Positive FCXM Research Articles

Intravenous Immunoglobulins Alone for the Desensitization of Lung Transplant Recipients with Preformed Donor Specific Antibodies and Negative Flow Cytometry Crossmatch.

The lack of evidence regarding optimal desensitization strategies for lung transplant candidates with preformed donor specific anti-human leukocyte antigen antibodies (DSAs) has led to varying approaches among centers towards this patient group. Our institution's desensitization protocol for recipients with preformed DSAs and negative flow cytometry crossmatch (FCXM) consists of intravenous immunoglobulin (IVIG) as the sole therapy. The study aimed to determine outcomes using this approach. This retrospective study included adults who underwent lung-only transplantation for the first time between January 2015 and March 2022 at a single center. We excluded patients with positive or missing FCXM results. Transplant recipients with any DSA ≥ 1000 MFI on latest testing within three months of transplant were considered DSA-positive, while recipients with DSAs <1000 MFI and those without DSAs were assigned to the low-level/negative group. Graft survival (time to death/retransplantation) and chronic lung allograft dysfunction (CLAD)-free times were compared between groups using Cox proportional hazards models. Thirty-six out of 167 eligible patients (22%) were DSA-positive. At least 50% of preformed DSAs had documented clearance (decrease to <1000 MFI) within the first 6 months of transplant. Multivariable Cox regression analyses did not detect a significantly increased risk of graft failure (aHR 1.04 95%CI 0.55-1.97) or chronic lung allograft dysfunction (aHR 0.71 95%CI 0.34-1.52) in DSA-positive patients compared to patients with low-level/negative DSAs. Incidences of antibody-mediated rejection (p=1.00) and serious thromboembolic events (p=0.63) did not differ between study groups. We describe a single-center experience of administering IVIG alone to lung transplant recipients with preformed DSAs and negative FCXM. Further studies are required to confirm the efficacy of this strategy against other protocols.

Impact of COVID-19 pandemic on transplant laboratories: How to mitigate?

A positive flow cytometry crossmatch (FCXM) due to donor specific antibodies (DSA) constitutes a risk for kidney transplantation; such a finding may indicates an unacceptable donor for this patient. However, positive FCXM in the absence of DSA is considered discordant and need further investigations. During COVID-19 pandemic, we observed 22% discordant results out of 445 FCXM performed during eight months period in our laboratory and another 7% were invalid due to high background negative control (NC). No study has addressed the impact of COVID-19 pandemic on FCXM and the overall pre-kidney transplant workups or described a solution to deal with these non-specific reactivities. Herein, we analyzed all FCXM results in SARS-CoV-2 seropositive patients and addressed how this pandemic affected significantly the pre-kidney transplant workups, highlighting both technical and financial implications. We also shared our modified FCXM procedures using dithiotheritol (DTT) sera treatment or blocking donor cells with negative control human serum (NCS) which we found to be successful to abrogate 98% of all discordant FCXM results and to validate all invalid results due to high background NC.In conclusion, COVID-19 pandemic has affected our HLA laboratory significantly by creating many false positive or invalid crossmatch results. Transplant laboratories must consider this before test interpretations and immune risk assessments. We recommend the use of DTT serum treatment to remove nonspecific bindings in the sera of kidney transplant candidates and the use of NCS-blocked donor cells to correct high background when performing FCXM in transplant candidates or donors with recent history of SARS-CoV-2 immunization respectively.

Desensitization using IVIG alone for living-donor kidney transplant: impact on donor-specific antibodies.

Sensitization to human leukocyte antigen is a barrier to. Few data have been published on desensitization using polyvalent human intravenous immunoglobulin (IVIG) alone. We retrospectively reviewed the of 45 patients with a positive complement-dependent cytotoxicity crossmatch (CDCXM) or flow cytometry crossmatch (FCXM) against living donors from January 2003 to December 2014. Of these, 12 were excluded. Patients received monthly IVIG infusions (2 g/kg) only until they had a negative T-cell and B-cell FCXM. During the 33 patients, 22 (66.7%) underwent living donor kidney transplantation, 7 (21.2%) received a deceased donor graft, and 4 (12.1%) did not undergo transplantation. The median class I and II panel reactive antibodies for these patients were 80.5% (range 61%-95%) and 83.0% (range 42%-94%), respectively. Patients (81.8%) had a positive T-cell and/or B-cell CDCXM and 4 (18.2%) had a positive T-cell and/or B-cell FCXM. Patients underwent transplantation after a median of 6 (range 3-16). The median donor-specific antibody mean fluorescence intensity sum was 5057 (range 2246-11,691) before and 1389 (range 934-2492) after desensitization (p = 0.0001). Mean patient follow-up time after transplantation was 60.5 (SD, 36.8) months. Nine patients (45.0%). Death-censored graft survival at 1, 3, and 5 years after transplant was 86.4, 86.4, and 79.2%, respectively and patient survival was 95.5, 95.5, and 83.7%, respectively. Desensitization using IVIG alone is an effective strategy, allowing successful transplantation in 87.9% of these highly sensitized patients.

Comparison of kidney transplant outcomes in HLA compatible and incompatible transplantation: A national cohort study.

Reports of HLA incompatible (HLAi) kidney transplant outcomes are inconclusive, especially in the context of lower level Donor Specific Antibodies (DSA). Multi-centre national cohort study of HLAi kidney transplant recipients matched in 1:2 ratio with HLA compatible (HLAc) kidney transplant recipients. HLAi defined as DSA identified by Luminex. Antibody mediated rejection (AMR) and transplant-survival were analysed using Kaplan-Meier plots. Propensity score (PS) matching was used to compare recipient and transplant survival between groups. We included 61 HLAi and 122 HLAc recipients; mean age 46 years; 60% female. MFIT0 : 3327 (IQR 1352-6458), 23 (38%) were Flow cytometry crossmatch positive (FC-XMPOS ). DSAPOS /FC-XMPOS transplantation carried an increased risk of AMR at 1year (52%) compared to DSAPOS /FC-XMNEG (27%) and HLAc (0%). Unadjusted death censored graft loss at 3 years was 13% (HLAi) and 8% (HLAc). Three-year patient survival was 95% in HLAc, 84% in DSAPOS /FC-XMNEG and 69% in DSAPOS /FC-XMPOS recipients; 58% of HLAi deaths were infection-related. HLA incompatibility was associated with a decreased 3-year survival in our PS-matched cohort. In kidney transplantation, DSA and positive FC-XM carries an increased risk of AMR. Despite inferior transplant and survival outcomes compared to HLAc transplantation, it remains a realistic option for highly sensitized patients facing prolonged waiting times and reduced survival on dialysis.

MO1002: Two-Step Desensitization Protocol Consisting of High-Dose Intravenous Immunoglobulin, Plasmapheresis and Rituximab in Highly Sensitized Kidney Transplantation

Abstract BACKGROUND AND AIMS High-dose intravenous immunoglobulin (IVIG) therapy has not only immediate effects but also gradual effects via Fc receptors. To optimize IVIG therapy, we adopted a two-step desensitization protocol in kidney transplantation (KTx) with positive flow cytometry crossmatch (FCXM). METHOD In the first step, rituximab 200 mg and IVIG 2 g/kg were administered 2 months before KTx. In the second step, patients received additional rituximab 200 mg and IVIG 2g/kg for 2 weeks prior to transplantation in combination with plasmapheresis. RESULTS Of the six KTx with positive FCXM, there was no rejection except one (16.7%) subclinical antibody-mediated rejection within 1 month after transplantation. Average graft function was acceptable (serum creatinine level of 1.17 mg/dL). CONCLUSION The two-step desensitization protocol is an effective option for achieving successful transplant outcomes in KTx with positive FCXM, which can spare more aggressive and expensive treatments, such as C5 inhibitors, proteasome inhibitors and imlifidase.

Determination of unacceptable antigens by summation of anti-HLA eplet antibody strength (MFI) based on single antigen bead assays: Excellent correlation with negative cell based cross matches

The reliability of single antigen bead (SAB) assays and their use in predicting a negative cell based cross match (CBXM) is essential in the era of expanded organ sharing. A wide range of accuracy (80–95%) in predicting negative CBXM has been reported. We hypothesized that in SAB assays an antibody against an HLA eplet that was common among a number of different HLA alleles would be distributed among all of the shared eplet positive SABs. This would reduce binding to the donor specific SAB resulting in an under-estimate of antibody strength. We tested this proposal in adsorption studies using, instead of lymphocytes, a novel reagent, single-SAB (sSAB). Properties of SAB assays were examined that provided a basis for conducting adsorption – elution experiments with the sSABs. We found that incubation of sera with sA*02:01 or sB*42:01 not only depleted reactivity to these alleles but also depleted reactivity to beads that shared the reactive eplet. Anti-eplet strength from SAB data (sum of the MFI of eplet positive SABs (MFI-s) was compared with CBXM out comes in two case studies and with 99 proficiency testing sera. In these validation studies, an MFI-s above 11,000 was associated with a positive FCXM. This approach was placed into clinical practice for listing unacceptable antigens that shared a common eplet. CDCXMs (n = 3261) and FCXMs (n = 1012) were performed on patients listed in UNOS for deceased donor kidneys. All CDCXMs were negative and all FCXMs except one were negative. We conclude that summation of eplet strength provides a highly reliable method of predicting prospective negative CBXMs resulting in substantial savings of time and effort. Based on shared eplet summation data, CMS/NYSDOH has accepted our bead based XM (BBXM) method (aka, virtual XM) performed prior to transplant as fulfilling the regulation that XM results be available before kidney transplantation.

Outcomes of Flow Cytometry Crossmatch-Positive Lung Transplants Using Peri-Operative Desensitization

<h3>Purpose</h3> Successful outcomes among virtual crossmatch (VXM)-positive lung transplant recipients (LTRs) following peri-operative desensitization have been previously reported. However, the subset of flow cytometry crossmatch (FCXM)-positive LTRs, who may be at higher immunologic risk, has not been studied specifically. We conducted a retrospective cohort study to determine the impact of a positive FCXM on allograft survival (death/re-transplant) and chronic lung allograft dysfunction (CLAD)-free survival among LTRs in our program. <h3>Methods</h3> All adult, first-time LTRs from January 2014 to December 2019 with complete crossmatch data were divided into 3 cohorts based on sensitization status at the time of transplant: VXM-negative, VXM-positive/FCXM-negative, and VXM-positive/FCXM-positive. Patients were followed until October 2021. VXM-positive LTRs were routinely desensitized using plasma exchange, intravenous immune globulin, and anti-thymocyte globulin peri-operatively. Patient characteristics were compared using the chi-square test, and Kaplan Meier estimates of allograft survival and CLAD-free survival were compared using the log-rank test. <h3>Results</h3> A total of 902 LTRs were included: 765 (85%) VXM-negative, 63 (7%) VXM-positive/FCXM-negative, and 74 (8%) VXM-positive/FCXM-positive. VXM-positive/FCXM-positive LTRs were more likely to be female (p<0.001) and less likely to have cystic fibrosis/bronchiectasis (p=0.022), but age and urgency status at listing did not differ from the other cohorts. There was no significant difference in allograft survival (p=0.9805) or CLAD-free survival (p=0.9306) among VXM-negative, VXM-positive/FCXM-negative, and VXM-positive/FCXM-positive LTRs (Figure 1). <h3>Conclusion</h3> In the largest study to date of FCXM-positive LTRs, we demonstrate that with our peri-operative desensitization protocol, outcomes are equivalent to other LTRs. Our protocol may be used to facilitate VXM-positive and FCXM-positive lung transplants safely.

Predictive Potential of Flow Cytometry Crossmatching in Deceased Donor Kidney Transplant Recipients Subjected to Peritransplant Desensitization.

Recipient sensitization is a major risk factor of antibody-mediated rejection (ABMR) and inferior graft survival. The predictive effect of solid-phase human leukocyte antigen antibody testing and flow cytometry crossmatch (FCXM) in the era of peritransplant desensitization remains poorly understood. This observational retrospective single-center study with 108 donor-specific antibody (DSA)-positive deceased donor kidney allograft recipients who had undergone peritransplant desensitization aimed to analyze variables affecting graft outcome. ABMR rates were highest among patients with positive pretransplant FCXM vs. FCXM-negative (76 vs. 18.7%, p < 0.001) and with donor-specific antibody mean fluorescence intensity (DSA MFI) > 5,000 vs. <5,000 (54.5 vs. 28%, p = 0.01) despite desensitization. In univariable Cox regression, FCXM positivity, retransplantation, recipient gender, immunodominant DSA MFI, DSA number, and peak panel reactive antibodies were found to be associated with ABMR occurrence. In multivariable Cox regression adjusted for desensitization treatment (AUC = 0.810), only FCXM positivity (HR = 4.6, p = 0.001) and DSA number (HR = 1.47, p = 0.039) remained significant. In conclusion, our data suggest that pretransplant FCXM and DSA number, but not DSA MFI, are independent predictors of ABMR in patients who received peritransplant desensitization.

The utility of cytolytic flow cytometry crossmatch before kidney transplantation

The donor/recipient matching in kidney transplantation is based on approved laboratory tests, which are complement-dependent cytotoxic crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM). Both have some disadvantages: CDC-XM has low sensitivity, whereas FCXM does not differentiate between lytic vs. non-lytic alloantibodies. To find an improved method, we have developed a new crossmatch technique of cytolytic flow cytometry crossmatch (cFCXM), which allows for sensitive detection of clinically relevant complement-binding antibodies. The cFCXM assay detects dead cells with viability dye that ensue from the binding of allospecific lytic antibodies. In our study, 135 unsensitized kidney transplant recipients were recruited based on the CDC-XM and FCXM results and the clinical utility of cFCXM was evaluated. The 5-year follow-up for acute rejection incidents revealed that cFCXM could verify the clinical relevance of positive FCXM results as recipients with positive FCXM but negative CDC-XM had the same risk of rejection as patients with both negative CDC-XM/FCXM results. These findings suggest that cFCXM assay may provide more precise immunological risk assessment in kidney transplant recipients.

Renal transplantation against a positive crossmatch due to HLA-DP donor-specific antibodies without prior antibody removal – Case report

Background: Antibodies directed against donor mismatched Human Leucocyte Antigens (HLA) are known to cause antibody-mediated rejection and affect the graft survival of transplanted organs. The influence of donor-directed antibodies against the products of HLA class I and II genes in renal transplantation are well described for class I (HLA-A, B and C), but still not clear for class II notably HLA DP. The clinical effects of donor-specific antibodies (DSA) directed against HLA-DP are still controversial.Methods: We report the outcome of kidney transplants in three highly sensitised individuals with significant sensitisation with donor-directed HLA-DP antibody. These recipients were on the waiting list for 6, 14 and 4 years, respectively and had kidney transplants from donors after brain death (DBD) with positive B cell flow cytometry crossmatch (FCXM). Two cases received induction therapy with a depleting antibody (Thymoglobulin®) while the third received Alemtuzumab followed by standard immunosuppression.Results: The clinical course of these three patients were different. The first patient developed transplant glomerulopathy, but the graft is still functioning with eGFR 29 mL/min/1.73 m2. The second patient did not have any adverse event with eGFR 26 mL/min/1.73 m2, while the third patient had severe antibody-mediated rejection (AMR), which was treated successfully with eGFR 32 mL/min/1.73 m2.Conclusion: Patients waiting for a deceased allograft with HLA-DP DSA and a positive FCXM can be transplanted successfully with depleting antibody or Alemtuzumab induction without prior antibody removal followed by standard immunosuppression.

P1791HLA DESENSITIZATION- INDIAN EXPERIENCE

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

Pre-Formed Donor-Specific Antibodies Impact Long Term Survival of Heart Transplants

We previously reported that heart transplant (HTx) recipients with pre-formed, anti-HLA antibodies at low titer had one-year survival that was comparable to patients without any anti-HLA antibodies. As a result, we defined unacceptable HLA antigens by MFI>1000 in serum at 1:16 dilution. The current study evaluates how pre-formed, low-titer, donor-specific antibodies (DSA) and positive crossmatch affect long term survival of HTx recipients. Total 495 adult recipients transplanted at our center between Jan. 2007 and Dec. 2016 were studied. Prior to HTx, recipients had HLA antibody screening (FlowPRA) and specificity testing (LABScreen). Unacceptable HLA antigens were defined by MFI>1000 in serum at 1:16 dilution. Acceptable donors were selected by virtual crossmatch using the criteria above, and flow cytometry crossmatch (FCXM) was performed peri- or post-HTx. Recipients transplanted across low titer DSA, positive in undiluted serum but negative at 1:16 dilution, received peri-operative plasmapheresis. We used log-rank testing to evaluate survival in the following groups compared to their controls: (1) presence of any pre-formed HLA antibodies; (2) presence of pre-formed DSA; and (3) positive FCXM. Of 495 HTx recipients, 179 (36%) had pre-existing HLA antibodies, and their overall survival was not different from patients without HLA antibodies (p=0.883). However, median survival time was reduced in patients with pre-formed DSA (n=62), compared to DSA-negative recipients (n=117): 9.1 years (95% CI 3.52 to 14.65) vs. 10.9 years (95% CI 6.13 to 15.70); p=0.02. The survival rates for 1-, 3-, 5-year of DSA-negative recipients were 91.5%, 85.4% and 79.5%, and the DSA-positive recipients were 83.9%, 66.1% and 61.7%, respectively. Total 62 recipients had pre-HTx DSA. Among them, 26 had a positive FCXM (46%). The survival rates of FCXM-positive cases were lower than FCXM-negative cases (n=36) at 1, 3, and 5 years: 80.8%, 57.9%, 52.1% vs. 86.1%, 71.4%, 67.6%. Our findings suggest that pre-formed DSA, even at low titer, negatively impacted HTx survival. Cases with positive FXCM had the worst outcomes. Avoiding all pre-formed DSA may improve survival for HTx patients, but may increase waitlist mortality. To improve the HTx survival, close post-transplant monitoring and proactive immunological management may be required when pre-formed DSA are present.

Significance of Luminex-based single antigen class II bead assay and its mean fluorescence intensity in renal transplant cases; a retrospective observation in 97 cases

ABSTRACTThe objective of this study was to determine the mean fluorescence intensity (MFI) values of class II Luminex single antigen bead (L-SAB) assay and compare these MFI values with cell-based complement-dependent cytotoxicity crossmatch with anti-human globulin (CDC-AHG-XM) and IgG-B-cell flow cytometry crossmatch (FC-XM) results and explore the near-accurate MFI-cutoff values of positive cell-based crossmatch results. This retrospective study was an analysis in 97 renal transplant recipients, who were tested for the presence of DSA by CDC-AHG-XM and IgG-B-cell-FC-XM methods with their corresponding donor as well as for anti-human leukocyte antigen (HLA) antibody detection using a sensitive L-SAB assay. In the group having DSA MFI values <1000, none of the patients showed positivity for FC-XM and CDC-AHG-XM; in the group having MFI values between 1000 and 3000, 35.48% showed positivity for FC-XM but none by the CDC-AHG-XM method. However, in the group having MFI values >3000, 83.33% of cases were positive for FC-XM. Further, in those groups with MFI values between 3000 and 6000, 38.09% were positive for CDC-AHG-XM, while 86.66% showed positivity in the group with MFI >6000. Our results indicated that Luminex-DSA MFI value >1995 (P < .0001) significantly correlated with IgG-B-cell-FC-XM positivity while Luminex-DSA MFI value of >4247 (P < .0006) was significantly correlated with positive CDC-AHG-XM. MFI cutoff of 1995 exhibited a diagnostic sensitivity of 97.56% and specificity of 89.29% for predicting positive IgG B-cell FC-XM and MFI cutoff of 4247 exhibited a diagnostic sensitivity of 90.48% and specificity of 97.37% for predicting positive CDC-AHG-XM. However, a cutoff MFI of >5000 and >7000 for SAB assay had a sensitivity and specificity of 100% in detecting a positive IgG B-cell FC-XM and CDC-AHG-XM, respectively.

Patients with immunological diseases or on peritoneal dialysis are prone to false positive flow cytometry crossmatch

Despite implementation of virtual crossmatches, flow cytometry crossmatches (FCXM) are still used by many transplant centers to determine immunological risk before kidney transplantation. To determine if common profiles of patients prone to false positive FCXM exist, we examined the demographics and native diseases of kidney patients tested with autologous FCXM (n = 480). Improvements to FCXM and cell isolation methods significantly reduced the positive rate from 15.1% to 5.3%. Patients with native diseases considered ‘immunological’ (vasculitis, lupus, IgA nephropathy) had more positive autologous FCXM (OR = 3.36, p = 0.003) vs. patients with all other diseases. Patients who were tested using our updated method (n = 321) still showed that these immunological diseases were a significant predictor for positive autologous FCXM (OR = 4.79, p = 0.006). Interestingly, patients on peritoneal dialysis (PD) also had significantly more positive autologous FCXM than patients on hemodialysis or waiting for pre-emptive kidney transplants (OR = 3.27, p = 0.02). These findings were confirmed in patients who had false positive allogeneic FCXM. Twenty of 24 (83.3%) patients with false positive allogeneic FCXM tested with updated method either had immunological diseases originally or were on PD. Our findings are helpful when interpreting an unexpected positive FCXM, especially for transplantation from deceased donors.

P010 Heterogeneous flow cytometry crossmatch reactions in the presence of patient sera with pan-dr antibodies

Aim During routine clinical evaluations of antibody profiles using single antigen bead (SAB) assays, several instances of widespread reactivity exclusively to DR antigens (‘pan-DR’ antibodies) were observed. Additional assays were performed to investigate reactivity of these pan-DR positive sera against different antigenic conformations present on bead products and donor cells. Methods Four serum samples taken two months apart from two hematopoietic cell transplant candidates were evaluated. Mixed bead and C1q assays (One Lambda, Inc) were performed to verify that the antibodies being detected were indeed against DR antigens and not cryptic epitopes that are present on SAB. Flow cytometry crossmatch (FCXM) was performed against two different donor cells. ELISA was performed to assess the involvement of AT1R antibodies in positive FCXM reactions based on reports of the capacity of anti- AT1R to cause positive FCXM. Results All sera were negative for Class II antibodies using mixed beads and C1q screening assays, contradicting the Pan-DR reactions with SAB. From patient#2, FCXM showed that serum #3 was T+/B + crossmatched against cell #1 but became T-/B- crossmatched against cell #2. From patient #1, serum #1 was borderline T+/B + when crossmatched against cell #1 but became T+/B- when crossmatched against cell #2. FCXM with all serum samples from both patients against cell#2 were negative. Additionally, auto-crossmatches for both patients were negative despite the presence of auto-antibodies. There was no association between AT1R antibody level and pan-DR observation. Conclusions The presence of antibodies against both naive and denatured antigens was indicated in pan-DR positive SAB/negative mixed beads, and in the positive T-cell FCXM and/or B-cell FCXM reactions. It is plausible that pan-DR antibodies described by the SAB data are not HLA antibodies at all but rather non-HLA antibodies with cross reactivity between cryptic HLA-DR antigens and yet unidentified non-HLA epitopes on lymphocytes.

P118 Reliability of virtual crossmatch: Reality of perception

Aim In this study we evaluated the concordance between vFXM and aFXM using SAB and a predetermined cut off point that has been established in our laboratory. Methods AXM: 773 crossmatches performed by FCXM were retrospectively analyzed. The cut off for T &B are 64, 100 respectively. This includes 573 crossmatches performed using PB/ or spleen deceased donor cells with potential recipients & 200 consecutive final crossmatches performed for living transplantation. VXM: Donor HLA- antigens were compared against the potential recipient’s antibody specificities. Prediction of vFXM was based on antibody identification using SAB. Normalized MFI values of 3000 were used to identify antibodies predicted to cause a positive FCXM for A, B, DR &5000 for HLA-C and DQ specificities when present as single antibody. A sum of multiple weak antibodies (MFI ⩽ 2000) that yield MFI of ⩾3000 was used to predict positivity. Results Only positive FCXM positive for B or B & T simultaneously are considered. The vast majority of aFXM were in concordance with vFXM with an overall concordance of 97%. Twenty-three predicted to be negative showed positive aFXM, 12 of them had 0% cPRA and 11 were found in patients with multiple non DSA- HLA- antibodies, dilution of these sera revealed weak DSA. Three predicted positive vFXM yield negative aFXM, two of them had allele specific antibodies. (Table 1). Because sera of highly sensitized patients are diluted we rarely deceived by prozone phenomenon. Therefore the only cause of false negative VXM is auto or non HLA- antibodies. Due to the extreme sensitivity of SAB, reporting false positive VXM remains a possibility and will continue to be a limitation for VXM. Conclusions vFXM based on precise characterization of antibody specificities detected by SAB using our cutoff point accurately predicted FXM in the majority of patients and can be used safely to allocate kidney offers without performing physical crossmatches in selected patients. Download : Download high-res image (61KB) Download : Download full-size image

P138 Clinical characteristics of patients who manifest unexplained crossmatch results for solid organ transplantation

Aim The flow cytometric crossmatch (FCXM) and the virtual crossmatch (VCXM) assess compatibility between solid organ donors and recipients. Theoretically, both methods should give the same results. Realistically, however, they can be discrepant due to limitations of assays used to detect alloantibodies, non-HLA alloantibodies, autoantibodies, and non-specific reactivity. We sought to identify clinical features of patients who manifest such discrepancies, hypothesizing an association with female gender, autoimmune disease, and potential heart recipients. Methods We retrospectively evaluated all living and deceased donor FCXMs (performed using BD FacsCanto flow cytometer, BD Biosciences, San Jose, CA) for solid organ transplant offers for patients at University of California San Diego from 2015 to 2017. Based on previous validation studies, a positive FCXM was coded as ”expected” when a single donor-specific HLA antibody (detected using LABScreen single antigen assay, One Lambda, Canoga Park, CA) demonstrated a mean fluorescent intensity of 3,000 or higher, or when the sum of multiple antibodies reached that threshold. Deceased donor typing was performed using LinkSeq real-time polymerase chain reaction kit (Linkage Biosciences, South San Francisco, CA). Living donor HLA genotyping was performed by SSO or NGS-based methods. FCXMs with unexpected positive results were investigated. Results 1594 FCXMs (1369 for kidney, 139 for heart, 47 for lung, and 39 for multiple organs) for 967 potential recipients (621 males, 346 females) were analyzed. Unexpected positive results occurred in 109 T cell FCXMs in 81 patients (8%) and 40 B cell FCXMs in 23 patients (2%). Analysis by both crossmatch and patient demonstrated correlations with female gender (T cell p Conclusions Unexpected positive FCXMs correlate with female gender, but not with transplant organ. In 5 cases with persistent unexpected positive FCXMs, we have successfully transplanted patients using the VCXM.

P139 Flow crossmatch thresholds: Where (and how) do we draw the line?

Aim Flow cytometry crossmatch (FCXM) is used to assess pre-transplant immunologic risk. Each laboratory must establish FCXM positive thresholds but the method to do so is not standardized. Many factors affect FCXM and its interpretation. Our aim was to compare methods of calculating FCXM thresholds and evaluate the impact of cell source and pronase. Methods Threshold study (TS) data were collected by FCXM with healthy control sera negative for HLA antibodies (n = 24) vs pronased, negatively selected T & B cells from either peripheral blood (PBL), fresh or frozen spleen (n = 264, 215, and 96, respectively). Non-pronased FCXM were tested in parallel (n = 72). Data normality tests were performed on this and previous TS data. Outlier values were assessed by 1.5 interquartile range (IQR) and standard deviation (SD). Thresholds were calculated by mean or median + varying SD as well as 97.5th percentile. Thresholds were assessed in expected positive FCXM (PBL & spleen) with/without pronase. Results Median channel values (MCV) were not normally distributed in either TS dataset (Fig. 1). Thresholds vary by cell source: fresh spleen Conclusions Non-parametric means of assessing FCXM thresholds may be required. Cell source affects FXCM sensitivity for class I antibodies, perhaps due to number of T vs B cells. We have previously shown that pronase increases class I detection on T cells; these FCXM data support this finding; preliminary thresholds calculated from non-pronased cells do not correct this reduced sensitivity. A thorough understanding of factors that affect FCXM will optimize determination of relevant thresholds and assist in the interpretation of this commonly used assay.

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

P011 HLA-B13 donor-specific antibodies yield atypical flow cytometry crossmatch outcomes

Aim To investigate observations of high rates of discrepancies between virtual crossmatch (VXM) positive and flow cytometry crossmatch (FCXM) negative cases in the presence of HLA-B13 DSA. Methods Sera with antibodies against HLA-B*13:01 and/or B*13:02 falling within a MFI ranging from 3286 to 11793, as determined by LABScreen Single Antigen beads (SAB) (One Lambda), were used in FCXM against cells expressing HLA-B*13:01, B*13:02 (sera n = 8, surrogate cells n = 5). Each serum sample was obtained from a unique donor. FCXM with non-B13 HLA class I DSA of a similar MFI were also conducted for comparison to B13 FCXM data. A linear regression analysis was conducted to determine correlation between MFI and MCS for both B13 and non-B13 DSA. Results Sera containing non-B13 DSA had a significant correlation between MCS and MFI values for both T (p 5000 producing positive reactions in only 66.7% of cases, compared to non-B13 DSA producing all positive FCXM reactions for DSA MFI > 5000. Additionally, of the five B13 sera tested on more than one cell, only two sera (#4 & #8) gave the same result (Fig. 1). Sera #1, 5 and 7 each resulted both negative and positive reactions. Conclusions High incidences of discrepancies in B13 FCXM indicate that the unpredictable nature of antigen availability at the cell surface occurs extensively in HLA-B13. One factor in these weak antigen-antibody interactions may be a lower salt bridge formation deduced from decreased protein-ligand interaction between B13 and KIR3DL1, which is unlike the other Bw4 + alleles. Another possibility, is that affinity level of anti-B13 reactions may be susceptible to certain eplets accessible only in naive antigen conformation. For certain specificities, including B13, it will be vital to evaluate FCXM reactions to avoid unnecessarily eliminating potential donors.