Method Description

Anti- human leukocyte antigens (HLA) fluorescein isothiocyanate/CD3 phycoerythrin monoclonal antibody reagent is added to human whole blood. The fluorochrome-labeled antibodies bind specifically to leukocyte surface antigens. The stained specimens are treated with lysing solution to lyse red blood cells, then washed and fixed prior to flow cytometric analysis. The flow cytometer is set up using BD 7 Color Setup beads and CS and T beads with FacsDIVA software and HLA-B27 calibration beads with the HLA-B27 software. The HLA-B27 software first identifies, on a forward scatter (FSC) versus fluorescence 2 (FL2) dot plot, the cluster of events with a uniformly bright CD3-positive signal (T-lymphocytes). During analysis, the median fluorescence intensity of the anti-HLA-B27 FITC signal is calculated for the events included in the FSC/FL2 gate. Specimens with a median fluorescence 1 channel result greater than or equal to the decision marker are considered HLA-B27 positive. Specimens with a median channel result lower than the decision marker are considered HLA-B27 negative. This decision marker is encoded in the suffix of the reagent lot number listed on the vial label.(Package insert: BD HLA-B27 Test Kit. BD Biosciences; 01/2018)