FLOW CYTOMETRIC HLA-B27 TYPING USING CD3 GATING AND MOLECULES OF EQUIVALENT SOLUBLE FLUOROCHROME (MESF) QUANTITATION

The determination of HLA-B27 (B27) status is important in the diagnosi s of ankylosing spondylitis, Reiter's disease, and other arthropathies . Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leu kocyte populations may manifest significant variation in staining inte nsity with B27 antibodies. Therefore, gating utilizing only light scat ter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3(+) cells were postulated as a mea ns to eliminate these anomalies. Furthermore, quantitative FCM measure ments, as afforded through use of molecules of equivalent soluble fluo rochrome (MESF) units, can minimize day-to-day and instrument-to-instr ument variabilities in fluorescence measurements. We compared CD3 gati ng to light scatter gating and MESF analysis on 123 specimens in a 4-m onth period and found: 1) CD3 gating gave the lowest nonspecific B27 a ntibody binding among B27-negative subjects; 2) there was no significa nt difference in MESF values between CD3 gating and light scatter gati ng of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) ident ification of patients with low 827 expression may require the use of C D3 gating in order to avoid costly confirmation testing; 5) fluorescen t standard beads are stable for routine use in a clinical flow cytomet ry laboratory. (C) 1996 Wiley-Liss, Inc.