HLA-B27 Typing: Evaluation of an Allele-specific PCR Melting Assay and Two Flow Cytometric Antigen Assays

Background: Human leukocyte antigen B27 (HLA-B27) is a major histocompatibility complex class 1 molecule that is strongly associated with the disease ankylosing spondylitis. Testing for HLA-B27 is of diagnostic value because 90% of patients with ankylosing spondylitis have the B27 antigen. Two commonly used HLA-B27 flow cytometric assays are commercially available.

Methods: An allele-specific polymerase chain reaction (PCR) melting assay for HLA-B27 was compared with two available antigen assays on 371 clinical samples. The accuracy of the assays was measured by receiver operating characteristic analysis using the PCR method and sequencing as the reference standard.

Results: When PCR results were compared with those of the antigen assays, complete concordance was observed except for five discrepant results that were resolved by sequence analysis. Using DNA sequencing as the gold standard, the sensitivity and specificity of PCR were 99.6 and 100.0, those of the best single antigen assay were 98.2 and 97.6, and those of a reflex combination of both antigen assays were 98.8 and 97.6.

Conclusions: The allele-specific PCR melting assay for HLA-B27 genotyping is easy to perform and has better sensitivity and specificity than antigen assays. The performance of the two flow cytometric antigen assays depends on the antibody used and the positive cutoff values assigned.