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HLA-B27 screening by flow cytometry
B Lingenfelter1, T C Fuller, L Hartung
1Department of Pathology, University of Utah, Salt Lake City, USA.
Insights
A new flow cytometry method accurately detects the lymphocyte Human Leukocyte Antigen B27 (HLA-B27) expression. This convenient assay shows high sensitivity and specificity for screening HLA-B27, aiding in clinical diagnosis.
Area of Science:
- Immunology
- Clinical Diagnostics
- Flow Cytometry
Background:
- Human Leukocyte Antigen B27 (HLA-B27) is a crucial marker in certain autoimmune diseases.
- Traditional microlymphocytotoxicity testing for HLA-B27 is time-consuming and complex.
- Development of rapid and accurate methods for HLA-B27 detection is clinically significant.
Purpose of the Study:
- To evaluate a novel two-color direct immunofluorescent flow cytometric assay for lymphocyte HLA-B27 expression.
- To compare the performance of the flow cytometric assay against traditional microlymphocytotoxicity testing.
- To determine the sensitivity and specificity of the flow cytometric HLA-B27 assay.
Main Methods:
- A two-color direct immunofluorescent assay using fluorescein-isothiocyanate (FITC)-conjugated anti-B27 and phycoerythrin (PE)-conjugated anti-CD3 antibodies.
- Whole blood samples from 209 clinical cases were analyzed using flow cytometry.
- Analysis involved gating on CD3 positive events and evaluating anti-B27 staining intensity via median channel fluorescence.
Main Results:
- The flow cytometric assay demonstrated high sensitivity (97.6%) and specificity (95.9%) for HLA-B27 detection.
- Median channel fluorescence correlated with HLA-B27 expression levels, distinguishing negative, B7 positive, and B27 positive samples.
- Cross-reactivity was observed with HLA-B7 (n=38), HLA-B37 (n=3), and HLA-B39 (n=3) antigens.
Conclusions:
- The flow cytometric assay for lymphocyte HLA-B27 expression is a convenient and useful screening tool.
- The assay offers high sensitivity and specificity, comparable to conventional methods.
- Confirmation with microlymphocytotoxicity or alternative monoclonal antibodies is recommended for positive flow cytometry results to maximize specificity.
Abstract:
A flow cytometric assay for lymphocyte HLA-B27 expression using a two-color direct immunofluorescent assay was compared to traditional microlymphocytotoxicity testing on 209 clinical samples. For the flow cytometric assay, whole blood was mixed with a monoclonal anti-B27 conjugated to fluorescein-isothiocyanate (FITC) and anti-CD3 conjugated to phycoerythrin (PE). The samples were analyzed with flow cytometry by gating on CD3 positive events and anti-B27 staining intensity was evaluated as median channel fluorescence of the histogram peak. The median channel fluorescence was least with B27 negative and B7 negative samples (84 +/- 17), intermediate with samples that were B27 negative but B7 positive (118 +/- 13), and greatest with samples that were B27 positive (155 +/- 13). In addition to cross-reactivity with the B7 antigen (n = 38), the monoclonal anti-B27 cross-reacted with HLA-B37 positive samples (n = 3) and HLA-B39 positive samples (n = 3). Using a median channel fluorescence cutoff of 136, 39 of the 40 B27 positive samples gave positive results in the flow cytometric assay for a sensitivity of 97.6%. The specificity was 95.9% with 7 false positives of 169 B27 negative samples. The flow cytometric HLA-B27 assay is a convenient, useful screening test. For greatest specificity, samples positive by flow cytometry should be confirmed by conventional microlymphocytotoxicity or by use of other monoclonal antibodies directed against B27.