A new strategy for systematically classifying HLA alleles into serological specificities: Update and refinement

HLA antigens were historically defined according to the unique reactivity pattern of cells expressing HLA molecules with distinctive clusters of allo-antisera and/or monoclonal antibodies. Subsequently, amino acid residues determining epitopes (DEP) in the HLA molecule were correlated with reactivity patterns. In current clinical practice, the presence of allo-antibodies is assessed using Luminex-based solid phase single antigen bead (SAB) assays for transplantation. Recently, novel antigens were proposed for HLA molecules with DEP patterns that do not match any serologically defined antigens recognised by the WHO Nomenclature Committee. To validate the antigens, mean fluorescence intensity values of SABs tested on >13,000 patients' sera were extracted from clinical databases and analysed by scatter plots using a linear regression model. We found that when two proteins were considered as the same antigen in the original study, for example, HLA-A*02:01 and -A*02:06, their correlation ranked among the highest values at each locus. In contrast, discrete asymmetric outliers were observed when there were different antigens, for example, HLA-A*30:01 and -A*30:02, allowing validation and confirmation of 20 novel antigens for HLA-A, -B, -C and -DR. The outliers were confirmed to be true or false by flow cytometric crossmatches. In addition to the previously defined residues for antigen assignments, findings suggest that further distinction should be made for common antigens by including the substitutions at residue 67 of HLA-B, 67 and 74 of -DR. These serologic analyses can be applied systematically to identify and confirm novel antigens. These developments will lead to designing optimal SAB panels and further improving virtual donor-specific antibodies assessment.