Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols |
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Authors: | Robert S. Liwski Anna L. Greenshields David M. Conrad Cathi Murphey Robert A. Bray Jorge Neumann Howard M. Gebel |
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Affiliation: | 1. Department of Pathology, Dalhousie University, Halifax, Nova Scotia B3H 1V8, Canada;2. Southwest Immunodiagnostics Inc., San Antonio, TX 78229, USA;3. Department of Pathology, Emory University Hospital, Atlanta, GA 30322, USA;4. Laboratory of Transplantation Immunology, Santa Casa Hospital, Porto Alegre, Brazil |
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Abstract: | 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. |
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Keywords: | 3SD 3 standard deviations CDC complement-dependent cytotoxicity DSA donor-specific HLA antibodies FCXM flow cytometry crossmatch FWB flow wash buffer HLA human leukocyte antigen MCF median channel fluorescence MCFS median channel fluorescence shift MFI mean fluorescence intensity NC negative control PBMC peripheral blood mononuclear cell PBS phosphate buffered saline PC positive control RT room temperature SAB single antigen bead SD standard deviation SFCXM standard flow cytometry crossmatch SWID Southwest Immunodiagnostics Inc. TAT turnaround time VXM virtual crossmatch XM crossmatch Flow cytometry cross match HLA antibodies Assay optimization Transplantation |
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