From the Guidelines
For quantitative results of HLA-B27, flow cytometry is the recommended testing method. This technique uses fluorescently labeled antibodies that specifically bind to HLA-B27 antigens on the surface of white blood cells, allowing for precise measurement of the presence and quantity of these markers. According to recent studies, such as the one published in Nature Protocols 1, caution should be taken when measuring HLA-related molecular phenotypes, including HLA gene and protein expression. When considering the method for testing HLA-B27, several factors come into play, including:
- The need for accurate measurement of HLA protein expression, as this is what plays a role in conditions like ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies
- The potential for bias in antibody-based methods due to differing binding affinities to the protein products of different HLA alleles, as noted in the study 1
- The importance of using a method that can provide quantitative results, as this is essential for understanding the relationship between HLA-B27 expression and disease severity or progression Flow cytometry provides superior sensitivity and specificity compared to other methods, with results typically available within 24-48 hours. The test requires a small blood sample (approximately 5-10 mL) collected in an EDTA tube. While molecular methods like PCR can detect the HLA-B27 gene, flow cytometry is preferred for quantitative assessment as it directly measures the protein expression on cell surfaces. This distinction is clinically relevant because it's the expressed protein that plays a role in conditions like ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies. Flow cytometry results are typically reported as percentage of cells expressing HLA-B27 and mean fluorescence intensity, providing both qualitative and quantitative information.
From the Research
Methods for HLA-B27 Testing
The following methods can be used for HLA-B27 testing:
- Flow cytometry: This method uses antibodies against HLA-B27 and can be used for routine typing 2, 3, 4, 5, 6
- Microlymphocytotoxic test (MLCT): This method uses a complete panel of anti-HLA-class I antibodies and can be used as a standard for HLA-B27 typing 2
- Enzyme immunoassay (EIA): This method uses serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) but may lead to typing errors 2
- Molecular biological methods: These methods, such as next-generation sequencing (NGS) and PCR melting assays, can be used for high-resolution HLA-B typing and can distinguish between pathogenic and nonpathogenic HLA-B27 variants 4, 5, 6
- DNA sequencing: This method can be used to identify HLA-B27 subtypes and cross-reactive subtypes 5
- DNA microarray: This method can be used for HLA-B27 allele detection and discrimination of AS/non-AS subtypes 5
Comparison of Methods
The studies compared the following methods:
- Flow cytometry vs. MLCT: Flow cytometry showed a complete concordance with MLCT when using a complete panel of anti-HLA-class I antibodies 2
- Flow cytometry vs. molecular biological methods: Molecular biological methods, such as NGS, were superior to flow cytometry for the unambiguous identification of HLA-B27 variants 4
- Flow cytometry vs. DNA sequencing: DNA sequencing can identify HLA-B27 subtypes and cross-reactive subtypes that may not be detected by flow cytometry 5
- Genetic sequence-based method vs. flow cytometry assay: The genetic sequence-based method and the flow cytometry assay gave consistent results in 99% of cases 6
Quantitative Results
For getting quantitative results of HLA-B27, the following methods can be used: