Flow cytometric HLA‐B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation

Abstract

The determination of HLA-B27 (B27) status is important in the diagnosis 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 leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; 2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; 5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory.

External quality assessment of flow cytometric HLA‐B27 typing

“…In conclusion, our 3.5-year experience with HLA-B27 EQA scheme has shown that flow cytometry can identify HLA-B27 pos samples (i.e., the subtype HLA-B*2705, prevalent in the caucasoid population), but that cross-reactivity of the commonly used HLA-B27 MoAb is a major problem that frequently leads to false-positive test results. HLA-B27 typing by flow cytometry may be further improved by (i) application of quantitative flow cytometry to standardize the distinction between relatively dim (cross-reactive) and strong (true positive) signals (32,33); (ii) complete documentation of the reactivity of all currently defined HLA-B27 alleles (i.e., B*2701 to B*2715) with the available anti-HLA-B27 MoAb; and (iii) rigorous analysis of the crossreactivity patterns of these MoAb with HLA-antigens other than HLA-B27. We feel that there will remain a place for cost-effective and simple screening methods for selected HLA alleles, such as HLA-B27, in the clinical diagnostic laboratory.…”

Gamma radiation induces CD69 expression on lymphocytes

“…Samples were analyzed using either a FACScan (Becton Dickinson Immunocytometry Systems, Inc., Mountain View, CA) or a Cytoron Absolute (Ortho Diagnostics) instrument. The daily quality control procedures with Calibrite (Becton Dickinson) and DNA-Check beads (Coulter Cytometry) were performed as previously described (10). The coefficient of variation (CV) of fluorescence measurements of the DNA-Check beads was less than 3% throughout this study.…”