An optimized method for routine HLA-B27 screening using flow cytometry.
Flow cytometry and monoclonal antibodies are promising tools for HLA-antigen detection. Previous approaches have been hampered by the lack of a carefully standardized system for calibration and sample analysis. A new system for HLA-B27 screening was developed using a FACScan flow cytometer, software for automated calibration and analysis, calibration beads, and the anti-HLA-B27-FITC/anti-Leu4-PE (CD3) monoclonal antibodies. The median fluorescence channel result for the HLA-B27-FITC signal of CD3+ T lymphocytes is compared to a decision marker. Values lower than this threshold are read as HLA-B27 negative and those above are recommended for retesting with the classic microcytotoxicity assay on the presumption of HLA-B27 positivity. The anti-HLA-B27 antibody reacts with all six HLA-B27 subtypes and shows a weaker binding to HLA-B7. The screening test results were compared with those from the microcytotoxicity assay for HLA-typing in studies involving several European centers. The observed sensitivity was 100% (95% Cl:98.6-100) and the specificity was 97.4% (95% Cl: 96.4-98.3). Other performance studies verified the reproducibility and reliability of results obtained with the screening system.
Hulstaert
F
F
Becton Dickinson Immunocytometry Systems, Erembodegem, Belgium.
Albrecht
J
J
Hannet
I
I
Lancaster
P
P
Buchner
L
L
Kunz
J
J
Falkenrodt
A
A
Tongio
M
M
De Keyser
F
F
Veys
E M
EM
D000925:Anticoagulants / Q000494:pharmacology; D001782:Blood Donors; D001793:Blood Preservation; D003601:Cytotoxicity Tests, Immunologic; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D015796:HLA-B27 Antigen / Q000097:blood*; D006801:Humans; D007106:Immune Sera; D008403:Mass Screening / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000925:Anticoagulants; D015796:HLA-B27 Antigen; D007106:Immune Sera