Converting an HLA‐B27 flow assay from the BD FACSCanto to the BD FACSLyric

Abstract

HLA‐B27 is a major histocompatibility complex (MHC) class I antigen which exhibits strong association (90%) with ankylosing spondylitis. HLA‐B27 detection in patients by flow cytometry is a widely used clinical test, performed on many different flow cytometer models. We sought to develop and validate a test conversion protocol for the HLA‐B27 test performed on the BD FACSCanto to BD's newer FACSLyric flow cytometers. The development and validation experiments were performed using anti‐HLA‐B27*FITC/CD3*PE antibody‐stained whole blood patient specimens. The anti‐HLA‐B27*FITC logarithmic median fluorescence (LMF) results on the BD FACSCanto were converted to median fluorescence intensity (MFI) values on the BD FACSLyric. Clustering of the HLA‐B27 positive and negative values, using a 3rd order polynomial equation, resulted in a conversion of the BD FACSCanto cutoff values, negative (<150 LMF) and positive (≥160 LMF), to negative (<4530 MFI) and positive (≥6950 MFI) on the BD FACSLyric. Accuracy was assessed by comparing the flow results obtained on the BD FACSCanto and BD FACSLyric to a molecular PCR based assay. Additional validation parameters (compensation verification, intra‐ and inter‐assay precision, and instrument comparison) were performed per the recommendations outlined in the Clinical and Laboratory Standards Institute (CLSI) H62 guidelines for validation of flow cytometry assays.