Improving the Sensitivity and the Specificity of HLA-B27 Typing by Replacing Serological Tests with a TaqMan-PCR Assay

HLA-B27 is a strong diagnostic biomarker as it is found in 90– 95% of ankylosing spondylitis. Routinely, the false-positive results generated by cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) with some other type of HLA-B antigens are problematic. The present study aims at improving the sensitivity and specificity of typing for HLA-B27 by using a more accurate DNA assay. A real time sequence specific primer polymerase chain reaction (PCR-SSP) is performed by using MGB TaqMan oligoprobe and an ABI PRISM^™ 7500 sequence detection system. The TaqMan PCR-SSP assay is compared with Immunophenotyping by flow cytometric antigen assay (BD HLA-B27-Kit, clone GS145.2). The results are finally confirmed by sequencing (ABI PRISM ® 3100). As summarised in the table, three methods are identical for clearly positive and negative results. There is 90% concordance for borderline negative results obtained by immunophenotyping with TaqMan PCR-SSC and/or sequencing. However, only 15.4% of borderline positives obtained by immunophenotyping are confirmed as true HLA-B27 positives by sequencing as well as TaqMan PCR-SSC. TaqMan PCR-SSP DNA assay completely correlates with sequencing (gold standard) with 100% PPV and NPV, compared to a PPV of 15% and a NPV of 98% for borderline results by immunophenotyping.

Conclusion: when compared with flow cytometric antigen assay, typing HLA-B27 by TaqMan PCR-SSP clearly demonstrates an advantage to better establish the genotype of the patient. Specifically, there is a marked difference for ambitious positive results from immunophenotyping. Moreover, compared to immunophenotyping, there is no need of fresh samples, can test a larger number of samples concomitantly, and reduces technical time as well as cost.