Positive Flow Cytometry Crossmatch Research Articles

Negative virtual crossmatch (vXM) with positive flow cytometry crossmatch (FCXM) in absence of prozone and DSA can be due to HLA-C epitope-specific antibodies

Negative virtual crossmatch (vXM) with positive flow cytometry crossmatch (FCXM) in absence of prozone and DSA can be due to HLA-C epitope-specific antibodies

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.

P100 HLA-Cw epitope analysis can resolve positive physical flow cytometry crossmatch in absence of donor-specific antibodies and prozone effect – A case study

P100 HLA-Cw epitope analysis can resolve positive physical flow cytometry crossmatch in absence of donor-specific antibodies and prozone effect – A case study

Kidney transplant outcomes in patients with antibodies to human neutrophil antigen 3a

BackgroundAntibody mediated rejection (ABMR) of kidney transplants has been shown to occur in the absence of a known donor specific antibody to human leucocyte antigen (HLA). Antibodies to the human neutrophil antigen (HNA) system have been detected in kidney transplant recipients and linked to ABMR in the absence of an HLA donor specific antibody (DSA), but there remains limited literature regarding this. MethodsCase series analysis was carried out examining three cases of HNA-3a antibody positive flow cytometry cross match (FC-XM) from two transplant centres in Scotland. ResultsAll patients included were female and had been sensitised as a result of pregnancy. One live donor recipient with HNA-3a antibodies identified prior to transplant received ATG induction and has had a good outcome. The remaining two patients received deceased donor transplants. HNA-3a antibodies were indicated following a retrospective flow cytometry crossmatch. Both patients received Basiliximab induction and both have experienced ABMR requiring supplementary immunosuppression. ConclusionsThe predicted rate of HNA-3a antibodies amongst patients awaiting kidney transplant in the UK is <1%. However, with increasing evidence to support a role for HNA-3a antibodies in the development of ABMR there may be value in screening at risk groups to allow for augmented immunosuppression to be considered at the time of kidney transplant.

#6257 PROGNOSTIC VALUE OF A POSITIVE CROSSMATCH BEFORE DESENSITIZATION IN A COHORT OF HIGHLY-SENSITIZED PATIENTS

Abstract Background and Aims Human Leukocyte antigen (HLA) sensitization in patients is a barrier for Kidney Transplantation (KT) access, and highly-sensitized patients remain on the waiting list longer than other patients which results in a higher morbidity and mortality. Desensitization may be an option to increase their access to KT. All patients transplanted in this context have a negative Complement-Dependent Cytotoxicity crossmatch (CDC-CM) the day of KT. However, the stratification of the long-term risk of graft loss according to flow cytometry crossmatch (FC-CM) and to CDC-CM during the desensitization period remains unknown. Method In this retrospective study, all highly-sensitized patients (Panel Reactive Antigen (PRA) ≥ 85) in our center were included. CDC-CM and FC-CM were performed on samples before and during the desensitization period and clinical follow-up data were retrieved. Pre-KT desensitization strategy consisted in two Rituximab infusions (375mg/m2) and a variable number of apheresis (mostly semi-specific immunoadsorption) associated with a classical immunosuppression-based regimen (tacrolimus, mycophenolate and steroids). Results A total of 183 highly-sensitized recipients received a KT between 2014 and 2022. Of these, 23 were desensitized prior to KT and among them 18 had a positive FC-CM before KT and 5 had no positive FC-CM on all assessed serums. Mean age was 52.9 ± 13 years. Mean follow-up was 5.2±4 and 3±2 years for non-desensitized and desensitized patients respectively. PRA before desensitization was higher in desensitized patients (98%) versus non-desensitized patients (95%) and the mean time on the waiting list was 5.8±4 versus 7.5±4 years respectively. Death-censored graft survival and patient's raw survival, using cox-survival analysis, were not statistically different between the desensitized kidney transplanted patients and the highly-sensitized patients without desensitization (p = 0.77 and p = 0.57 respectively). In sub-group analyses, the positivity of FC and/or CDC-CM did not add an additional risk of graft loss or patient mortality. Conclusion Desensitization allows highly-sensitized patients to access KT with an HLA-incompatible graft (with a positive CDC or FC-CM on the serum collected prior to desensitization), with the same graft survival as similarly sensitized patients transplanted with an HLA-compatible graft without desensitization.

Desensitizing With Temporary Donor Splenic Transplant: Hope for the Sensitized Patients on Pancreas and Kidney -Pancreas Transplant Waitlist

Sensitized patients on a waitlist with donor specific antibodies (DSA) or positive flow cytometry cross match (FXM) to deceased donor organ have few pretransplant desensitization options due to increasing graft cold ischemia time. Herein, sensitized simultaneous kidney/pancreas recipients received temporary splenic transplant from the same donor under the hypothesis that spleen would function as a DSA graveyard and provide a safe immunologic window for transplant. We analyzed presplenic and postsplenic transplant FXM and DSA results of 8 sensitized patients who underwent simultaneous kidney and pancreas transplantation with temporary deceased donor spleen between November 2020 and January 2022. Pre-splenic transplant, 4 sensitized patients were both T-cell and B-cell FXM positive; one was only B-cell FXM positive and 3 were DSA positive/FXM negative. Post-splenic transplant, all were FXM negative. Pre-splenic transplant class I and class II DSA were detected in 3 patients, only class I DSA in 4 patients, and only class II DSA in 1 patient. Postsplenic transplant, class I DSA was eliminated in all patients. Class II DSA persisted in 3 patients; all showed a marked decrease in DSA mean fluorescence index. Class II DSA was eliminated in one patient. Donor spleen functions as a DSA graveyard and provides an immunologically safe window for kidney-pancreas transplantation.

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.

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.

T-Cell-Positive, B-Cell-Negative Flow Cytometry Crossmatch: Frequency, HLA Locus Specificity, and Mechanisms Among 3073 Clinical Flow Cytometry Crossmatch Tests.

A T-cell-positive and B-cell-negative flow cytometry crossmatch result remains a conundrum since HLA class I antigens are expressed on both T and B cells. We investigated the frequency, donor HLA specificity of the antibodies, and mechanisms for these crossmatch results. We analyzed 3073 clinical flow cytometry crossmatch tests performed in an American Society of Histocompatibility and Immunogeneticsaccredited histocompatibility laboratory. The sera associated with the T-cell positive and B-cell negative flow cytometry crossmatches were also tested for donor HLA immunoglobulin G antibodies using LABScreen single antigen assays. Among the 3073 test results, 1963 were T-cell negative and B-cell negative, 811 were T-cell negative and B-cell positive, 274 were T-cell positive and B-cell positive, and 25 were T-cell positive and B-cell negative. The LABScreen single antigen assay detected HLA class I immunoglobulin G donor-specific antibodies in 23 of 25 sera associated with a T-cell positive and B-cell negative flow cytometry crossmatch result, and donorspecific antibodies directed at not only HLA-Cw but also at HLA-A or HLA-B were observed. In addition, we identified that the B-cell channel shift threshold used to classify a B-cell flow cytometry crossmatch was a potential contributor to a T-cell-positive and B-cellnegative flow cytometry crossmatch result. Our analysis of 3073 flow cytometry crossmatches, in addition to demonstrating that HLA antibodies directed at the HLA-A, -B, or -Cw locus were associated with a T-cell-positive and B-cell-negative result, identified mechanisms for the surprising T-cell-positive and B-cell-negative flow cytometry crossmatch result.

Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw antibodies in kidney transplantation.

Anti-denatured HLA-Cw antibodies are highly prevalent, whereas anti-native HLA-Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti-HLA-Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti-denatured and anti-native HLA-Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA-Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty-three (31.6%) were anti-denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ=0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ=0.289). We studied 52 kidney recipients with preformed anti-HLA-Cw donor-specific antibodies. Those with anti-native HLA antibodies experienced more acute and chronic antibody-mediated rejections (P=.006 and .03, respectively), and displayed a lower graft survival (P=.04). Patients with anti-native HLA-Cw antibodies more frequently had previous sensitizing events (P<.000001) or plausibility of their antibody profile according to known anti-native HLA-Cw eplets (P=.0001). Anti-native but not anti-denatured HLA-Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA.

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.

P151 Eliminating false positive flow cytometric crossmatches due to monoclonal antibody therapy

Aim Biologics are increasingly used in autoimmune, cancer, and transplant patients. Unfortunately they can also interfere in laboratory tests. Monoclonal antibodies, such as Rituximab, are well known to cause positive flow cytometry crossmatches (FCM) independent of donor specific HLA antibody (DSA). We assessed the ability of blocking antibodies to Rituximab (RTX) and Alemtuzumab (ALM) to negate interference by these biologics in the FCM. Methods FCM was performed with serum from patients receiving RTX and/or ALM. Some sera were pretreated with anti-Rituximab ( α RTX) and/or anti-Alemtuzumab ( α ALM) blocking antibodies (Bio-Rad, Inc). HLA DSA was determined using multiplex bead arrays (LabScreen Single Antigen, Thermofisher, Inc.). FCM channel shifts (CS) were compared between treated sera and sera treated with PBS as a control. Results Blocking antibodies were titrated in serum to determine the working dilution to use in subsequent studies. Sera from patients receiving RTX and/or ALM were treated with the predetermined dilution of blocking antibody. Blocking antibody pre-treatment reduced FCM CS with surrogate donors to levels comparable to PBS controls: RTX blocking: pre-treatment CS = 23 T and 302 B; post-treatment CS = 35 T and 29 B; ALM blocking: pre-treatment CS = 262 T and 236 B; post-treatment CS = 34 T and 2 B. A highly sensitized (cPRA = 99%; DSA ;= B35, Cw4, DR17, DR52, DQ5) pediatric renal transplant candidate (RTX and ALM treated) was crossmatched with their living donor using pre-transplant and 4 day post-transplant (post-RTX and ALM treatment) sera. Post-treatment CS were increased without blocking but reduced with blocking antibody treatment. Blocked FCM CS results correlated better with reduced DSA MFI summed values in contrast to the unblocked serum (Table 1). Conclusions Antibodies to RTX and ALM effectively block interference by these biologics in FCM resulting in the ability to provide useful FCM data for patient management.

P142 Pronase treatment can cause false positive T-cell flow cytometry crossmatches in patients with autoimmune diseases: two clinical cases

Aim Pronase treatment of lymphocytes for flow cytometry crossmatching is used to reduce non-specific B-cell reactivity due to the binding of immune complexes to Fc receptors. However, pronase has been shown to produce false-positive T-cell crossmatches in patients with HIV. We have also encountered this phenomenon with patients suffering from autoimmune diseases. In two cases, the observed T-cell reactivity decreased significantly when sera were crossmatched against unpronased cells. Methods Crossmatches were performed according to standard flow cytometry protocols. Antibodies were assessed with One Lambda Luminex single antigen with a positive cutoff of 2000 MFI. Results Case 1 is a 15 year old African-American male listed for kidney transplant. He has a primary diagnosis of SLE and no sensitization history. Pre-transplant medication history is notable for prednisone and cyclosporine. Sera were crossmatched against pronased deceased donor cells, with T-cell positive results in the absence of DSA. This reactivity disappeared when the crossmatch was repeated with unpronased cells. Case 2 is a 40 year old African-American female listed for kidney/pancreas transplant. She has a primary diagnosis of type 1 diabetes and a sensitization history of 2 pregnancies and moderate transfusions, none recent. Pre-transplant medication history does not include any immunoactive regimens. She is mildly sensitized to Class I and not sensitized to Class II. Sera across several years exhibited positive T-cell crossmatches against multiple cells of type A2. She has A2 antibodies with low titers (2600 MFI). No other DSA was involved. T-cell reactivity was reduced to negative ranges when unpronased cells from A2 donors were used. Conclusions In both cases, auto flow cytometry crossmatches were negative. Therefore, autoreactive antibodies recognizing cryptic epitopes that might be exposed after pronase treatment does not fully explain these results. Neither patient received rituximab at any time. Patients suffering autoimmune diseases may exhibit false positive T-cell crossmatches against pronased cells, in the same manner that has been documented for HIV patients. This reactivity is not attributable to DSA and disappears or significantly decreases when crossmatching is performed with unpronased cells.

Intravenous Immunoglobulin and Rituximab in HLA Highly Sensitized Kidney Transplant Recipients

IntroductionHLA-sensitized patients are penalized both in the access to kidney transplantation (KT) and, once transplanted, in the incidence of rejections and long-term allograft survival despite aggressive induction and maintenance therapy. MethodsThis study retrospectively evaluates the impact of combining T- and B-cell–depleting agents and intravenous immunoglobulin for induction therapy in 45 highly sensitized KT patients (anti-panel reactive antibodies >60%, positive flow cytometry crossmatch or donor specific antibodies at the time of transplantation). The outcome data included the occurrence of biopsy-proven acute rejection, new-onset proteinuria, development of leukopenia, incidence of poliomavirus infection (BK or JC virus), fungal or bacterial infection after KT, de novo neoplasia, graft function, graft loss, or death with functioning KT. ResultsThe average panel reactive antibody was 62.5%; 41 patients (91.1%) had ≥3 HLA mismatches with the donor and 91.1% of patients had class I or II anti-HLA antibodies. Fourteen patients (31.1%) presented pre-KT donor-specific antibodies and 6 patients (13.3%) had a positive flow cytometry cross-match at the time of transplantation. The incidence of acute rejection in the first 6 months was 24.4% and the cumulative incidence was 37.8%. Two patients were diagnosed with leukopenia in the first 6 months after KT. Two patients (4.5%) had cytomegalovirus disease, 17 patients (37.8%) were diagnosed with bacterial infections. Cutaneous neoplasms were identified in 5 patients (11.1%) and solid tumors in 4 (8.9%). The death-censored graft survival was 100% in the first 6 months and 93.5% at the last evaluation. Patient survival in the same periods was 97.8% and 93.3%, respectively. ConclusionsInduction immunosuppressive therapy with intravenous immunoglobulin and rituximab is effective; outcomes demonstrate an excellent patient and allograft survival and allograft function over the follow-up period.

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.

P199 A successful lung transplantation in two patients with positive donor specific antibodies and positive crossmatch

Introduction Sensitization to HLA antibodies has been associated with worse outcome in lung transplant recipients. Many lung transplant centers avoid transplanting patients with positive flow cytometry crossmatch (FCXM). However, delaying the transplant in these patients may increase their mortality on the waiting list. We are reporting two cases of crossmatch positive lung transplantation. Case scenario A 37-year old lady diagnosed with pulmonary fibrosis. Patient sensitized to HLA class I and II at time of transplant.The FCXM was positive T-cell (+78 MCS) and B-cell (+165 MCS). She had multiple DSA: Anti A2 (2795 MFI), Anti DR51 (2215 MFI), Anti DR52 (2088 MFI), Anti DQB1:05:02 (655 MFI), Anti DR14 (1411 MFI), anti DP2 (813 MFI). Second case is a 58-year old lady with pulmonary fibrosis secondary to Sjogren’s syndrome. Patient sensitized to HLA class II only at the time of transplant. The T-cell crossmatch was negative while, B-cell was positive (+318 MCS). She had multiple DSA: Anti DRB1∗10:01(14776 MFI), Anti-DR53 (4796 MFI), Anti-DP1 (1942 MFI), Anti- DQB1∗05:01(3384 MFI), Anti-DP2 (790 MFI). In view of patients’ deterioration, lung transplantation was preceded despite of positive crossmatch results, using combination of plasmapheresis, Intravenous immunoglobulin (IVIG) and Thymoglobulin. Both patients did well with no evidence of graft rejection. However, the repeated HLA antibodies screening during follow up showed persistent DSA. in the first patient: Anti-A2(1377 MFI), Anti-DRB1∗14 (4864 MFI), Anti-DR52 (3044 MFI), Anti-DR51 (1708 MFI), Anti-DP2 (1993 MFI). In the second patient: Anti-A1 (3561 MFI), AntiA24 (712 MFI), Anti-DQB1∗05:01(5837 MFI), Anti-DP1 (692 MFI), Anti-DRB1∗10:01 (643 I). Conclusion Lung transplantation is possible in patients with crossmatch positive, using perioperative desensitization protocol. Desensitization treatment did neither eliminate DSA nor prevent de novo DSA formation. However, follow up of these patients showed no evidence of graft rejection. Long-term data is needed.

P235 Positive T-cell and negative B-cell flow cytometry crossmatch due to HLA-B donor-specific antibodies

Aim We describe here three cases with T-cell positive and B-cell negative flow cytometric crossmatch (FCXM) in the presence of HLA-B donor-specific antibodies (DSA). Methods A standard three-color FCXM was performed. FCXM reactivity was assessed as a ratio of test serum to negative control (Fluorescence Index = FI). The result was considered positive if the FI was > 1.5 for T-FCXM and > 2.0 for B-FCXM. HLA antibody specificity was determined by single antigen beads (Luminex or FlowPRA). Results Case 1: A kidney transplant candidate with a negative alloantibody status had an unexpected positive T-FCXM (FI = 2.4) with deceased cadaver donor. The B-FCXM was negative (FI = 1.23). An auto FCXM was performed but no auto-antibody reactivity with T (FI = 1.1) and B (FI = 1.3) cells was observed. The patient had a history of alloimmunization (4 pregnancies and 5 transfusions). Therefore, the positive allogeneic T-FCXM was interpreted as most likely due to donor-specific alloreactivity, which was proven by the detection of DSA in the new serum against the mismatched HLA-B56 (MFI = 1541). Case 2: The current serum of a patient was positive for T-FCXM (FI = 1.75) and negative for B-FCXM (FI = 1.27). Historic serum, containing weak HLA-B49 DSA (MFI = 1832) also demonstrated T-cell alloreactivity (FI = 1.65). Case 3: A patient was evaluated for living-donor kidney transplantation. DSA were not identified during the routine testing, although HLA class I antibodies were detected. The T-FCXM was positive with the current serum (FI = 1.66) and negative with a historic serum (FI = 1.06). The B-FCXMs were negative. The new serum contained antibodies against the mismatched HLA-B18 antigen. Conclusions According to our policy a positive T-FCXM is a contraindication for transplantation. Therefore, it is very important to clarify for each patient the reasons for an unexpected T-cell positive and B-cell negative FCXM. Acknowledgements Partly supported by grant 8509/12.2016, Medical University, Sofia, Bulgaria.

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

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

P152 Prozone phenomena may skew the strength of Donor Specific Antibody (DSA)

Aim Positive Complement Dependent Cytotoxicity (CDC) and Flow Cytometry Crossmatch (FCXM) usually correlate with high titer of Donor Specific Antibodies (DSA). Now a days assignment of relevant DSA is based on solid phase assay (SPA), in which false low/negative results can be observed due to prozone effect. Here we present three cases underwent a workup for living related kidney transplant that their crossmatch data does not correlate with the single antigen (SA) Median Fluorescent Intensity (MFI) value. Method B cell and/or T cell AHG-CDC, FCXM and antibody identification by SPA were performed for three living related pairs. Results Two patients show positive T cell AHG-CDC crossmatch and one shows positive B cell CDC crossmatch with 8 reaction strength at 1:8 dilution and positive FCXM with very high Median Channel Shift (MCS). However the DSA result was negative for one patient and the other two patients showed A2 (891 MFI) and DQ6 (1299 MFI) respectively. The SA testing indicated possible skewed MFI value, therefore all serum were diluted 1:4 to overcome a prozone effect if present. Conclusion MFI value of the SPA was not correlated with the high MCS and positive CDC crossmatch in undiluted serum due prozone phenomena of high antibody concentration. Therefore serum dilution should be considered if strong crossmatch reactivity observed.

P007 Utility of autologous crossmatches in resolving unexpected positive flow cytometry crossmatches

Aim Flow cytometry crossmatches (FCXM) are a sensitive method for the detection of HLA antibodies bound to donor lymphocytes. As a means of risk assessment at the time of transplant, a positive FCXM can be interpreted as high immunologic risk if donor-specific HLA antibody (DSA) is also present. FCXM can also be positive in the absence of DSA. One way to investigate a potentially false positive FCXM is to perform an autologous (auto) FCXM to assess inherent serum interferences. Our goal was to investigate the utility of the auto FCXM in resolving unexpected positive FCXM in the absence of DSA. Methods All FCXM performed between July 2013 and February 2016 were reviewed (n = 1641). At our centre, a positive FCXM triggers an auto FCXM. There were 123 cases with a positive FCXM and corresponding auto FCXM results. All FCXM included pronase treatment and all recipients’ FCXM sera were tested by single antigen beads as per our testing algorithm. Results 59% of the positive FCXM were from recipient sera lacking DSA. Within this population, 63% also showed negative auto FCXM reactivity. Of the 41% of cases with DSA identified in the pre-transplant serum, 14% were resolved by auto reactivity alone; the DSA median fluorescence intensity was below the expected FCXM sensitivity level and the auto FCXM median channel values were similar to the reactivity of the FCXM. Both DSA and auto interference likely contributed to the positive FCXM in another 14% of cases. All others were DSA positive and auto FCXM negative with expected FCXM reactivity. One case with three sera dates tested against both donor and auto cells showed identical reactivity in all sera against the donor cells but varying reactivity in the auto FCXM, thus suggesting that the results of the auto FCXM may fluctuate over time and can be independent of donor FCXM reactivity. Conclusions The auto FCXM can sometimes be a useful tool to aid in the resolution of an unexpected positive FCXM but the ability of the auto FCXM to help resolve unexpected positive reactivity appears limited in the context of FCXM at our centre. With the current tools available to assess HLA antibody and the urgency for results at the time of transplant, performing a STAT single antigen bead assay rather than an auto FCXM may provide more clarity for clinicians when assessing immunologic risk in the face of a positive FCXM.