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Diagnostic Accuracy of Flow Cytometric Detection of HLA-B27 Using ABCm3 Antibody Compared with that of PCR-SSP in
Mustajab Alam1,2, Hamid Nawaz Tipu1,2, Muhammad Hussain1,2
1Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan.
Objective:
To assess the diagnostic accuracy of flow cytometry (FCM) using the ABCm3 monoclonal antibody for HLA-B27 detection in patients with clinically suspected ankylosing spondylitis (AS), using PCR-SSP as the molecular gold standard, and to determine the optimal median fluorescence intensity (MFI) cut-off values via receiver operating characteristic (ROC) analysis.
Study Design:
A cross-sectional study. Place and Duration of the Study: Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from August 2024 to January 2025.
Methodology:
Eighty patients aged <70 years clinically suspected of AS were enrolled. FCM using FITC-conjugated ABCm3 antibody was performed on fresh EDTA blood samples and analysed with FACSDiva software. PCR-SSP with SC1/SC2 primer sets served as the gold standard. The MFI was compared between the PCR-positive and negative groups. The ROC analysis was used to determine optimal cut-off values, sensitivity, specificity, and diagnostic accuracy.
Results:
Of the 80 patients, 39 (48.8%) met ASAS criteria for AS. PCR-positive patients showed significantly higher median MFI (1,678; IQR: 1,525-1,876) compared to PCR-negative individuals (840; IQR: 340 -1,408) (p <0.001). ROC analysis revealed an AUC of 0.914. At an MFI cut-off of 1,384, sensitivity was 94.9%, and specificity was 73.2%. At 1,422 MFI, specificity increased to 85.4% with a minor reduction in sensitivity (89.7%).
Conclusion:
FCM using the ABCm3 antibody shows strong concordance with PCR-SSP and provides a reliable, cost-effective alternative for rapid HLA-B27 screening in suspected AS.
Key Words:
Ankylosing spondylitis, HLA-B27 antigen, Flow cytometry, Polymerase chain reaction, Diagnostic accuracy.
