FCXM Assay Research Articles

Evaluation of rapid optimized flow cytometry crossmatch (Halifaster) in living donor kidney transplantation.

Kidney transplantation is often the preferred treatment for end-stage renal disease. However, the presence of preformed donor-specific antibodies (DSA), including those against HLA, can lead to antibody-mediated rejection and significantly impact transplant outcomes. The Flow Cytometry Crossmatch (FCXM) is a crucial tool in kidney transplantation, as it also enables the measurement of low levels of anti-HLA DSA antibodies. However, current methodologies for detecting these antibodies, however, are time-consuming and require extensive reagents. In this study, we analyzed the performance of the Halifaster FCXM protocol in 133 consecutive living kidney donor pairs, correlating these results with single antigen-based anti-HLA DSA results. Anti-HLA DSA was identified in 31 patients (23.3%). Both T and B lymphocyte FCXM assays demonstrated high sensitivity and specificity in detecting anti-HLA DSA. Furthermore, a Tree model to determine the levels of anti-HLA DSA to produce a flow crossmatch positivity, was developed offering an accuracy of 93% and 90% for T and B lymphocytes, respectively. Both approaches point to a thresh old of 1000-2000 MFI for T lymphocytes and 3000 MFI for B lymphocytes. Our findings indicate that the Halifaster protocol facilitates fast and efficient FCXM testing without compromising accuracy, marking a significant advancement in the field of kidney transplantation. The inclusion of HLA-specific antibody analysis underscores the protocol's comprehensive approach to improving transplant outcomes.

Novel Flow Cytometry-Based Method for Detection of Anti-HLA Complement Activating Donor-Specific Antibodies in Renal Transplant Recipients and Its Comparison With Other Conventional Detection Methods

Presence of preformed donor specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC-XM) is a strong contraindication for transplant. However, it has limitations including its sensitivity and its inability to distinguish between HLA-specific and other non-HLA-specific antibodies. In this study, we standardized CDC-XM by flow cytometry and determined its relevance by comparing its results with other methods of DSA detection, such as routine CDC-XM, antibody binding assay by flow cytometry (FC-XM), and Luminex-based crossmatch assays, such as Luminex crossmatch (LXM) and virtual crossmatch (VXM). A total of 79 serum samples were tested for DSAs by the flow cytometric complement-dependent cytotoxicity crossmatch assay (FC-CDC-XM) and then the results of FC-CDC-XM were compared with other detection methods such as CDC-XM, FC-XM, LXM, and VXM. We found that the FC-CDC-XM assay is more sensitive than routine CDC-XM. Out of total 79 sera, 24 sera were detected positive (T cells positive: 1 case and B cells positive: 23) by FC-CDC-XM as compared with 3 sera using CDC-XM; these 3 sera also showed positivity by FC-CDC-XM. After FC-XM assay, 23 samples were positive by FC-XM and out of these 23 samples, 13 were also positive by FC-CDC-XM. On comparing the FC-CDC-XM results with VXM and LXM, 10 sera of 24 FC-CDC-XM positive had HLA class II antibodies detected on a Luminex platform. The FC-CDC-XM is a more sensitive and specific method for detection of HLA-specific complement-fixing antibodies than CDC-XM and FC-XM. FC-CDC-XM should be used in tissue-typing laboratories after intra- and inter- laboratory validation.

Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays.

Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is "prozone effect," which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called "positive concoction" in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.

Phasing out the pre-transplant cytotoxicity crossmatch: Are we missing something?

Introduction: The anti-human globulin-enhanced complement-dependent cytotoxicity crossmatch (AHG-CDCXM) assay has been used to assess the presence of donor-specific antibodies (DSA) in recipient’s serum before kidney transplantation. The flow cytometric crossmatch (FCXM) assay was first introduced as an additional test. The aim of this study was to clinically validate the single use of the FCXM assay.Methods: This study compared the outcomes of a cohort of kidney transplant patients that underwent FCXM only (FCXM group) versus a cohort of kidney transplant patients that underwent AHG-CDCXM (control group).Results: Ninety-seven patients in the FCXM group and 98 controls were included. All crossmatches in the control group were negative. One patient in the FCXM group had a positive B cell crossmatch. One year after transplantation, there were no significant differences in patient survival (p = 0.591) and graft survival (p = 0.692) between the groups. Also, no significant difference was found in the incidence of Banff ≥ 1A acute cellular rejection episodes (p = 0.289). However, acute antibody-mediated rejections occurred in 3 controls (p = 0.028).Conclusion: The results showed that discontinuing the AHG-CDCXM assay does not modify the clinical outcomes in a 1-year follow-up.

Flow cytometry crossmatching to investigate kidney-biopsy-proven, antibody-mediated rejection in patients who develop de novo donor-specific antibodies

IntroductionThe appearance of de novo donor-specific anti-human leukocyte antigen antibodies (dnDSAs) after kidney transplantation is independently associated with poor long-term allograft outcomes. The objective of the present study was to evaluate the predictive value of a flow cytometry crossmatching (FC-XM) assay after the appearance of dnDSAs related to antibody-mediated allograft rejection (ABMR) after kidney transplantation. Materials and methodsA total of 89 recipients with dnDSAs after transplantation were included. The crossmatching results were compared with the dnDSA profile (the mean fluorescence intensity (MFI), the complement-binding activity, and the IgG subclass profile) and the biopsy's morphological features. ResultsOf the 89 patients, 59 (66%) were positive in an FC-XM assay, 17 (19%) had complement-binding DSAs, 55 (62%) were positive for IgG1 and/or IgG3 in a solid phase assay, and 45 (51%) had morphological biopsy features linked to ABMR. ConclusionAn FC-XM assay was unable to discriminate between cases with or without ABMR on biopsy findings; it had a low positive predictive value (<70%) and a low negative positive predictive value (<42.9%), taking into account the sensitivity of our assay (limit of detection: DSAs with an MFI >3000). In this context, the height of the MFI of the dnDSAs might be enough for a high positive predictive value for ABMR and additional testing for complement binding activity can remain optional.

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.

P156 Optimization of a rapid plate flow cytometry crossmatch assay on the cytoflex platform: The baylor protocol

Aim The Flow Cytometric Crossmatch (FCXM) assay has become the standard method for immunologic risk assesment of donor/recipient pairs. There is growing interest in streamlining the assay to expedite results and reduce performance costs without compromising assay accuracy or sensitivity. The introduction of powerful benchtop instruments, such as the Beckman Coulter CytoFLEX system, combined with the previously published Halifaster FCXM by Liwski et al., allowed our group to optimize and simplify the crossmatch procedure. Methods Instrument analytical performance was defined to validate use of the CytoFLEX, a research use only platform, for high complexity clinical testing. The instrument was evaluated for precision, stability, sensitivity, carryover, and accuracy. Performance characteristics were defined by Median Fluorescence Intensity (MFI), counts, and percentages, and compared to the current cytometer platform. The FCXM assay was optimized for time reduction (centrifugation cycles, incubation times, plating and washing), reduction of sample and reagent volumes, and minimization of cell loss during processing (plate type, pronase treatment). Results Instrument Performance: The instrument analytical performance is summarized in the following table. The CytoFLEX showed increased MFI precision and sensitivity when compared with current instrumentation. Assay Optimization: Each optimized component of the Baylor Protocol FCXM is summarized in the following table. The resulting process reduces assay completion time by 2 1/2 h with optimal cell recovery. Conclusions In this report we describe a model for step-by-step performance characterization of a RUO insturment and optimization of every component of a clinical test. We provide a method for CytoFLEX standardization, which may be reproducible with minimal variation across labs. We also demonstrate that the Baylor Protocol FCXM is a sensitive, fast, and cost effective method for use in clinical transplant labs. Download : Download high-res image (892KB) Download : Download full-size image

Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols

The flow cytometric crossmatch (FCXM) assay, which detects the presence of donor specific HLA antibodies in patient sera, is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time, we developed and validated two modified crossmatch procedures, namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60% and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification, employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63% vs 69 ± 19.06%), reduce cell isolation time (by ∼40%) and conserve FCXM assay reagents. Importantly, linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98–0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally, a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7% and 96.8% specificity and sensitivity, respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care.

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.

OR14 Next-generation HLA antibody reagents

Aim A humanized monoclonal antibody (Ab) pan-reactive for HLA Class I is useful for many HLA testing applications, including validation and confirmation of HLA Ab assays and platforms. By engineering the Ab so it is also available in subtype-specific (IgG1-4) and allotype-specific forms, it becomes useful for validating and characterizing Ab assays dependent upon complement-fixation, and validating the specificity of secondary Abs used as detection reagents. Methods The IgG1-4 and allotype-specific forms of this Ab were tested with the detection reagents for our flow cytometric crossmatch (FCXM) assay and single-antigen bead (SAB) assay. The IgG1-4 Abs were also tested in the CDC assay, with and without AHG. The IgG1 Ab was tested at dilutions against cells in the FCXM. The IgG1-4 Abs were tested at multiple dilutions in the SAB and C1q assays, and with and without heat-inactivation and EDTA treatment of sera. Results All lots of AHG used as detection reagents in the FCXM and SAB assays were able to detect the IgG1-4 Abs, and the FCXM reagents detected all 29 isoallotypes. No false-reactivity was seen in the Class II SAB assay. In the CDC assay, IgG1-3 were positive both with and w/out AHG, but IgG4 was only positive when AHG was added. When tested against cells from 15 different individuals, the IgG1 Ab provided consistent reactivity, regardless of the HLA type of the donor cells. When serial dilutions of the IgG1-4 Abs were tested in the SAB and C1q assays, a distinct hierarchy of sensitivity in the C1q assay was apparent, with IgG3 > IgG1 > IgG2. IgG4 was negative in the C1q assay. Conclusions These IgG1-4 monoclonal Abs, directed against a monomorphic Class I epitope, are valuable reagents for validating and characterizing the multiple HLA antibody assays typically employed in a modern HLA laboratory. In Red Cell serology, it has been demonstrated that some IgG isoallotypes are NOT detected by the secondary AHG detection reagents, making these Abs useful to confirm detection with different lots of AHG. These antibodies are also useful as controls in antibody assays that rely on complement-fixation (i.e. CDC and C1q). Finally, when titrated appropriately, these Abs can give consistent low, medium, or strongly-positive reactivity in the SAB and FCXM assays, making them useful as a reagent to standardize and/or normalize results.

P099 The impact of lymphocyte purity on flow cytometry crossmatch (FCXM) assay. It’s not purely theoretical

Aim Lymphocyte flow cytometric crossmatch (FCXM) is used in pre-transplant risk assessment to detect donor specific antibodies (DSA). Many labs use density medium centrifugation to isolate donor cells for FCXM, which yields variable lymphocyte purity (15–90%) depending on the original concentration of lymphocytes and other leukocytes in donor blood, as well as sample quality and age. However, it is unclear whether variation in lymphocyte purity can effect FCXM assay. The goal of this study was to assess the impact of donor lymphocyte purity on FCXM results. Methods Enriched lymphocytes (Ly; >97%), neutrophils (Nu; >98%) and monocytes (Mo; >92%) were isolated from 5 volunteer donors using subset specific EasySep Direct kits (STEMCELL). Whole leukocyte (WL) cell preps were obtained by mixing enriched Ly, Nu and Mo in equal proportion (approx. 33% each) to mimic poor quality cell preps. Ly and WL cell preps were treated with pronase (2.35 kunitz units/ml) or control (PBS) and used as targets in FCXM against negative control (NC) and several dilutions of positive control (PC) sera. Median channel fluorescence (MCF) shifts from NC obtained with Ly vs WL target cell preps were compared. Results T and B cell FCXM NC reactivity was similar with Ly and WL cells. Pronase treatment was equally effective at reducing B cell background in Ly and WL cell preps (191 vs 198 MCF shift). Interestingly, both T and B cells FCXM performed with WL targets showed significantly reduced PC reactivity compared to Ly cells (Fig. 1). This was true at all PC concentrations (1/50–1/400) and was seen with both pronase treated and untreated cells. Average MCF decrease in pronase treated Ly vs WL preps was 49 MCF for T and 68 MCF for B cell FCXM, and in untreated preps it was 41 MCF for T and 61 MCF for B cell FCXM. Conclusion Lymphocyte purity has significant impact on FCXM results. Weak DSA could be missed if low purity lymphocyte preps are used in FCXM assay. Use of highly enriched lymphocytes will improve detection of DSA and reduce variability of FCXM results. Download : Download high-res image (220KB) Download : Download full-size image

P058 Validation of anti-human antibodies used in flow cytometric crossmatching

Aim Flow cytometric crossmatches (FCXM) are widely used to detect recipient alloantibodies against donor tissue to assess compatibility for transplantation. Polyclonal anti-human globulin (AHG) is the reagent typically utilized for the detection of these alloantibodies. Humans produce four primary subtypes of IgG, designated as IgG1–4. Here, we describe a system to determine whether the AHG is able to detect all IgG subtypes. Methods Humanized monoclonal antibodies of each IgG subtype, specific for a red blood cell (RBC) antigen were produced. Briefly, the cDNA for the complimentarity-determining regions (CDR) of the heavy and light chains specific for the Kell RBC antigen were ligated into expression vectors for the separate IgG subtypes 1–4 and kappa light chains, co-transfected into CHO cells, and purified. These monoclonal IgG1–4 antibodies were then tested against RBCs, and three different lots of AHG were used to detect the antibodies bound to the RBCs in a standard FCXM assay. An anti-kappa light chain antibody that recognizes an epitope common in all IgG subtypes was used as a positive control. The AHG alone was used as a baseline control. Results As shown in Fig. 1, each lot of AHG was able to detect all four subtypes of IgG. In addition, titration experiments were performed to determine whether there was preferential detection of any IgG subtype(s). These experiments demonstrated that AHG does exhibit preferential binding to different IgG subtypes. In one specific lot of AHG, the hierarchy of binding/detection was IgG3 > IgG1 > IgG2 > IgG4. Download : Download high-res image (264KB) Download : Download full-size image Conclusion These data provide a proof-of-principle for a method to characterize and validate AHG reagents used in FCXM assays. This is important, because the inability of AHG reagents to detect IgG subtypes may lead to false-negative FCXM results. These animal-derived AHG reagents are polyclonal, and there may be lot-to-lot variability in their ability to detect different IgG subtypes. J. Zimring: Grant/Research Support; Company/Organization; Immucor, Inc.. Scientific/Medical Advisor; Company/Organization; Rubius Therapeutics. Employee; Company/Organization; Bloodworks NW.

OR12: AUTOMATED FLOW CYTOMETRY CROSSMATCH USING THE BIOTEK ELx50 MICROPLATE WASHER

Aim Our laboratory currently uses the “tube” method to perform the flow cytometry crossmatch (FCXM). This method is time and labor intensive and, in some instances, contributes to delayed organ allocation. We have used a microplate washer to automate the Luminex-based bead assays performed with filter bottom plates. The goal of this study was to investigate the use of this instrument toward the automation of the FCXM. Methods 166 sera were run in parallel using the “tube” method and the “microplate” method using a BioTek ELx50 microplate washer (BioTek Instruments) with a MultiScreen 96-well filter plate with a 1.2 μm low protein-binding Durapore membrane (Millipore). Lymphocytes were isolated by means of negative selection using the RoboSep Automated Cell Separator (StemCell Technologies). Then, 3-color FCXM were performed and analyzed on a Becton–Dickinson FACSCalibur instrument (BD Biosciences). Statistical analyses were performed by linear regression and Fisher’s exact tests. Results Linear regression showed a significantly high correlation between the “tube” and the “microplate” T- and B-cell FCXM (R2 = 0.9869 and 0.9899, respectively, p Fig. 1 ). Fisher’s exact test indicated that the “plate” method is significantly more sensitive than the “tube” method. Sensitivity and specificity of the “tube” T-cell FCXM as compared to the “microplate” FCXM were 84% (95% CI = 73–92%) and 99% (95% CI = 95–100%), respectively. Sensitivity and specificity of the “tube” B-cell FCXM as compared to the “microplate” FCXM were 85% (95% CI = 75–93%) and 98% (95% CI = 93–100%), respectively. In addition, the time of the “microplate” FCXM was significantly shorter that the “tube” FCXM (2.5 h vs. 4.0 h, respectively). Conclusions We have demonstrated the ability of the BioTek microplate washer to automate the FCXM assay. Automation using the BioTek microplate washer speeds processing, improves washing efficiency, increases sensitivity and improves accuracy by eliminating tech to tech variability.

19-P

Aim Our laboratory is moving from a tube to tray based flow cytometry crossmatch (FCXM). We investigated the use of a vacuum manifold rather than a flick wash (FW) to perform the wash steps. Methods Crossmatches were performed as previously described using the rapid optimized FCXM method (Liwski et al. ASHI 2011, abstract 45-P). Our laboratory adopted this method but used a vacuum manifold to remove the supernatant. This manifold has 96 height adjustable probes and is attached to a vacuum source, in this case the hospital’s central vacuum system. The probes were lowered into the wells and the supernatant was removed. The cell button was vortexed before adding the next wash. We tested this vacuum manifold wash FCXM method in parallel with the FW FCXM method with 2 different cells. Negative serum (NS) samples as well as 4 dilutions of positive controls (PC) were included in these FCXM. The lymphocytes were isolated using the StemCell™ EasySep™. Reproducibility was evaluated based on the standard deviation (SD) of 14 duplicates for each wash method. The median channel values (MVC) shifts for T and B cell FCXM were also determined for each PC dilution in each wash method by subtracting out the NS value. Results The SD of the vacuum washed duplicates was 4.4 vs. 8.9 in the FW. The MCV shift was 1.8-2.0% more sensitive in the vacuum washed FCXM.[figure1]Fig. 1: T and B cell MCV shifts in vacuum vs wash FCXM trays. Conclusions The vacuum manifold wash method for tray based FCXM assay was as least as effective and reproducible as the FW method. The vacuum manifold also avoids the creation of potentially hazardous aerosols in the laboratory. We have adopted this method for performing FCXM wash steps.

P34 Multicenter prospective study of evolution and outcome of patients with recently diagnosed systemic lupus erythematosus from Mumbai

The flow cytometric crossmatch (FCXM) assay, which detects the presence of donor specific HLA antibodies in patient sera, is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time, we developed and validated two modified crossmatch procedures, namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60% and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification, employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63% vs 69 ± 19.06%), reduce cell isolation time (by ∼40%) and conserve FCXM assay reagents. Importantly, linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98–0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally, a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7% and 96.8% specificity and sensitivity, respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care.

Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation

The anti-HLA antibody can be detected using either a complement-dependent lymphocytotoxicity (CDC) assay or a flow cytometry crossmatch (FCXM) in renal transplantation. Discordant results are often obtained because the two methods detect different reaction phases between the donor lymphocytes and the recipient sera. This study was intended to confirm that antibody binding and cytotoxicity to the lymphocytes can be detected simultaneously in a single FCXM assay, cytotoxic FCXM. In the cytotoxic FCXM, the antibody binding to the lymphocytes was measured using anti-IgG-FITC, and the cytotoxicity using 7-aminoactinomycin D (7-AAD) after adding complement. For an evaluation of two test parameters, the cytotoxicity test moiety (dead-cell percentage) was compared with the anti-human globulin (AHG)-CDC, and the antibody-binding test moiety (sample/control fluorescence ratio) with the conventional FCXM in 77 positive and 30 negative crossmatches. In the cytotoxic FCXM, both antibody binding and cytotoxicity could be assessed in a single anti-IgG-FITC/7-AAD plot. Regarding the correlation between the presence of HLA antibodies and the test result, the cytotoxicity parameter (r = 0.55) appeared to be more suitable than that of the AHG-CDC (r = 0.50) but the antibody-binding parameter (r = 0.83) was worse than that of the conventional FCXM (r = 0.93). The sensitivity of both parameters of the cytotoxic FCXM was not significantly different from each conventional counterpart (P = 0.33 and P = 0.22, respectively). The simultaneous detection of Ab binding and cytotoxicity was possible by the cytotoxic FCXM with the test efficiencies similar to the conventional counterparts. If this new assay is improved through the further studies to optimize the critical assay variables, this may be used as an alternative to the conventional assays to acquire more information on the characteristics of the recipient's HLA alloantibodies.