Positive Virtual Crossmatch Research Articles

Heart transplantation across preformed donor-specific antibody barriers using a perioperative desensitization protocol

Heart transplantation across preformed donor-specific HLA-antibody barriers is associated with impaired short- and long-term survival. Therefore, in recipients with preformed anti-HLA antibodies, waiting for crossmatch-negative donors is standard practice. As an alternative strategy, recipients with preformed anti-HLA donor specific antibodies have been managed at our institutions with a perioperative desensitization regimen. A retrospective analysis was performed comparing heart transplant recipients with preformed donor-specific HLA-antibodies to recipients without donor-specific antibodies. Recipients with a positive virtual crossmatch received a perioperative desensitization protocol including tocilizumab intraoperatively, plasma exchange and rituximab followed by a six-month course of IgGAM. Among the 117 heart-transplanted patients, 19 (16%) patients underwent perioperative desensitization, and the remaining 98 (84%) patients did not. Cold ischemic time, posttransplant extracorporeal life support for primary graft dysfunction, and intensive care unit stay time did not differ between groups. At 1-year follow-up, freedom from pulsed steroid therapy for presumed rejection and biopsy-confirmed acute cellular or humoral rejection did not differ between groups. One-year survival amounted to 94.7% in the treated patients and 81.4% in the control group. Therefore, heart transplantation in sensitized recipients undergoing a perioperative desensitization appears safe with comparable postoperative outcomes as patients with a negative crossmatch.

Eight-Year Results of an IgA- and IgM-Enriched Human Immunoglobulin-Based Therapy for Early Detectable Anti-HLA Donor Specific Antibodies After Lung Transplantation

<h3>Purpose</h3> The development of anti-HLA donor specific antibodies early after lung transplantation (eDSA) has been associated with antibody-mediated rejection (AMR) and poor graft survival. At our institution, since 2013, successive infusions of IgA- and IgM-enriched human intravenous immunoglobulins (IgGAM, first infusion: 2gr/kg, then 0.5gr/kg once every 4 weeks for a maximum of 6 months) formed the backbone of eDSA therapy. Actually, patients with only evidence of eDSA (possible subclinical AMR) are treated with IgGAM only. Patients with concomitant graft dysfunction (possible clinical AMR) or preformed DSA (positive virtual crossmatch) receive additionally plasmapheresis (PE) or immunoabsorption before the first IgGAM dose, and a single dose of anti-CD20 antibody (Rituximab) thereafter. Aims of this study were to present the 8-year results of the IgGAM-based therapy. <h3>Methods</h3> Records of patients transplanted between 02/2013 and 10/2021 were reviewed. Outcomes were compared between patients with eDSA and treated with IgGAM (IgGAM group) and without eDSA (control group)<i>.</i> Median (IQR) follow-up was 45 (21-72) months. <h3>Results</h3> During the study period, among the 994 transplanted patients, 250 (25%) patients formed the IgGAM group and 721 (73%) the control group. The remaining 23 (2%) patients (14 patients with eDSA but not treated, and 9 patients treated only with tPE and Rituximab) were excluded. Fifty-Five IgGAM patients (22%) showed pre-formed eDSA and 41 (16%) patients showed possible clinical AMR. Median time to eDSA detection was 14 days after transplantation. At follow-up end, treatment was completed in 229 (92%) patients (still on treatment, n=8; in-hospital deaths, n=4; treatment interrupted earlier as intended by protocol, n=9). In these patients, IgGAM treatment cleared eDSA in 208 (91%) patients, 29 (14%) patients showing eDSA recurrence a median of 12 months after treatment end. In IgGAM vs. control patients and at 5- and 8-year follow-up, respectively, graft survival (%) was 77 vs. 76 and 68 vs. 67 (p=0.53), and freedom from CLAD was 73 vs. 72 and 65 vs. 62 (p=0.76), respectively. <h3>Conclusion</h3> After lung transplantation, a treatment protocol for eDSA based on IgGAM yielded high eDSA clearance. Patients with eDSA and IgGAM-treatment have good 8-year graft survival similar to control patients.

First North American Experience with Lung Transplantation from Donation After Medical Assistance in Dying

<h3>Purpose</h3> In 2016, medical assistance in dying (MAID) became legal in Canada. Organ donation after MAID can provide donors with the opportunity to fulfill their dying wishes, and end-stage lung patients with the opportunity for lung transplantation (LTx). The objective of this study was to report on the logistics of this process and to assess the short- and intermediate-term outcomes of LTx with lungs from MAID donors. <h3>Methods</h3> This is a retrospective study of LTx using lung donation from MAID and non-MAID donors at our institution from 02/2018 to 07/2021. <h3>Results</h3> A total of 745 lung grafts were recovered (MAID, 38 (5.1%); non-MAID, 707 (94.9%)). MAID donors included those with neurodegenerative diseases (n=33) and end stage organ disorders (n=5). In the MAID group, 13 cases (34%) were transplanted directly, whereas 25 (66%) cases were evaluated on Ex Vivo Lung Perfusion (EVLP) and 19 (76%) of those were used for transplantation (non-MAID; EVLP: 369 (52%), transplanted: 239 (65%)). Two patients had MAID procedure and death declaration at home prior to be transferred to the hospital for organ recovery. Overall, 33 patients received lungs (one double-lung was given to two single lung recipients) from MAID donors, and 565 patients received lungs from non-MAID donors. MAID donors were significantly older than non-MAID donors (56 (51-64) vs. 49 (32-61) yr; p = .0009). Recipient characteristics were similar in both groups, except for a higher incidence of positive virtual crossmatch rate in the MAID group (MAID, 40%; non-MAID, 16%; p = .015). Positive actual crossmatch rate was not different. Short and intermediate-term outcomes were similar between the two groups (Table 1). <h3>Conclusion</h3> In this first North American report with LTx using donors after MAID, we demonstrate that recipient outcomes are excellent and similar to patients receiving grafts from non-MAID donors. Therefore, where this practice is permitted, donation after MAID should be strongly considered for LTx as an option to increase the organ donor pool.

Perioperative Desensitization for Sensitized Lung Transplant Candidates

Purpose Sensitized lung transplant (LTx) candidates have longer waiting times, decreased likelihood of transplant, and increased risk of death while on waitlist. To mitigate this and to increase the access to donor pool, for sensitized candidates, we modified and implemented The Toronto perioperative strategy to treat DSA-positive recipients with positive virtual crossmatch (VCM) donor offers. We hypothesize that such perioperative desensitization strategy can be safely implemented with acceptable outcomes and help decrease the waitlist mortality. Methods We performed a retrospective chart review of sensitized candidates who underwent LTx between June 2017 - June 2020 and required perioperative desensitization. We collected the baseline demographics and post-transplant variables. The protocol was implemented if the calculated panel reactive antibody (cPRA) was > 30 % and had positive VCM. Desensitization protocol included preoperative plasma exchange (PLEX) with 1.5 volume, intra operative basiliximab induction, followed by five sessions of PLEX, intravenous immunoglobulin (IvIg, 1 gm / kg) and Anti Thymocyte globulin (dose 3 mg/kg for VCM positive vs 5 mg/kg for positive actual crossmatch (ACM)). We used tacrolimus (12 - 15 ng/ml), mycophenolate and prednisone for maintenance immunosuppression. Results 25 patients required desensitization. 76% (n=19) of them are females. Median LAS score is 41.3 (IQR 33.87-60.33). Median wait time was 28.9 days (IQR 12.6-65). All patients underwent bilateral lung transplantation. ACM was positive in 24% of patients (n=6). 76% (n=19) completed the protocol. Incidence of Primary graft dysfunction at any time point was 12% (n=3). Acute cellular rejection >= grade 2 was 8% (n=2). 8% (n=2) had chronic lung allograft dysfunction. Overall survival of this cohort is 96% with a median follow up of 17 months (IQR 5 - 25.5). Conclusion Perioperative desensitization can be safely implemented with acceptable outcomes in sensitized LTx candidates. On comparison of our SRTR data, before and after desensitization implementation, we noted a decline in the waitlist mortality (11.4% to 7.6%) and 50th percentile median wait times (8.5 months to 1.3 months).

Management of Positive Prospective Cross Matches in Lung Transplant Recipients: Outcomes from a Single-Center Experience

Purpose Highly sensitized recipients remain a challenge in lung transplantation. There are no standardized protocols among lung transplant centers to guide management of these patients. Some centers choose not to transplant highly sensitized patients due to the risk of graft dysfunction and death. We describe our center's experience and outcomes managing lung transplant recipients with positive flow-cytometric prospective cross matches (FXM). Methods We retrospectively analyzed patients who underwent lung transplantation with positive FXM from August 2015 to November 2019. Outcomes included overall length of stay, 1-year survival, graft function at one year, and incidence of antibody mediated rejection (AMR) and CLAD (BOS). Results There were 11 patients with FXM (6 females, 5 males; 6 with restrictive lung disease, 3 with obstructive lung disease, 1 with cystic fibrosis, and 1 with pulmonary vascular disease). 7 (63%) had concomitant positive virtual cross matches. 10 (91%) were managed with high-dose IVIg alone immediately post-op. 3 patients additionally received intra and/or post-op plasma exchange, rituximab and/or bortezimab. Median length of hospital stay was 50 days. Median length of follow up is 30 months (range 11-63 months). KM estimate of one-year survival was 90%. Clinical episodes of AMR requiring treatment occurred in 1 (9%) patient at 3 years post-transplant. DSA cleared in 8 patients (73%). Infections occurred in 5 patients (45%). None were life threatening. CLAD grade ≥ 1 occurred in only 2 patients (18%), one at 1.5 years and the other 3 years post-transplant. FEV1 at one year was >89% of personal best in all patients. Conclusion Successful lung transplantation of highly sensitized recipients is feasible. Using our center's approach results in longer hospital stay but excellent one-year outcomes. Further study is needed to identify patients at risk for graft dysfunction and death due to AMR who benefit from early aggressive treatment, and to avoid unnecessary treatment in lower risk patients.

Clinical Outcomes of Perioperative Desensitization in Heart Transplant Recipients.

Background.Sensitization remains a barrier to heart transplantation (HT). Perioperative desensitization strategies have been described; however, a paucity of evidence exists to demonstrate efficacy and safety in HT.Methods.This single-center, retrospective study consisted of adults who received an HT. Perioperative desensitization was initiated if virtual crossmatch or flow-cytometry crossmatch was positive. Therapy consisted of plasmapheresis, intravenous immunoglobulin, and rabbit antithymocyte globulin. Historical controls received standard immunosuppression or induction. The primary endpoint was survival at 12 mo. Secondary endpoints included freedom from acute rejection, cardiac allograft vasculopathy (CAV), and infectious complications.Results.Of the 104 patients included, 48 received no induction, 46 received induction, and 10 underwent perioperative desensitization. No differences were observed in the primary endpoint at 12 mo (90.0% versus 97.9%, P = 0.25 for desensitization versus no-induction; 90.0% versus 100%, P = 0.72 for desensitization versus induction). Rates of acute rejection were lower with induction and desensitization compared with no-induction. There were no significant differences in CAV between the groups. Infectious complications were also similar among the groups (10.0% versus 16.7%, P = 0.62 for desensitization versus no-induction; 10.0% versus 30.4%, P = 0.34 for desensitization versus induction).Conclusions.This study suggests that a perioperative desensitization strategy triggered by positive virtual crossmatch or flow-cytometry crossmatch allows for successful transplantation of sensitized HT recipients and results in acceptable rates of survival, rejection, CAV, and infection at 12 mo.

Treatment of Anti-HLA Donor-Specific Antibodies in Heart Transplantation: A Single-Center Experience

The presence of anti-HLA donor specific antibodies (DSA) after heart transplantation is associated with antibody-mediated rejection (AMR) and poor graft survival. Aim of this study was to present the results of our treatment protocol of DSA in heart transplantation. Since late 2016, at our institution, heart-transplanted patients with DSA but without graft dysfunction (Group 1) were treated with IgA- and IgM-enriched intravenous immunoglobulins (IgGAM) only (first infusion: 2g/kg, then 0.5g/kg once every 4 weeks for a maximum of 6 months). Patients with DSA and graft dysfunction (Group 2, clinical AMR) were treated with additional 5 sessions of plasmapheresis (PE) before IgGAM and a single a single dose of anti-CD20 antibody (Rituximab, 375mg/m2) after the first IgGAM infusion. Highly allosensitized (PRA>50%) patients showing preformed DSA (pfDSA), i.e. a positive virtual crossmatch (Group 3), were treated with an intraoperative single dose of the IL-6 receptor antibody Tocilizumab (10mg/kg), in addition to 5 sessions of PE, IgGAM infusions and a single Rituximab dose, as aforementioned. Among the 72 patients transplanted between 10/2016 and 10/2019, 3 (4%) patients formed Group 1, 8 (11%) patients Group 2, and 12 (17%) patients Group 3. Group 1 patients developed de novo DSA 31, 145 and 28 days after transplantation, were treated with IgGAM, which cleared DSA in all patients, were alive and did not show any episode of biopsy-confirmed rejection (ISHLT Grade R>1) at the end of follow-up. Group 2 patients developed de novo DSA at a median of 207 days after transplantation and showed graft dysfunction requiring ECMO support in 4 cases. Five (63%) patients died in-hospital. Among the 3 surviving patients, DSA were cleared in one patient. Group 3 patients showed pfDSA with >50% pre-transplant PRA. Post-transplant, patients did not show severe primary graft dysfunction, but one patient died in-hospital of pneumonia. pfDSA were cleared in 5 (42%) patients. At follow-up, all discharged patients survived, and 3 patients showed at least one episode of ISHLT Grade R>1 rejection or of minimal AMR (pAMR 1+). In heart transplantation, clinical AMR showed a dismal prognosis, despite treatment. Early diagnosis and preemptive treatment of DSA should be pursued. Transplantation across positive crossmatch barriers can be feasible and safe.

Treatment and Pattern of Resolution of Antibody-Mediated Rejection with Hemodynamic Compromise Resistant to Standard Therapy after Heart Transplantation with a Positive Virtual Crossmatch

Antibody-mediated rejection (AMR) with hemodynamic compromise (HC, heart failure requiring inotropic support) after heart transplantation (HT) confers a significant risk of allograft failure and poor survival. Standard therapy (plasmapheresis and thymoglobulin) to mitigate and/or treat AMR has been variably effective. In January 2018, we embarked on a prospective treatment strategy using Bortezomib, Eculizumab, and consolidation therapy using total lymphoid irradiation (TLI) for standard therapy-resistant AMR (ST-R AMR) with HC. Bortezomib was given in conjunction with plasmapheresis every 3 days x5 doses over 2 weeks and Eculizumab was given weekly x4 doses then spaced to monthly. Consolidation therapy with TLI (2x/week for 5 weeks) was started when rejection episode was resolving defined as improving hemodynamics (filling pressures and echocardiogram) and improvement in histological features of AMR. Total protocol duration was 44-68 days. Donor-specific antibody (DSA) specificity and mean-fluorescence intensity (MFI) was determined using Luminex. Antibody strength was assessed by change in neat and 1:25 dilution MFI. Four virtual crossmatch-positive patients with ST-R AMR and HC were treated. Three of four patients were CDC-crossmatch positive. Median time to AMR diagnosis was 6 days (4-13) and initiation of the protocol was 13 days (8-22) post-HT. Median times to reduction in DSA was 27 days (27-29), improvement in cardiac function was 31 days (7-42), and time to start of TLI was 38 days (21-56). Median time to resolution C4d staining was 64 days (50-69). Three of four patients survived and underwent consolidation with TLI and none have had recurrence of AMR after treatment (6-19 months post-HT). One patient who died 38 days post-HT had persistent histological evidence of endothelial damage on biopsy despite clearance of C4d deposition, persistently elevated filling pressures, progressive systolic dysfunction, rebound increase in DSA, and multi-organ failure. This pilot study shows this protocol may be an effective treatment of ST-R AMR with lasting effects many months post-HT. Initial improvement in DSA and cardiac function can be seen in weeks, but complete resolution of C4d staining may take additional weeks to months.

P151 Correlating positive virtual crossmatch predictions with flow cytometric crossmatch outcome

P151 Correlating positive virtual crossmatch predictions with flow cytometric crossmatch outcome

Cardiac Transplantation across Preformed HLA-Antibody Barriers: Results of a Peritransplant Desensitization Protocol

Purpose A growing number of patients awaiting heart transplantation presents with preformed anti-HLA antibodies. This allosensitization prolongs waiting times and increases morbidity and mortality. In 2017, we designed aprotocol for peritransplant desensitization of heart-transplanted patients showing preformed anti-HLA donor specific antibodies (pfDSA), i.e. a positive virtual crossmatch. The protocol included: an intraoperative single dose of the IL-6 receptor antibody Tocilizumab (10mg/kg) just before releasing the aortic clamp; 5 sessions of plasmapheresis (PE, 1 before transplantation, 1 during transplantation and 3 thereafter); an infusion (2g/kg) of IgA and IgM-enriched human immunoglobulins G (IgGAM) after the last PE; and a single Rituximab dose (375mg/m2) after IgGAM. IgGAM are repeated every 4 weeks (0.5g/kg) for a maximum of 6 months, if DSA are still positive. Aim of this study was to present the preliminary results of this peritransplant desensitization protocol. Methods Records of heart-transplanted patients at our institution between 01/2017 and 10/2018 were reviewed. Patients with pfDSA (positive virtual crossmatch) formed the case group, remaining patients the control group. Median (IQR) follow-up amounted to 9 (5, 16) months. Results During the study period, among the 42 transplanted patients, 9 (21%) patients formed the case group and the remaining 33 (79%) the control group. All 9 case patients showed pfDSA with >50% pre-transplant panel reactive antibodies (PRA). Pretransplant recipient and donor characteristics did not differ between groups. Post-transplant, no case patients showed severe primary graft dysfunction, but 6 (19%) control patients did (p=0.19). At treatment end, pfDSA were cleared in 4 (44%) patients. At 1-year follow-up, survival was 100% vs. 75% in case and control patients (p=0.18); freedom from biopsy-confirmed rejection (Grade >1R) was 89% vs. 92% in case (n=1) and control (n=2) patients (p=0.78); and from steroid-pulsed therapy 89% vs. 82% in case (n=1) and control (n=5) patients (p=0.76), respectively. One case patient showed minimal AMR (pAMR 1+). Conclusion The present study showed that heart transplantation across positive crossmatch barriers can be feasible and safe. The present peritransplant desensitization protocol yielded good survival and rejection-free survival.

Cardiac transplantation across preformed HLA-antibody barriers

Cardiac transplantation across preformed HLA-antibody barriers

Renal Transplant with Positive Virtual Crossmatch in Patients with High Immunological Risk

Introduction In the past few years the number of patients in the waiting list with anti-HLA antibodies has been increased. This makes it more difficult to find an appropriate immunological donor for these receptors. For that reason, we decided to perform in our hospital renal transplants with positive virtual crossmatch in certain patients. Materials and Methods We analyzed the renal transplants performed in our hospital since October 2014 with positive virtual crossmatch and a specific donor antibody value greater than 20,000 SFI. A total of 27 patients have been included so far. In all cases the crossmatch test by cytotoxicity was negative at the time of transplantation. The induction treatment is with Anti-Thymocyte Globulin at doses of 4.5 mg/kg associated with corticoids, tacrolimus and mycophenolate mofetil. Since July 2016 the patients who have a sum of specific donor antibodies greater than 80.000 SFI or PRA greater than 98% receive a plasmapheresis cycle with human immunoglobulin, starting at day + 5 post-transplant. In this paper we present the clinical and immunologic data of patients who have completed 1 year of follow up. Results Of the 27 renal transplants performed with this protocol 20 patients have completed the 1 year follow up. The average age of the donors it is 64 years old and that of the recipients is 54-year-old. 5 patients received plasmapheresis and human immunoglobulin in immediate post-transplant prophylactically. In table 1 and 2 will expose the immunological data previous and posterior to renal transplant.In terms of rejection cases, 5 humoral rejections were diagnosed (20%) of which 1 was the cause of the graft loss and the other 4 responded to the treatment. 4 of these patients were of the group without prophylaxis with plasmapheresis. The survival of the graft a year is 75% with 1 case of humoral rejection (5%) 2 for Pyelonephritis, 1 case of immediate thrombosis and one of death with a functioning graft. The patient's survival is 95% per year. Discussion: Renal transplantation with specific donor anti-HLA antibodies is an option for hyperimmunized patients, as only one of the patients in our series lost the graft by humoral rejection. Greater number of patients required and more follow-up to obtain more conclusive data. The realization of plasmapheresis with human immunoglobulin after transplantation as prophylaxis, it seems to us a possibility to reduce the risk of humoral rejection in these patients. As a main complication of the increase in immunosuppression we found infections, which in 2 cases led to graft loss. Conclusion The renal transplant with virtual positive crossmatch is a valid option in patients with high antibody titers. Adding plasmapheresis in induction therapy may decrease the risk of humoral rejection.

Cincinnati Protocol for Preoperative Screening and Donor Selection for Ocular Surface Stem Cell Transplantation.

To describe our process for preoperative screening and donor selection for ocular surface stem cell transplantation (OSST). A 7-year retrospective chart review was performed on limbal stem cell deficiency patients. The inclusion criterion was all patients who underwent an OSST procedure. The exclusion criterion was eyes with unilateral disease in which an autograft was performed. Data for human leukocyte antigen (HLA) typing, virtual crossmatching, donor-specific antibody, and panel reactive antibody level were obtained. Of the included 142 eyes (104 patients), 19 patients had no recorded living donor availability data, and HLA typing was not performed on 16 patients. A total of 94 donors (mean 1.4 donors/patient, range 1-6) were tested for 67 recipients. For 2 patients with graft-versus-host disease, no further HLA typing was needed, as the donors were known HLA-identical donors. For 47 patients, only 1 donor was tested, whereas multiple donors underwent HLA typing for 20 patients. There were 73 ABO (blood group)-compatible matches for the 61 tested recipients, and only 1 recipient did not have any ABO-compatible donor. For the virtual crossmatch, there were 5 patients who did not have a compatible donor (positive virtual crossmatch). The best available donor match was a sibling for 41 recipients (65%), a parent for 19 recipients (30%), and an offspring for 3 recipients (5%). Our protocol for OSST preoperative screening and donor selection minimizes the antigenic burden for transplanted tissue by selecting the best available donor match.

Desensitization in Lung Transplant Recipients with a Positive Virtual Crossmatch

Sensitized lung transplant recipients (LTR) are at an increased risk of acute rejection and mortality. Studies comparing outcomes between LTR with a positive virtual crossmatch (P-VXM) who undergo desensitization vs. LTR with a negative VXM (N-VXM) are limited. The purpose of this study is to compare the incidence of biopsy proven acute rejection (BPAR) between these groups.

Charges and resource utilization for pediatric heart transplantation across a positive virtual and/or cytotoxicity crossmatch.

There is growing acceptance of transplantation across a positive crossmatch for highly allosensitized pediatric HT candidates. While survival may be similar to patients transplanted across a negative crossmatch, costs are unknown. Among 60 HT recipients at our center from 5/07 to 6/12, we analyzed hospital charges and length of stay from the day of HT to discharge and through the first year after transplant. Median age at HT was 6.2years (15days-20.5years). Charges in the first year post-HT were greater for crossmatch-positive patients ($907678 vs $549754; P=.017), with a trend toward higher charges for the HT hospitalization ($537640 vs $407374; P=.07). Plasmapheresis was more common in crossmatch-positive patients during the HT hospitalization (80% vs 4%, P<.001). In the first year after HT, crossmatch-positive patients had a greater number of endomyocardial biopsies (10 vs 7.5, P=.03) and episodes of treated rejection (2 vs 0, P=.004). Pediatric HT across a positive crossmatch is associated with higher first-year costs, including increased use of plasmapheresis and care around an increased number of rejections. These novel data will help inform decision and policymaking regarding care practices for the growing population of highly sensitized pediatric HT candidates.

P167 A case of positive virtual crossmatch and negative crossmatch: Role of fetal calf serum

Case A 69-year-old male with chronic progressive renal failure was evaluated for living donor kidney transplantation. Two prior donors were turned down due to donor specific anti-HLA antibodies (DSA) resulting in a positive flow crossmatch (FXM). Laboratory Testing Low to medium resolution HLA typing was performed on the donor-recipient pair by reverse SSO method. Recipient serum was screened for anti-HLA antibodies by single antigen beads (SAB, LabTYPE, One Lambda) and FXM was performed using donor lymphocytes. Results Donor is 4/6 mismatch with the recipient at HLA-A,B and DR (Donor DR 4,11 and recipient DR 11,13). SAB demonstrated antibodies to HLA-DR4 (DSA) and DR16 with MFI >2000 which is predicted to result in positive FXM at our center. However, FXM was T and B cell negative. Investigations To understand the clinical significance of this DSA, additional investigations were performed. Donor typed as DRB1∗04:04. Surrogate crossmatches with two additional DRB1∗04:04 donors using the same recipient sera were negative. To rule out the possibility of rare donor null allele, crossmatch with the donor cells and surrogate sera with high DRB1∗04:04 MFI, resulted in a positive crossmatch. As the HLA antigens on the beads are derived from cell lines that have been cultured in fetal calf serum (FCS), one could speculate that this reactivity maybe due to binding of naturally occurring heterophile antibodies to beads. To test this hypothesis serum was tested after pre-treating the beads with FCS. Indeed testing recipient sera after FCS treatment resulted in removal of the ‘spurious’ anti-HLA reactivity (figure). Download : Download high-res image (416KB) Download : Download full-size image Conclusions With increasing use of VXM to screen donors, this case adds another exception when positive VXM does not result in positive FXM due to spurious bead reactivity. Such false positive VXM may result in declining a donor for transplantation. Pre-treating the beads with FCS provides target antigens that block this spurious reactivity. We have observed this reactivity specifically to DR4-DR16 beads only.

P117 Limitation of donor specific antibody mean florescent intensity values in predicting transplant compatibility: The saga continues

Aim A positive flow cytometry crossmatch (FCXM) can be predicted when the presence of strong donor specific antibody (DSA) is detected by Luminex single antigen bead (SAB) assays. We present the case of a 23 year-old Hispanic male kidney transplant candidate with a strong HLA-B45 DSA to his potential living donor and consistent negative crossmatches. Methods DDT-treated sera was tested to detect HLA antibodies using LABScreen PRA beads and LABScreen Single Antigen beads (One Lambda, Inc.). Acid treatment of the SAB was used to denature the HLA Class I antigens. The sera was also tested using SAB from a different manufacturer (Immucor, Inc.). FCXM was performed with pronase-treated peripheral blood lymphocytes isolated by negative selection using the RoboSep Automated Cell Separator (StemCell Technologies). FCXM were performed with 3 surrogate HLA-B45-positive cells. CDC crossmatch was performed with potential donor. Results Strong HLA-B45 DSA (MFI = 5052) gave consistent negative crossmatch results with all HLA-B45-positive cells ( n = 3). The strong HLA-B45 antibody disappeared upon testing with acid-treated SAB (One Lambda, Inc.), implying presence of a true DSA. Testing with PRA beads (One Lambda, Inc.) gave positive results. Additional testing with the SAB from a different vendor (Immucor, Inc.) demonstrated the presence of a weak HLA-B45 DSA (MFI = 1426) that correlated better with the negative crossmatch results. Conclusions Relying on MFI values from a single vendor to predict a positive virtual crossmatch and influence clinical decision making may unnecessarily disadvantage the patient. This study indicates that the use of the LABScreen SAB assay as the sole method for unacceptable antigen determination can lead to a significant disadvantage for selected patients. The use of 2 different SAB platforms or 2 different bead assays (PRA and SAB) in the HLA antibody testing algorithm would prevent selected patients displaying high reactivity to certain specificities from being inappropriately excluded from receiving an organ due to aberrantly highly positive SAB results. Moreover, this case highlights the necessity to perform prospective crossmatches to confirm strong antibody specificities detected on SAB.

P250 Reporting potential donor specific antibodies (DSAS) to aid clinicians’ choice of recipients for deceased donor kidney transplant

Aim A virtual crossmatch determines which recipients are eligible for a deceased donor kidney based on UNET unacceptable antigens. The MFI threshold for unacceptable antigens is often higher than what most labs consider to be positive (e.g. 1000 MFI). Clinicians are interested in knowing whether DSAs below the threshold are present so immunosuppression protocols can be altered or, if several are present, they should rule out a patient for transplant. What is the mechanism for reporting to clinicians the presence of potential DSAs during deceased donor kidney allocation?. Methods The Laboratory of Immunogenetics & Transplant (LIT) serves three kidney transplant centers and the local organ procurement organization. Waitlist patients are screened yearly by One Lambda Single Antigen I/II beads. Unacceptable antigen (UA) cutoffs are 3000-5000MFI, while single antigen beads >1000 MFI are assigned as positive with a few exceptions. During deceased donor evaluation, LIT technologists complete HLA-specific data entry into DonorNet and perform the matchrun. Using LIT-specific Histotrac programming, LIT runs exclusion algorithms for local protocols, choses the top candidates to crossmatch, and creates a Virtual XM report. Two centers use a protocol to avoid unacceptable antigens with additive MFIs >3000. We reviewed matchruns, virtual crossmatch exclusions, and transplants from March 2015 to Febuary 2017. Results In two years of cases, 332 kidney matchruns were evaluated: 286 local matchruns were performed while 46 import donors were evaluated. Consequently, 2288 patient-donor pairs were reviewed by virtual crossmatch. Patients were ruled out due to additive DSAs in 17 (0.7%) patient-donor pairs. Two-thirds of patients who were transplanted with a deceased donor kidney had a 0% cPRA while 5% had cPRA 98–100%. Positive virtual crossmatches prompted additional review of UA and antibody history. Conclusions The laboratory staff is responsible for completing deceased donor matchruns and generating a virtual crossmatch report, thus providing a streamlined approach to deceased donor evaluations. The virtual crossmatch report is essential for clinicians’ center-specific algorithms, induction protocols, and assessing risk for kidney candidates in the presence of potential DSA.

OR6 The positive virtual crossmatch. Correlation between HLA DSA and flow cytometry crossmatch results

Aim Virtual crossmatch (VXM) is a good tool to expedite and streamline pre-transplant recipient selection and facilitate national organ sharing. However, VXM can be a barrier to transplantation, based on donor specific antibodies (DSA) detected by single antigen bead (SAB) testing, especially when low MFI cutoffs are used to assign unacceptable antigens. Recent studies report good clinical outcomes in patients transplanted across DSA with a negative FCXM, emphasizing that the choice of VXM cutoffs should be based on careful correlation with FCXM. The purpose of this study was to assess the correlation between MFI of DSA and FCXM results. Methods The results of 2843 deceased donor FCXM performed at our center between 03/2015 and 03/2016 were analyzed and correlated with cumulative MFI of locus specific DSA. Donors were typed (HLA-A, B, C, DR, DQ) by SSP method. FCXM were performed as per Halifax protocol (Liwski et al., ASHI Q 2015) with spleen or lymph node derived donor cells. SAB testing (LABScreen) was performed with heat inactivated sera to prevent prozone. DSA were assigned based on 1000 (1K) MFI cutoff. All sera with DP antibodies were excluded. Results Of 1859 VXM −ve samples (no DSA), 56 were FCXM +ve (3% false positive rate). Of 318 VXM +ve (DSA; >1K MFI) samples, only 214 (67.3%) were FCXM +ve. The degree of concordance, VXM vs FCXM, was locus and MFI dependent (Fig. 1). HLA-A and B DSA predicted a +ve FCXM (T and/or B) in 80%, 93% and 100% of cases using 1K, 2K and 5K cutoffs respectively. HLA-DR DSA predicted positive B cell FCXM in 54%, 89% and 100% of cases with same MFI cutoffs. HLA-C and DQ DSA predicted positive FCXM reliably only when 10K cutoff was used (100% and 95% respectively; Fig. 1). Conclusions Concordance between VXM and FCXM depends on MFI cutoff and DSA locus. 1K cutoff is not useful for FCXM prediction regardless of DSA specificity. For HLA-A, B and DR DSA, 2–5K cutoff can be used to reliably predict FCXM, while for HLA-C and DQ only 10K cutoff is predictive. Download : Download high-res image (138KB) Download : Download full-size image

In Kidney Transplant Recipients With a Positive Virtual Crossmatch, High PRA was Associated With Lower Incidence of Viral Infections

There is little information on the incidence, risk factors, and outcomes associated with CMV and BK infections in sensitized patients. We examined 254 consecutive kidney transplant recipients with positive virtual crossmatch and negative flow crossmatch. A total of 111 patients (43%) developed CMV disease or BK infection or nephropathy (BKVN). Specifically, 78 patients (30.7%) developed BK infection, 19 (7.5%) had BKVN, and 33 (12.9%) presented with CMV disease. Four patients (1.5%) developed both infections. Mean time from transplant to diagnosis for BK and CMV was 4.07 ± 3.10 and 8.35 ± 5.20 months, respectively. African American (HR, 2.64; 95% CI, 1.37-5.07; P = 0.003), thymoglobulin induction (HR, 2.18; 95% CI, 1.38-3.43; P = 0.0008), DSA greater than 500 MFI at transplant (HR, 1.64; 95% CI, 1.05-2.57; P = 0.03), history of diabetes (HR, 1.62; 95% CI, 1.01-2.60; P = 0.04), CMV D+/R- (HR, 2.30; 95% CI, 1.06-5.01; P = 0.03), and acute rejection (HR, 1.49; 95% CI, 0.99-2.24; P = 0.05) were associated with increase incident of BK/CMV, whereas rituximab (HR, 0.47; 95% CI, 0.24-0.91; P = 0.02), peak PRA greater than 80% (HR, 0.48; 95% CI, 0.27-0.84; P = 0.01), and living donor transplant (HR, 0.57; 95% CI, 0.36-0.87; P = 0.01) were associated with a lower likelihood of infection. Thymoglobulin induction (HR, 2.50; 95% CI, 1.02-6.13; P = 0.04), and peak PRA greater than 80% (HR, 0.45; 95% CI, 0.23-0.86; P = 0.02) remained significant predictors of infection after multivariate adjustment. Although more than 40% of patients with a positive virtual crossmatch presented with BK infection/CMV disease, high PRA greater than 80% seemed to be protective.