Positive FXM Research Articles

Risk Stratification Before Living Donor Kidney Transplantation in Patients With Preformed Donor-specific Antibodies by Different Crossmatch Methods.

Preformed donor-specific HLA antibodies (DSA) are a well-known risk factor in kidney transplantation. There is still considerable debate, however, about the optimal risk stratification among patients with preformed DSA. Additionally, data on the prognostic value of different crossmatch assays in DSA-positive patients are scarce. DSA-positive living kidney transplant recipients were selected from a multicenter study examining 4233 consecutive renal transplants. An additional 7 patients from 2 further centers were included. Flow cytometric crossmatches (FXM), Luminex-based crossmatches, and virtual crossmatches based on C1q- and C3d-binding antibodies (C1qXM and C3dXM) were performed retrospectively using pretransplant sera and lymphocytes isolated from fresh samples. These samples were obtained from 44 donor and recipient pairs from 12 centers. Clinical outcome data and the control group without DSA were compiled from the previous study and were supplemented by data on 10-y death-censored graft survival (10yGS). Between 19% (C3dXM) and 46% (FXM) of crossmatches were positive. Crossmatch-positive patients showed high incidences of antibody-mediated rejection (AMR) within 6 mo (up to 60% in B-cell FXM+ patients). The incidence of AMR in crossmatch-negative patients ranged between 5% (FXM-) and 13% (C1qXM-). 10yGS was significantly impaired in patients with positive T-cell FXM and total FXM compared with both patients without DSA and those with DSA with negative FXM. Especially FXM are useful for risk stratification, as the outcome of DSA-positive, FXM-negative patients is similar to that of DSA-negative patients, whereas FXM-positive patients have both more AMR and decreased 10yGS. Because of their lower sensitivity, the significance of Luminex-based crossmatches, C1qXM, and C3dXM would have to be examined in patients with stronger DSA.

The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.

Temporary Deceased Donor Splenic Transplant Prior to Intestinal Transplantation: A New Strategy for Desensitization?

Intestinal transplantation is a therapeutic treatment option for patients with irreversible intestinal failure. The presence of donor-specific antibodies (DSAs) has been associated with increased antibody-mediated rejection and allograft loss for recipients of all the solid organ transplants. This case report describes the posttransplant course in the first year of a patient who received a T-cell and B-cell flow cross-match (FXM) and complement-dependent cytotoxicity cross-match positive intestinal transplant in the presence of several class I and class II DSAs who underwent a "temporary desensitization" using the donor spleen. The temporary donor splenic transplant removed several class I and II DSAs as demonstrated by the negative subsequent T-cell FXM, the decreased mean channel shift of the positive B-cell FXM with a significant decrease in DSA mean florescence intensity post temporary splenic transplant. The patient experienced an isolated incidence of acute rejection, which responded to therapy. He had no infectious or cancerous sequelae from the immunosuppression modalities. He was able to discontinue total parenteral nutrition and gained weight after the procedure. Long-term effects are not able to be determined from this approach; hence, further research is warranted to better evaluate the real efficacy of this strategy.

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.

Dose-Dependent Response to Desensitization in Pediatric Heart Transplant Candidates Assessed by Single Antigen Bead Technology

Purpose The efficacy of desensitization, the process of reducing anti-HLA antibodies in sensitized patients awaiting heart transplantation (HT), is unclear in pediatric HT candidates. Methods To evaluate the impact of DS in pediatric HT candidates, we performed a retrospective analysis of all patients listed for HT between 1/1/10 - 6/30/18 at our institution. Sensitized patients were defined as having a panel reactive antibody (PRA) ≥ 10% and ≥1 strong positive antibody with mean fluorescence intensity (MFI) ≥ 5500. Patients were identified as nonsensitized (NS, n = 55), sensitized but no therapy (S-NT, n = 18), and sensitized undergoing desensitization therapy (S-DS, n = 14). Response to desensitization was defined as ≥25% reduction in the MFI for ≥90% of strong positive antibodies for whom follow up PRA testing was performed prior to delisting, HT, or death (n = 13). Results DS consisted of IVIG and rituximab for all patients; 2 also received bortezomib. DS responders, defined by reduction in antibody burden (n = 8), received on average more doses of IVIG than nonresponders (9 vs 3, p = 0.04). All five S-DS patients that received HT during the study period had a negative flow crossmatch (FXM) and most (80%) would have had a positive FXM with historical serum. All S-DS had history of single ventricle congenital heart disease and homograft exposure. Prior VAD was associated with sensitization severity (mean positive antibodies 75 vs 26, p = 0.01) but not response to DS, and history of ECMO was associated with neither. S-DS compared S-NT were on average more sensitized (mean number of high MFI antibodies 45 vs 21, p = 0.04), waited longer for HT (569 vs 148 days, p = 0.005), and trended towards higher waitlist mortality (36% vs 11%, p = 0.09). NS patients were less likely to have homograft exposure (p Conclusion A favorable response to DS was observed in most patients in this cohort. Homograft and VAD, but not ECMO, were associated with severity of sensitization. IVIG appears to have some benefit, with a dose-dependent effect. Post-HT outcomes for S-DS, S-NT, and NS patients were similar in this study.

P135 Living donor kidney paired donation transplantation: First report from Kingdom of Saudi Arabia

Aim Kidney paired donation (KPD) is an effective way to transplant patients with willing but incompatible donors. We report our experience with KPD through development of local Registry for incompatible pairs. Methods 62 recipients & their incompatible donors plus 3 non-directed donors were entered into KPD registry. Incompatibility was due to ABO-incompatibility (ABOi) in 25% & HLA-incompatibility (HLAi) in 75%. Patients with HLAi had cPRA between 60–99%. Antibodies with MFI of ⩾ 3000 by SAB assay were listed as unacceptable antigens. We used balanced, unbalanced & non-directed anonymous donors (NDAD). ABOi donors were accepted if patients had low titer ( ⩽ 16) of Isohemagglutinins. Amenable DSA for desensitization are those weak, single or multiple that yielded negative or weakly positive flow cross match ( Results 23 candidates (37%) were transplanted, enabling pre-emptive transplant in 1 patient. The longest chain was seven -ways domino chain initiated by a NDAD & ended in transplant of deceased donor listed patient with multiple access failure. Additional desensitization was used for 9 patients due to DSA and/ or positive FXM. 1 patient received TPE due to anti ABO antibody titer of 16. We had one case of DGF, AMR occurred in 2 patients & 1 patient had ACR. Graft function was good through follow-up time of 6–18 months but one patient died 8 months post-transplant due to refractory sepsis. Conclusions Although our series is early with 62 patients, transplant rate is excellent compared to other old programs(Netherlands, UK, & Canada) where the transplant rate were 37%, 29%, 44% respectively. This might be attributed to involving NDAD, accepting ABOi & combining desensitization for low level DSA in selected patients. Considering that 75% of the patients were highly sensitized yet received living transplants with excellent results, our experience demonstrates promising short-term outcomes. However, longer follow-up is needed to assess the impact of KPD on organ shortage & the outcome of highly sensitized patients transplanted through KPD program.

P047 A cold case: When single antigen beads assay do not respond

Case history: We evaluated a live donor (D1) and recipient (P1) pair for potential kidney donation however their flow crossmatches (FXM) continuously result in B-cell positive. P1 was tested negative by single antigen beads and no donor specific antibody (DSA) was evident. Patient has been transfused once in 2014 and treated with adalimumab for ankylosing spondylitis in the past. Methods: Autologous FXM of D1 was conducted. A series of FXM with surrogate donors was conducted. Supplemental single antigen beads, FlowPRA beads and Luminex-based mixed screening beads were also conducted but results are questionable. Results: After the first allo FXM, a repeat was done to rule out B cell positive FXM whether it was reproducible. Since patient was screened negative by single antigen beads assay and supplemental beads, absence of DSA could not explain why B cell FXM can be positive. When patient serum subject to two other surrogate donors (D2 “O” and D3 “A”) interestingly B cell FXM has become negative. To rule out blood type issue and non–HLA factors, autologous FXM of D1 and allo-FXM with zero PRA serum (P2 “A”), and with positive PRA serum (absence of DSA, P3 “A”) were performed and all were found negative, but remained positive with diluted serum of P1. Patient P1 was tested again in 3 months however the same result remains. The final FXM was then focused on testing with surrogate donors (D4 and D5) who carry the same class II antigens as D1. The result became positive with D5. Although no clear evidence can confirm the antibody to DR11 to be allele specific, the collective results infer that suspected DSA DR11 is not distinguishable by FlowPRA screening beads and mixed beads and not detectable by single antigen and supplemental beads. Conclusion: This is the first case in our experience to discover such a situation with “cold bead” or specificity-unproven DSA. Transplantation is not recommended. Correlation between different assays and diligent testing to clarify unreasonable positive FXM is very important especially in cases of autoimmune disease.

P111 Can a virtual crossmatch be used for final donor selection for kidney transplantation?

Aim Our transplant center has used virtual crossmatch (vXM) for donor selection for heart and lung transplantation for over 10 years with great success. The UNET match run for kidney transplant is used to select potential donor for final prospective crossmatches (fXM). Therefore, the match run can also be considered as a vXM process. A question has been raised if the vXM can be used for final donor selection to replace the fXM for kidney transplants. Routinely typing all of HLA gens including DQA1, DPB1 and DPA1 has become a standard practice in many HLA labs in the past a couple of years which may reduce the rate of unexpected positive fXM. This study was to retrospectively review fXMs performed in 2016 for kidney transplantation and to determine if a positive fXM could be predicated by the vXM. Methods 358 fXMs performed for kidney transplantation in 2016 were included in the study. Their fXMs were performed by flow cytometry. The donor cells were routinely treated with pronase to reduce background bindings. vXMs were performed either based UNET match run or by manually checking presence/absence of DSA detected in patients. Results Among these 358 fXMs, 19 (5%) fXMs were positive. Of these 19 positive fXMs, 14 positive could be predicated by vXM because of presence of DSA. The other 5 positive fXMs were non-HLA specific because of absence of detectable DSA. One (1) of the 5 non-specific positive fXMs was T cell positive and B cell negative caused by pronase treatment and it became negative with cells without pronase treatment. Another (1) T cell positive and B cell negative fXM might have been caused by presence of non-HLA antibodies because of absence of detectable DSA. fXM with non-pronase treated cells remained T cell positive and B cell negative. The other 3 positive fXMs were from the same patient who was HIV positive with no detectable DSA. It is well known that non-specific positive crossmatches occurred in HIV positive patients. Conclusions Based on the findings of our study, we can conclude that performing vXM can avoid positive fXMs caused by HLA antibodies and can be safely used for donor selection for kidney transplantation without performing a fXM.

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.

P172 Examination of the roll of DTT and/or molecular weight spin columns in evaluating positive antibody screens and positive flow corss matches in heart transplantation

Aim To study the short and long term effects of transplanting hearts over positive B-cell flow crossmatches to patients with positive DSAs that become negative when DTT or molecular weight spin columns are used. Methods Recipient and donor HLA typing was performed by rSSO-Luminex. Anti-HLA class-I and II antibodies by Single Antigen ((SAB) LABScreen Single Antigen Class-I and II) with and without molecular weight spin columns (MWSCs) or DTT. For identification of complement-fixing anti-HLA antibodies, the C1q test was used. Donor crossmatching was performed within 24 h of transplantation by flow cytometry (FXM) using serum drawn immediately before transplant. Patient allograft heart biopsy results and patient health were followed. Results Nine heart allograft recipients between 2012 and 2017 were identified that had either positive FXM and/or positive SAB I/II results that became negative when either DTT/MWSC were used (Table 1). In most, at least one of the antibodies identified was DSA. In 8, the DSA was also known to be a cryptic antigen. Some of the MFI values for DSA were below our cut-off of 3000 MFI. Patients were followed for rejection. Only one patient died from unrelated causes and never had any signs or biopsy results of cellular or antibody mediated rejection (AMR). The remaining patients are all alive and doing well. No patients ever had any biopsied signs of AMR. Several did have some cellular rejection that resolved with treatment however, rejection was not higher than in other heart transplant patients. This phenomenon was also observed in kidney transplantation. Conclusions The use of MWSCs to evaluate positive FXM, SAB1, SAB2, and DSA in heart transplant candidates is effective in eliminating interference and cryptic antigens as well as determining which antibodies are “real” and pose a risk to the recipient. Here we show that use of MWSCs allows clarification of test results and gives one additional data on which to decide to transplant across DSA. Notably, all patients did well following transplant with no AMR. Download : Download high-res image (411KB) Download : Download full-size image

P056 Cellular crossmatch (CXM) by flowspot predicts cellular rejection in kidney transplantation

Aim To evaluate newly developed CXM assay in prediction of cellular rejection of kidney transplantation. Methods: CXM: Irradiated auto or donor PBMC were co-incubated with recipient PBMC and Th1 (IFNγ, IL-2) and Th2 (IL-4, IL-5, IL-10) cytokine capture beads for overnight. After cell lysis, the captured cytokines on each bead were detected by PE-labeled 2nd antibody on a flow cytometer. The percentage of positive beads (Flowspot %) were counted which proportionately correlated with numbers of donor specific responding cells. The MFI level correlated with the cytokine secretion capability of responding cells. DSA-FXM: Stanford method; Standard methods: T/B cell FXM, Luminex IgG/C1q SAB. Results PBMC from two cellular rejection kidney transplantation clinical cases were tested by CXM Flowspot assay. Case 1 LMX IgG/C1q DSA was negative in pre-14 days patient serum. T/B FXM results showed auto positive and allo positive and DSA FXM also detected auto- and allo- CI and CII DSA in the same serum. Patient had cellular rejection on 19th day after kidney transplantation (Tx), proved by biopsy (ACR 1B-2). The retrospective CXM on patient PBMC (14 days before transplantation) showed that the recipient had increased secretion of IFNγ and IL-2 to donor cells. De novo LMX-IgG DSA B44, B57, DQ6, DP3 (MFI: 948–2512) were found in post-11 and 18 days sera; and LMX-C1q DSA B44, B57, DQ6 (MFI: 2302–11551) in post-18 days serum. Case 2 LMX-IgG detected possible DR52 (MFI: 816) DSA but none complement fixing antibody in pre-8 days serum. T/B FXM results were negative and no DSA was detected by DSA-FXM in the same sample. However, patient got cellular rejection post-transplantation (ACR 1A). The retrospective CXM by Flowspot on patient PBMC (pre-43 days) showed strong donor specific IFNγ and IL-2 secretions. Conclusions CXM by Flowspot is able to detect donor specific immunity in recipient. It potentially can be used to predict cellular rejection in kidney transplantation. Download : Download high-res image (292KB) Download : Download full-size image

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

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

P004 The challenge of virtual crossmatching in the presence of weak donor specific antibodies

Aim Our laboratory uses a normalized 2500 MFI cut off in the Luminex single-antigen bead assay to define unacceptable antigens. Antibodies (Abs) between 1000 and 2500 MFI are considered weak positive, but are not used to strictly rule out potential donors. The challenge is to accurately predict flow crossmatch (FXM) results in the presence of weak donor-specific Abs (DSA). Method We identified 62 FXM performed in the presence of weak DSA. For simplicity we considered only the B-cell FXM results. We analyzed the data based on the DSA MFI values or DSA epitope profile (DSA against shared epitopes (SE), DSA against a single antigen (Ag), and multiple weak DSA (M)). Results 39/62 (64%) were positive and 22/62 (36%) were negative. All (10/10) FXM with multiple weak DSA were positive. The majority (13/17) of FXM in the presence of weak DSA against a single antigen (Ag) were negative (76%). On the other hand, most (25/34) FXM with weak DSA that shares epitopes with other antigens (SE) were positive (74%). Table 1 summarizes these results. When we analyzed the FXM results based on DSA MFI (excluding multiple DSA cases), we observed that DSA with MFI of 1,000-2,500 resulted in a positive FXM in 50% of cases (Table 2). Unexpectedly, we got more positive FXM (14) than negative (7) when the DSA was under 1,000 MFI. Further analysis revealed that 13/14 of these positive FXM were caused by Abs with reactivity to SE and in particular Abs against HLA-B and -DRB1. Conclusion MFI values of weak DSA are not a reliable predictor of FXM results. When performing a virtual XM in the presence of weak DSA, the Ab profile is a better predictor for FXM reactivity (PPV for DSA with SE is 73.5, independent of the MFI value). Abs that target broadly-shared epitopes frequently cause positive FXM. Download high-res image (105KB) Download full-size image Download high-res image (104KB) Download full-size image

P050 DSA-FXM distinguish auto-Ab from Allo-Ab in FXM and detect true donor specific HLA-Abs

A false Allo-FXM positive caused by non-specific Auto-Ab from a recipient is often observed. A positive Auto-FXM is most likely to result in a positive Allo-FXM (52%) and makes the interpretation of final FXM troublesome. A novel DSA-FXM was developed and is able to distinguish HLA-Ab from Non-HLA-Ab, Auto-DSA from Allo-DSA and eliminate false positive Allo-FXM results caused by non-HLA-Abs. DSA-FXM: 0.2×106 donor PBMCs were incubated with recipient serum at RT for 30′, after washes, the beads conjugated with anti-HLA class-I (CI) and -II (CII) MoAbs were added. The cells were lysed with lysis buffer containing PE-anti-IgG and the complexes of HLA-Ag/DSA/PE-anti-IgG were captured by CI or CII beads. The beads were finally acquired on a flow cytometer. The PE intensities on CI and CII beads were proportionately correlated with the HLA-DSA levels in the serum. FXM: General IgG FXM procedure Luminex Single Antigen Beads (L-SAB): One Lambda method A total of 30,364 T&B FXMs including 15460 (51%) auto-FXM have been performed in our lab during last 5 years. 51% auto-FXM (N = 15460) were performed and 22% were positive (N = 3317); 52% auto-FXM+ (1726) accompanied by Allo-FXM+. Of total 80 both auto- and allo-FXM positive samples, LMX-DSA were detected in only 13 (16%) and 26 (33%) in auto- and allo-FXMs respectively; and DSA were identified positive by DSA-FXM in 15 (19%) and 28 (35%) in above two groups. Furthermore, DSA positive by both LMX-DSA and DSA-FXM were only 7 (9%) and 19 (24%). The concordance of LMX-DSA and DSA-FXM were 83% and 80% in auto- and allo-FXM groups.Download : Download high-res image (108KB)Download : Download full-size image Most of allo-FXM positive with auto-FXM positive were caused by non-HLA-Ab and auto-HLA-Ab (Auto-DSA). DSA was identified by DSA-FXM has a good concordance with DSA called by LMX virtual XM. DSA-FXM is able to specifically detect the true DSA against native HLA-Ag on donor cell surface and eliminate false positive FXM results.

C1Q Assay Results in Complement-Dependent Cytotoxicity Crossmatch Negative Renal Transplant Candidates with Donor-Specific Antibodies: High Specificity but Low Sensitivity When Predicting Flow Crossmatch.

The aim of the present study was to describe the association of positive flow cross match (FXM) and C1q-SAB. Methods. In this observational, cross-sectional, and comparative study, patients included had negative AHG-CDC-XM and donor specific antibodies (DSA) and were tested with FXM. All pretransplant sera were tested with C1q-SAB assay. Results. A total of 50 donor/recipient evaluations were conducted; half of them had at least one C1q+ Ab (n = 26, 52%). Ten patients (20.0%) had DSA C1q+ Ab. Twenty-five (50%) FXMs were positive. Factors associated with a positive FXM were the presence of C1q+ Ab (DSA C1q+ Ab: OR 27, 2.80–259.56, P = 0.004, and no DSA C1q+ Ab: OR 5, 1.27–19.68, P = 0.021) and the DSA LABScreen-SAB MFI (OR 1.26, 95% CI 1.06–1.49, P = 0.007). The cutoff point of immunodominant LABScreen SAB DSA-MFI with the greatest sensitivity and specificity to predict FXM was 2,300 (sensitivity: 72% and specificity: 75%). For FXM prediction, DSA C1q+ Ab was the most specific (95.8%, 85–100) and the combination of DSA-MFI > 2,300 and C1q+ Ab was the most sensitive (92.0%, 79.3–100). Conclusions. C1q+ Ab and LABScreen SAB DSA-MFI were significantly associated with FXM. DSA C1q+ Ab was highly specific but with low sensitivity.

Optimizing transplantation of sensitized heart candidates using 4 antibody detection assays to prioritize the assignment of unacceptable antigens

The virtual crossmatch relies on the assignment of unacceptable antigens (UAs) to identify compatible donors. The purpose of our study was to identify an algorithm for assignment of UAs such that a negative complement-dependent cytotoxicity (CDC) crossmatch and concomitant negative or weakly positive flow cytometric crossmatch (FXM) are obtained. We used 4 antibody methods: (1) Luminex single antigen (LSA), (2) LSA with a 1:8 serum dilution, (3) C1q LSA, and (4) CDC panel. The UAs were prioritized in the following order: (1) all C1q+/CDC+, (2) LSA 1:8 >7,500 median fluorescence intensity, and (3) LSA >10,000 median fluorescence intensity. Of 295 heart transplants that were performed at our center, 69 (23%) recipients had detectable human leukocyte antigen specific antibody at the time of transplant. All donor specific antibodies (DSAs) were avoided for 44 of 69 (64%) (DSA-). There were 25 recipients who had DSA at the time of transplant: 12 (48%) had negative FXM (DSA+/FXM-), and 13 (52%) had positive T-cell and/or B-cell FXM (DSA+/FXM+). Lower freedom from antibody-mediated rejection was observed for the DSA+/FXM+ group compared with the DSA- group (p < 0.0001). DSA remained detectable after transplant in the sera of 14 recipients, and de novo DSA was detected in 32 recipients. Freedom from antibody-mediated rejection was comparable for both groups (p = 0.53) but was lower than the DSA- group (p < 0.0001). Survival was comparable for all groups at 1,200 days post-transplant. Strategic prioritization of UA assignment has allowed transplantation of highly sensitized patients across the DSA barrier with survival rates comparable to DSA- heart transplant recipients.

Reasons for failing offers in a Kidney Paired Donation program: A 7-year analysis

Aim Computer-identified possible Kidney Paired Donation (KPD) exchanges are presented as offers to participating transplant programs and eventually converted into transplants. We evaluated reasons why these offers failed to progress into successful transplants. Methods/Results The Alliance for Paired Donation (APD) has an optimization algorithm to generate possible KPD exchanges, which are presented to transplant centers for their review as offers. Each offer may be composed of one to multiple 1-way exchanges. We tracked the progress in moving from offers to transplants and recorded the reason for failure. Between January 2008 and August 2014, the APD made 981 offers. Of these offers 181 resulted in transplants while 800 failed, namely 268 failed for specific reasons while 529 failed as they were dependent on the same 268 1-ways. Overall, specific reasons accounted for 57% of all failures with: 25% positive flow crossmatch (FXM); 13% competing offer outside the APD; and, 19% declined of donors by transplant centers (Fig. 1). Our detail analysis showed that the 25% positive FXM with negative virtual crossmatch (VXM) correlated with an increased number of highly sensitized patients: the pool of sensitized patients with a PRA >80% has increased from 31% in 2008 to 47% in 2014. Since many patients choose to enroll in multiple KPD registries some offers resulted in futile exchanges with patients already committed to alternative exchanges. Finally, donors were declined by transplant centers. To avoid these specific reasons for failure, the APD has begun pre-review consulting about donors and recipients prior to making a formal offer. Conclusions To prevent specific reasons for failure the APD has implemented the following changes: (1) testing of all donors for HLA-DQA, DPA and DPB; (2) expansion of the donor pool to increase options for highly sensitized patients; and (3) improved coordination with other KPD programs to avoid competing offers. Download : Download full-size image

A 6-Year Experience of a Kidney Paired Donation Centralized Lab, Performing Crossmatches Resulting in 220 Transplants.

INTRODUCTION: Flow crossmatch (FXM) results with donor cells were compared to computer-based virtual crossmatch (VXM) results in patients stratified by their calculated PRA (cPRA) values to evaluate the accuracy of both crossmatch methods. METHODS: A total of 1949 VXM tests (with HLA-A, B, C, DR, and DQB) were performed. A single antigen bead assay was used for patients with 1-100% cPRA to create a list of unacceptable HLA antigens; VXM results were used to perform daily match runs. Selected donor/recipient pairs were tested by FXM with donor cells. Weekly match runs were performed to identify compatible pairs with negative VXM results. RESULTS: Between 2007 and 2012, there was an increase in the number of sensitized (1-79% cPRA) and highly sensitized (80-100% cPRA) patients (Figure 1). The accuracy of negative VXM ranged from 70.5% in 2007 to 74% in 2012 (Figure 2). False negative VXM results ranged from 26% in 2012 to 29.5% in 2007. When all negative VXM results were divided by cPRA, patients with 0% cPRA had no false negative results, those with 1-79% cPRA had 20% positive FXM, and those with 80-100% cPRA had 45% positive FXM. Analysis of the class I and II false negative VXM results revealed that the majority were falsely negative in both class I and II HLA (47%), rather than in class II alone (40%) or in class I alone (13%).Figure: No Caption available.Figure: No Caption available.CONCLUSION: Significant (˜ 25%) false negative VXM (especially for high PRA and Class II sensitized patients) when assessed by actual FXM demonstrates that data reported to centralized repositories by multiple transplant centers results in higher false negative VXM than can be achieved by single centers.

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To develop a FXM for detecting donor specific HLA antibody (DSA) to A, B, C, DR, DQ, DP. DSA-FXM: 0.2X10 6 donor PBMCs were incubated with recipient serum at RT for 30’, washed, and then beads conjugated with anti-HLA class-I (CI) and -II (CII) MoAbs were added. The cells were lysed for 30’ and the complexes of HLA-Ag-Ab were captured by CI or CII beads; and detected by PE-anti IgG. The final washed beads were acquired on a flow cytometer. The PE intensities on CI and CII beads were proportionately correlated with the HLA-DSA levels in the serum. FXM: IgG FXM procedure. LMX-SAB: One Lambda method. Pooled HLA-Ab positive serum (PPS) in various dilutions was tested against different numbers of cells by DSA-FXM, FXM, and LMX-IgG. Results showed that DSA-FXM done with 25,000 cells has comparable sensitivity to FXM. DSA-FXM was able to detect low levels of LMX-DSA <500 MFI. The CI or/and CII DSAs including Cw, DQ, and DP were clearly identified in 75 of 117 samples and 87% of the DSAs were confirmed by LMX-SAB. 8 false positive LMX-DSAs were proven by both FXM and DSA-FXM. Interference by Rituximab and auto-Abs was completely eliminated by DSA-FXM. [Figs. 1 and 2] The DSA-FXM is a novel solid phase XM for selectively detecting HLA-DSA to all HLA loci in recipient serum. It eliminates false positive FXM caused by Non-HLA Ab and is truly donor HLA specific.

Clinical Significance of a Positive Cross Match in the Setting of Non-Complement Fixing Donor Specific Antibodies in Adult Heart Transplant Recipients

Purpose The presence of donor specific antibodies (DSAs) increases the risk of antibody mediated rejection (AMR) and coronary vasculopathy in heart transplant recipients. Identifying non complement binding DSAs may allow successful transplantation of highly sensitized patients. We analyzed clinical outcomes of adult heart transplant recipients with a positive flow cytometric cross match (FXM) but non complement fixing antibodies as determined by the C1q assay. Methods and Materials Charts of transplant recipients at our institution were reviewed from January 2009 to July 2012 to identify patients with DSAs and a positive FXM and to analyze outcomes. Results Seventy patients were transplanted during this time; five had a positive prospective FXM followed by transplant if their DSA mean fluorescent intensity (MFI) was figure 1 ] Conclusions Differentiating complement and non-complement fixing antibodies using the C1q assay may help in the risk assessment of transplanting a patient with a positive FXM. PreTx PRA (class I/II) # of DSAs # of rejected donors FXM (T/B) AMR Death 1 47/0 1 2 +/- 0 0 2 0/96 2 11 -/+ 0 0 3 100/45 1 1 +/+ n/a YES 4 67/9 2 13 +/+ 0 0 5 98/72 2 12 -/+ 0 0