical defect was suspected. The OPO consented to allocate the second kidney from the same donor; it was transplanted within hours, and the same events transpired. Three days later, the recipient had bleeding and pain at the graft site, and donor vein and artery grafts had to be removed. We sought to identify the factors which caused the allografts loss. Both kidneys were sent for microscopic and C4d evaluation. Recipient HLA antibodies were tested by FlowPRA and Luminex Single Antigen Beads (LSAB). XM were performed by CDC and flow cytometry. Recipient serum was tested for MICA antibodies by LSAB, endothelial cell (EC) antibodies by XM, AT1R antibodies by ELISA, and non-ABO blood group antibodies. Molecular blood typing of the donor DNA was performed. Histopathology identified karyorrhectic nuclear debris in the glomeruli, small fibrin thrombi, and neutrophilic infiltrates, consistent with hyperacute rejection, although IHC failed to show definite C4d deposition. All FlowPRA, XM, LSAB, MICA, and EC XM tests were negative. The AT1R result was 14 U/mL (at risk). Molecular testing confirmed the donor as Group O. Testing ruled out the rare Bombay phenotype, and the recipient was negative for all non-ABO blood group antibodies, except for weak cold agglutinins only seen at 4 °C. The events during the surgery and the pathology findings are consistent with humoral hyperacute rejection. Confoundingly, the recipient has no HLA antibodies and had negative CDC and flow XM with the donor. However, none of the other testing has been able to identify an antibody with a strength that would explain hyperacute rejection. The only identified antibodies thus far are anti-AT1R and cold agglutinins. We are continuing to investigate more non-HLA antibodies that may have a synergistic impact on graft rejection.

High strength HLA-DPA1 donor-specific antibodies cause positive CDC B-cell crossmatch but weak positive B-cell flow crossmatch

DPA1 antibodies were shown to be a risk of antibody-mediated rejection (AMR). We present a case showing high MFI DPA1 donor-specific antibodies (DSA) causing +ve CDC crossmatch (CxM), but -ve flow xM (FxM). At the time of deceased donor offer, we perform prospective FxM if the intended candidate displays strong DP DSA. This particular case received first kidney transplant in 2004 from a 8/10 mismatched donor. Luminex single antigen bead assay (One Lambda) revealed strong antibodies to all mismatched donor’s HLA and CREG, and thus had 100% CPRA. The patient also displayed strong antibodies to several DPs including to the alpha chains, DPA1∗01 and DPA1∗03. The patient received a deceased donor offer in May 2015. Virtual xM (VxM) identified 3 strong DSAs directed to DPA1∗01(MFI = 16282), DPA1∗03(MFI = 13167) and DPB1∗04(MFI = 19568), and predicted positive B FxM. Contrary to the prediction, the donor-specific FxM was -ve (T cell MCS = 29; B cell MCS = 110). Kidney offer was declined because of multiple high MFI DSAs. To have better insights on our failed VxM prediction, we performed pronase as well as CxMs. The pronase B FxM was weakly +ve with a MCS = 209 (+ve cutoff > 120 MCS), which is within the range relates to -ve CxM. Unexpectedly, the B CxM with and without DTT-treated sera were turned to be strongly +ve. Additional xMs with a surrogate donor cells expressing a single target DPA1∗01:03 also produced similar results, i.e., +ve pronase B FxM with MCS=178 and +ve B CxM. We confirmed DP antibodies assignment using Immucor single antigen beads, and typed previous donor and patient for DPA1 and DPB1 loci. These results ruled out DPB1∗04:01 antibody, but cannot confirm DPB1∗04:02, because DPB1∗04:02 beads in both venders’ systems were paired with DQA1∗01:03. It is more likely the patient has only DPA1∗01 and DPA1∗03 antibodies. Unambiguous assignment of DPA1 antibodies will remain challenging until the single antigen bead array includes multiple DP antigen series with different alpha and beta chain pairs. In conclusion, comprehensive antibody analyses by single antigen beads, and high-resolution typing of DPB1 and DPA1 of donor and recipient are necessary to define DPA1 antibodies. CDC xM is a critical test to determine the risk of hyper acute AMR in patients having strong DPA1 DSAs, and FxM may yield false negative results in these cases.

P115 : PRIMARY GRAFT DYSFUNCTION AFTER 0 MM KIDNEY TRANSPLANTATION SECONDARY TO ACUTE CELL MEDIATED REJECTION

Aim To present a highly sensitized patient with end stage renal disease who received his third kidney transplant from HLA-A, -B, -DR zero mismatched (0 MM) deceased donor (DD) and developed early acute cellular rejection (ACR – BANFF 2A) in absence of DSA. Methods Donor and recipient were HLA typed by RSSOP Luminex assay for HLA loci A, B and C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1. HLA antibody identification were performed with One Lambda Luminex single antigen bead assay. Sera were tested untreated, treated with heat inactivation (HI) and 1:8 dilutions. CDC (with and without HI) and flow XM for T and B cells were tested at the day of transplant. Results A 40 years old man, highly sensitized (CPRA 100%) with a history of two previous renal transplants (lost due to non-immunological cause) with multiple class II DP antibodies with high MFI. The patient received a local zero mismatched donor was offered and with confirmatory testing an antigen level match for all HLA including DP. CDC and flow XM for T and B cells were negative. No DSA was detected pre or post-transplant. In post-operative day 1, the patient presented with anuria and hyperkalemia that mandated dialysis, and underwent dialysis throughout the rest of hospitalization as needed. Due to delayed graft function, he had a biopsy on post-operative day 8 that showed ACR – BANFF 2A – with negative C4d, peritubular capillaritis, and DSA. Treatment with anti-thymo globulin (ATG) did not show improvement. Consequently the patient received 3 sessions of plasmapheresis plus IvIG but the patient remained dialysis dependent. In post-operative day 50, biopsy showed chronic active T cell mediated rejection with no DSA. The graft remained non-functioning and graft nephrectomy was performed. This case suggests that highly sensitized kidney transplant candidates are at increased risk of cell mediated rejection and graft loss even in the context of 0 MM, negative DSA and flow crossmatch.

Do Pre or Postransplant Anti-DP Antibodies Impact Kidney Graft Survival?

Solid-phase assays on Luminex platform employing beads with purified HLA antigens assess the presence of antibodies against classical HLA antigens A, B and DR, but also C, DQ or DP. DP antibodies have been considered less immunogenic than other class II antibodies. There is not much information about the frequency and impact on graft survival of DP antibodies in kidney transplantation (KT). We have evaluated the prevalence of DP antibodies in our cohort of transplant patients and the impact of DP antibodies in patients with and without donor-specific antibodies (DSA) Methods Prospective study of 440 KT recipients (performed 1979-2012 across negative CDC XM) recruited I/2008-III/2012. Pre and postransplant serum samples were tested with Luminex Lifecodes LifeScreen y LSA Class II (Gen-probe,Stanford,CT) kits. Raw-MFI values >1000 for any single antigen bead were considered positive. Results 291 KT patients had preTR samples tested: 68 (23.4%) had HLA class II antibodies, DSA in 36 cases (52.9%). DP antibodies were present in 27/68 (39.7%) patients with pretransplant HLA antibodies and in 17/36 (47.7%) with DSA. Death-censored graft survival of pretransplant DSA-II patients (median 76.5 months) was worse than HLA non-DSA patients (p=0.005). The presence of DP antibodies did not influence graft survival (with or without DSA)[Figure 1].346 KT patients were monitored after KT (median: 54 months): 59 (17.1%) had HLA class II antibodies and 33/59 (56%) with DP antibodies. Twenty-three patients had DSA-II (39%) and 14/23 (60.9%) with anti-DP. At 40 months, death-censored graft survival was worse in patients with DSA II (p=0,022). DP antibodies did not change these results.Figure: No Caption available.Conclusions DP antibodies are detected in around half KT recipients with pretransplant DSA and 61% after transplantation. Detection of DSA class II with single antigen beads before or after transplantation associates with worse graft-survival, but the presence of DP antibodies does not modify the impact.

Long-term outcomes of kidney transplantation across a positive complement-dependent cytotoxicity crossmatch.

More than 30% of potential kidney transplant recipients have pre-existing anti-human leukocyte antigen antibodies. This subgroup has significantly lower transplant rates and increased mortality. Desensitization has become an important tool to overcome this immunological barrier. However, limited data is available regarding long-term outcomes, in particular for the highest risk group with a positive complement-dependent cytotoxicity crossmatch (CDC XM) before desensitization. Between 2002 and 2010, 39 patients underwent living-kidney transplantation across a positive CDC XM against their donors at our center. The desensitization protocol involved pretransplant immunosuppression, plasmapheresis, and low-dose intravenous immunoglobulin±rituximab. Measured outcomes included patient survival, graft survival, renal function, rates of rejection, infection, and malignancy. The mean and median follow-up was 5.2 years. Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years. Uncensored graft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years. Twenty-four subjects (61%) developed acute antibody-mediated rejection of the allograft and one patient lost her graft because of hyperacute rejection. Infectious complications included pneumonia (17%), BK nephropathy (10%), and CMV disease (5%). Skin cancer was the most prevalent malignancy in 10% of patients. There were no cases of lymphoproliferative disorder. Mean serum creatinine was 1.7±1 mg/dL in functioning grafts at 5 years after transplantation. Despite high rates of early rejection, desensitization in living-kidney transplantation results in acceptable 5-year patient and graft survival rates.

171-P: VIRTUAL CROSSMATCHING: IT’S NOT AS SIMPLE AS IT SEEMS

Aim Use of the virtual crossmatch (XM) has increased the efficiency of organ allocation and reduced the number of organs declined based on unexpected positive XMs (Cecka JM., et al. Am J Transplant. 2011). Despite these benefits, a cell based XM still provides a final safeguard before transplantation. False negative XM predictions may arise when reactivity is due to both antigen specific antibodies and antibodies to crossreactive epitopes. Methods HLA-specific antibodies were assessed using class I or class II phenotype panels and IgG or C1Q single antigen bead assays. (Lifecodes Immucor; Labscreen, One Lambda,Inc). One wash CDC B cell XMs were performed. Results Luminex IgG tests yielded reactivity with DR53 phenotypes ranging from 3,000-10,000 MFI with DR53 heterozygotes and >10,000 MFI with homozygotes. Reactivity with DR10 phenotypes were 2,000-3,000 MFI. Single antigen beads yielded reactions with DR53 of 20,000 MFI with undiluted serum and 17,000 MFI with serum diluted 1:8. Reactions with DR10 were 15,000 and 2,000 MFI with undiluted and 1:8 diluted serum, respectively. MFI values given above would predict negative XMs with DR10+/DR53- cells and positive XMs with DR10-/DR53+ cells. The C1q assay yielded MFI values of 28,000 with DR53 and 300 with DR10. These C1Q results substantiate the prediction of a negative CDC XM result with a DR10 cell. CDC XM tests yielded the following results: + with a titer of 2 with a DR10+/DR53- cell, + with a titer of 32 with a DR10-/DR53 homozygote cell, and negative with DR53 heterozygotes and with cells bearing other antigens crossreactive with DR10 (DR1 and DR15/51). Conclusions We propose that the CDC+ XM with the DR10 cell was due to contributions from the DR10 antibodies and the much stronger antibodies to DR53 that shares one or more epitopes with DR10. These data show that accurate prediction of crossmatches must take into account the possible contributions of antibodies positive for antigens crossreactive with the target antigen.