Flow Cytometric Crossmatch Results Research Articles

A new flow cytometry assay identifies recipient IgG subtype antibodies binding donor cells: increasing donor availability for highly sensitised patients

ObjectivesThere are four immunoglobulin (IgG) subtypes that have varying complement‐activating ability: strong (IgG3 and IgG1) and weak (IgG2 and IgG4). The standard flow cytometric crossmatch (FCM) assay does not distinguish between the various subtypes of the IgG molecule. This study outlines the development and use of a novel cell‐based IgG subtype‐specific FCM assay that is able to detect the presence of and quantitate the IgG subtypes bound to donor cells.MethodsA six‐colour lyophilised reagent was designed that specifically detects the four IgG subtypes, as well as distinguishes between T cells and B cells in the lymphocyte population. To test the efficacy of this reagent, a retrospective evaluation of a group of highly sensitised patients awaiting heart and kidney transplant was carried out, who, because of positive standard FCM results, had been deemed incompatible with numerous prior potential donors.ResultsObservations in this study demonstrate that the positive standard FCM results were mainly because of the presence of noncomplement‐activating IgG2 or IgG4 antibodies. The results were supported by the absence of C3d‐binding donor‐specific antibodies (DSA) and a negative complement‐dependent cytotoxicity crossmatch (CDC).ConclusionPreliminary data presented in this study demonstrate the reliability of the novel IgG subtype assay to detect the presence of pretransplant, complement‐activating antibodies bound to donor cells. The knowledge gained from the IgG subtype assay and the C3d‐binding specificities of DSAs provides improved identification of donor suitability in pretransplant patients, potentially increasing the number of transplants.

OR34 Flow and virtual cross match exchange – A new component of the International Cell Exchange

Aim Single antigen bead assays provide investigators with a means to accurately identify the presence and relative strength of HLA antibodies through the use of MFI (Mean Fluorescence Intensity) values. However, inter-laboratory variation to the interpretation of MFI values creates the potential for discordant results. The aim of this study was to provide a medium to compare and correlate MFI data together with flow crossmatch outcomes. Methods In 2014, the International Cell Exchange program introduced the UCLA Flow and Virtual Cross Match Exchange. In three exchanges, a total of 6 cells and 12 sera were tested by 17 laboratories around the world. T and B flow cytometric crossmatch results, along with class I and class II single antigen data were reported and analyzed. Fifteen laboratories used a Luminex single antigen bead assay (LSAB) and 2 used a flow single antigen bead assay (FSAB). To further investigate the correlation between MFI values and positive flow crossmatch results, the strength of donor-specific antibodies were compared with their crossmatch outcomes. In addition, coefficients of variations (%CV) were calculated using the MFI reported for each DSA and by each laboratory. We also examined the prediction of positive T/B virtual crossmatches in relationship to the strength of DSAs identified. Results The mean class I and class II DSA MFI of the positive T and B flow crossmatches were 15355 and 14641, respectively. Among labs reporting LSAB MFI, the mean %CV was 26%. Overall, 16 (67%) T-cell and 8 (33%) B-cell flow crossmatch challenges were reported among the labs without discordance. When restricting analysis to DSA MFI greater than mean values, the T flow concordance rate improved to 79%, while B flow concordance remained poor at 31%. In contrast, high concordance rates were observed for virtual crossmatch predictions: 92% (22/24) for T- and 88% (21/24) for B-cell crossmatches. Conclusions The data suggests that the variability of cell based assays is higher than that observed in solid phase SAB test. It also suggests that variability exists in the sensitivity of flow crossmatch testing among laboratories and that this variability is correlated with DSA strength. As such, educational activities should continue to provide standards for quality control of antibody and crossmatch testing methods.

Flow cytometric crossmatch results using lymphocytes isolated from donor peripheral blood and spleen tissue on five consecutive days

A negative flow crossmatch (FXM) is considered as a “go ahead” to transplant solid organs at many centers. FXM is a biologic assay where donor lymphocytes are reacted with recipient sera to identify donor specific antibodies (DSA). Lymphocytes can be derived from different sources like peripheral blood (PB), lymph node or spleen. Mix of cellular components from all three sources are different and also may have different HLA expression depending on the age of the sample. We present our data to understand this variability. Lymphocytes were isolated from PB and spleen of the same donor on five consecutive days using a different sample aliquot. Lymphocytes from PB were isolated by Ficoll–Hypaque technique. Lymphocytes from spleen tissue were isolated using a cell separation kit (Stemcell Technologies). Recovered cells were treated with 1mg/2mL pronase and re-suspended for testing in PBS containing 2% fetal calf serum. FXM was performed in a tray after mixing 3×105 cells with 20 μl of serum. After washing, cells were labeled with FITC goat anti-human IgG, anti-CD3 PerCP and anti-CD19 PE then acquired on a FACS Calibur to detect DSA. A comparison of the FXM mean channel shift (MCS) results for PB and spleen with a cutoff of 52 for T-cells and 106 for B-cells were made for 5 consecutive days. The negative control mean fluorescence intensity (MFI) increased with specimen age for PB and spleen (Figure A), which caused a decrease in MCS in the positive control over time for both lymphocyte sources (Figure B). Patient samples with FXM results close to the T-cell and B-cell cutoffs were discrepant based on lymphocyte source and age of specimen (Table 1). Older samples have a higher negative control which results in a decrease in the MCS and may cause borderline positive results to be reported as negative. The isolation of lymphocytes from PB by day 5 proved to be difficult as the prep was contaminated with whole blood components. Our data showed that the interpretation is dependent on the donor sample source and quality.Download : Download full-size image

LVAD Implant as a Bridge to Heart Transplantation Is Associated with Allosensitization as Measured by Single Antigen Bead Assay

Left ventricular assist devices (LVAD) as a bridge (BTT) to heart transplantation (HTX) may be limited by the formation of anti-human leukocyte antigen antibodies. Whether sensitization occurs with continuous axial flow LVAD implant as assessed by single antigen bead (SAB) assay is unknown. Cytotoxic panel-reactive antibody (PRA) and SAB assays were analyzed in HTX recipients undergoing LVAD implant as a BTT. Sensitization was defined as peak anti-human leukocyte antigen antibody values of more than 2000 mean fluorescence intensity because these values have been found to correlate with flow cytometric crossmatch results. LVADs were implanted as BTT in 30 patients. There were 7% (2 of 30) of patients before LVAD implant and no patients after LVAD implant with PRA more than 10%. However, 20% (6 of 30) of patients before LVAD and 53% (16 of 30) after LVAD were sensitized as measured by SAB (P=0.024). At HTX, 47% (14 of 30) of patients remained sensitized. A positive virtual crossmatch was observed in 28% (4 of 14) of the sensitized patients at HTX. There was no difference between the sensitized and nonsensitized groups (P>0.4 for all) in usage of blood products (6411 vs. 6339 units) and time to HTX (28,663 vs. 25,748 days), and 1 year after HTX, there were no differences in rejection (total rejection score 0.30 vs. 0.37) and survival (93% vs. 88%). Allosensitization after LVAD is common despite cytotoxic PRA being negative. One year after HTX, this sensitization does not translate into increased acute cellular or antibody-mediated rejection or reduced survival.

Partially Mismatched Transplantation and Human Leukocyte Antigen Donor-Specific Antibodies

The presence of donor human leukocyte antigen (HLA)-specific antibodies (DSA) increases engraftment failure risk in partially HLA-mismatched, or HLA-haploidentical, allogeneic marrow (alloBMT) transplantation. As pre-existing sensitization to HLA antigens is not well characterized among candidates for HLA-haploidentical alloBMT, we retrospectively evaluated both the incidence and relative strength of DSA in this patient population. Based on correlations of solid-phase antibody assays on the Luminex (Luminex, Austin, TX) platform with actual crossmatch tests, DSA were characterized as weak for results that were consistent with negative flow cytometric crossmatch results or as moderate-to-strong for results consistent with positive flow cytometric or cytotoxicity crossmatches. We evaluated 296 alloBMT candidates; 111 (37.5%) were female. DSA were detected in 43 (14.5%) candidates, mostly among female candidates (42.9% female versus 12.5% male). Moderate-to-strong DSA strength was more frequently encountered when directed against haploidentical donors as compared with mismatched unrelated donors. DSA were most commonly detected in female patients directed against their children. Because the presence of DSA has been considered prohibitive for HLA-mismatched alloBMT, we additionally report a desensitization methodology used to reduce DSA to negative or weak levels, ie, levels well below those detectable in a flow cytometric crossmatch. Nine patients without other available donors underwent desensitization. Eight who reduced their DSA to negative or weak levels proceeded to alloBMT and achieved full donor engraftment. These data support routine DSA evaluation in all patients considered for mismatched alloBMT; however, for patients with no other viable options, desensitization to weak or negative DSA levels may afford the opportunity for successful transplantation.

Donor Specific Anti-HLA Antibody Detection in Heart Transplantation: Comparison of a Donor-Specific Bead-Based Crossmatch Technique with Flow-Crossmatch and Single-Antigen Bead Methodology

Introduction: Progress in assays detecting and characterizing anti-human leukocyte antigen (HLA) antibodies have significantly improved outcomes in thoracic organ transplantation. However, due to the complicated nature of humoral alloresponses practices are still evolving in laboratories to determine the best combination of assays that can be used for graft allocation in prospective heart allograft (HTx) recipients, as well as for post-transplant monitoring of sensitized patients. In the present study we analyzed the efficiency of three different laboratory methodologies for the detection of donor-specific anti-HLA antibodies (DSA), their degree of correlation and their clinical relevance. Methods: The results of a Luminex donor-specific crossmatch (DSA-LX), in which donor-isolated HLA antigens are coated onto capture microbeads (Gen-Probe Transplant Diagnostics, USA), were compared with the results of a flow-cytometric crossmatch (FCM-X) for the same donor-recipient combination and with the results of a Luminex single-antigen bead methodology (SA-LX) in which recombinant HLA class I or class II molecules are coated onto microbeads (One Lambda, USA). A total of 46 sera samples obtained pre- (27 samples) and post-transplant (19 samples) were included in the study. All 46 samples were tested with the DSA-LX and SA-LX methodology, while the 27 pre-transplant samples were also analyzed by the FCM-X methodology. Results: For the pre- transplant sera, the results of the three methods were in agreement for class I DSA in 21/27 (77.8%) samples and for class II DSA in 17/27 (63.0%) samples. When the results of the two bead-based methodologies (DSA-LX and SA-LX) were compared to the results of the FCM-X methodology, the SA-LX results showed better correlation with the flow-cytometric crossmatch results than the DSA-LX results both for class I and for class II antibodies. The results of the two bead-based assays SA-LX and DSA-LX, coincided for 40/46 (87.0%) and for 33/46 (71.7%) of sera tested for class I and class II HLA-DSA respectively. Furthermore, in the group of transplanted patients the SA-LX results had more clinical relevance than the DSA-LX results, particularly when samples were tested positive by SA-LX and negative by DSA-LX. The methodology reproducibility score for both the SA-LX and the FCM-X was 100% whereas for DSA-LX that score was 88.9%. Conclusions: The results of the DSA-LX methodology do not have a good correlation with the results of the more established FCM-X and SA-LX techniques for the detection of DSA, particularly as far as class II antibodies. Furthermore, in comparison to FCM-X and SA-LX, the DSA-LX methodology has a lower reproducibility performance and a lower clinical relevance which renders it unsuitable for reliable graft allocation and patient monitoring.

Serum Flow Cytometric C1q Binding Antibody Analysis of Renal Recipients with Low Levels of Sensitization

AimPatients displaying flow cytometric crossmatch results within the grey zone of positivity are hard to evaluate, especially if they are undergoing their first transplantation. For these patients assays of donor-specific anti-HLA (human leukocyte antigen) antibodies with complement-fixing properties to cause cell lysis are important for antibody-mediated rejection and graft failure. The aim of this study was to detect the relevance of serum C1q-binding antibodies detected in renal recipients with grey zone crossmatch reactivity who were considered to show low levels of sensitization against their potential donors. MethodThis study includes 114 patients who were admitted for their first renal transplantation between September 2009 and August 2011, including 33 subjects considered by flow cytometric cross-match to be the sensitized group, whereas the remaining 81 recipients had negative results. We analyzed the accumulation of serum the immunoglobulin (Ig)G bound C1q on HLA-coated flowcytometric panel reactive antibody (FlowPRA) beads. The serum samples were retrospectively analyzed with [C1q]FlowPRA (HLA class I and II), which were collected during the pretransplantation period every 6 months and every week posttransplantation within the first month and every 3 months thereafter. All serum samples were analyzed for the presence of anti-FlowPRA IgG alloantibody. We compared the C1q FlowPRA-positive and-negative groups for the number of posttransplantation days that the serum creatinine level was below <2 mg/dL as a metric of graft function. ResultsWith a mean follow-up of 492 ± 84 days, there was a significant difference between flow cytometric crossmatch results and creatinine decrease rate (P = .02). The serum creatinine decrease rates of the 9 C1q-positive versus the 15 C1q-negative subjects showed significant difference (P < .05). ConclusionC1q-binding antibody analysis shows the presence of serum antibodies capable of complement binding and antibody-mediated rejection, which could be useful to assess rejection risk among the “grey zone” of renal recipients with low levels of sensitization against their donors.

Evaluation of Flow Cytometric Crossmatch Results in Comparison with Donor-specific Antibodies Detected by Luminex-PRA Tests in Organ Transplantation Patients

Seon Young Kim, M.D., Bok Youn Han, M.T., Jungwon Hyun, M.D., Shin Young Joo, M.D., Eun Young Song, M.D. and Myoung Hee Park, M.D.. J Korean Soc Transplant 2012;26:92-100. https://doi.org/10.4285/jkstn.2012.26.2.92

14-P Discordance between flow cytometric crossmatch results and the detection of donor-specific antibody of the “B4402/4403/45/76/82” specificity group by luminex single antigen

14-P Discordance between flow cytometric crossmatch results and the detection of donor-specific antibody of the “B4402/4403/45/76/82” specificity group by luminex single antigen

Flow-cytometric screening of platelet antibodies with previously frozen cells

In this study the flow-cytometric crossmatch results were compared between fresh cells and cells processed by various cryopreservation and storage methods. Platelets from healthy donors were incubated with 12 sera containing platelet reactive antibodies as well as with 62 control sera from blood donors. Direct comparisons were made between fresh platelets and platelets after freezing at −28 °C, −40 °C and −80 °C and in liquid nitrogen, using 6% DMSO as cryoprotectant. In addition, the effects of using controlled-rate freezing were evaluated. Finally we evaluated the application of the cryoprotectant ThromboSol. The best results were obtained after cryopreservation of the platelets with ThromboSol at −80 °C with controlled cooling rates. Using ThromboSol cryopreserved platelets, the sensitivity for the detection of incompatible platelets was 100% and the specificity was 97.1%, using the previous results obtained with flow-cytometry, MAIPA and LCT as a reference. Conclusion Platelets can be frozen using ThromboSol as the cryoprotectant, with controlled rate freezing and storage at −80 °C for the screening of platelet antibodies and for flow-cytometric crossmatch procedures. This system yields a reproducible and logistically simple method for platelet crossmatching that yields results superior to fresh cells and can be easily incorporated into standard clinical laboratory practices.

Flow cytometric crossmatch and early pregnancy loss in women with a history of recurrent spontaneous abortions who underwent paternal leukocyte immunotherapy

The purpose of our study was to evaluate the clinical significance of flow cytometric crossmatch in managing women with unexplained recurrent spontaneous abortions who have been immunized with paternal leukocytes. Two-color flow cytometric crossmatch was performed after immunotherapy in 69 women with recurrent spontaneous abortions to detect maternal immunoglobulin G antibodies against paternal T lymphocytes. A positive flow cytometric crossmatch was determined by a > or = 20 channel increases (256 channel linear scale-log amplification) in the fluorescence intensity of the T-cell peak. We analyzed the clinical correlation between flow cytometric crossmatch results and subsequent pregnancy outcome in those patients using the Fisher exact test. In 46 subsequent pregnancies among 69 immunized women the incidence of early subsequent pregnancy wastage was only 6 (17.1%) of 35 in flow cytometric crossmatch-positive patients and 7 (63.6%) of 11 in flow cytometric crossmatch-negative patients (p < 0.01). These data indicate that a two-color T cell flow cytometric crossmatch predicts subsequent pregnancy outcome in patients undergoing immunotherapy for recurrent spontaneous abortion.

Survival, function, morphology and serological aspects of rat renal allografts. Effect of short-term treatment with cyclosporine A, anti-CD4 and anti-interleukin-2 receptor monoclonal antibodies.

The aim of this study was to investigate the effect of short-term treatment with cyclosporine A (CyA) combined with anti-CD4 (OX-38) and anti-interleukin-2 receptor (OX-39) monoclonal antibodies (MAbs) on graft survival, graft function, morphology, and anti-donor antibody levels in a BN-to-LEW rat kidney transplantation model. Spontaneous rejection occurred at 9.3 days (range 9-10 d). Administration of CyA (12.5 mg/kg/d) for 14 days prolonged graft survival to 33 days (range 23-40 d, P less than 0.02). Supplementing with OX-38 and OX-39 100 micrograms/kg/d, given i.p. from days 0 to 7, further prolonged graft survival to 70 days (range 38- greater than 100 d, P less than 0.02 vs controls and CyA group). One of seven recipients had good graft function for more than 100 days. A three-fold increase of the MAb dosage did not improve mean graft survival (53.5 d), but three of eight recipients had well functioning grafts for greater than 100 days. Kidney function was characterized by reduced creatinine clearance, also in the recipients with long-term graft survival, and a defect in concentrating urine creatinine with subsequent pronounced increase in urinary output. Graft histology showed a complex pattern of interstitial alterations including mononuclear cell infiltration, fibrosis, tubular atrophy and vascular damage with intimal/endothelial cell hyperplasia and perivascular inflammation. In nine of 10 MAb-treated recipients with graft survival greater than 60 days, granular deposits of immunoglobulins and C3 were found by immunofluorescence microscopy (IFM). The deposits were localized in the glomerular capillaries and mesangium. IFM in MAb-treated control animals could not demonstrate any deposits. Flow cytometric evaluation of posttransplant serum samples against donor target cells showed increasing amounts of anti-donor antibodies until the time of rejection, while recipients with long-term graft function had moderately positive cross-matches up to two months after transplantation. Hereafter antibody titres decreased and cross-matches at the time of sacrifice were again negative. The morphological findings and the flow cytometric cross-match results seem to indicate a postponed antibody-mediated type of rejection. The reason why some kidney recipients showed decreasing antibody titres and stable long-term graft function is unclear.