Rapid rare ABO blood typing using a single PCR based on a multiplex SNaPshot reaction

Background: ABO subgroups would be considered when discrepancies in ABO grouping occur. Serological methods including adsorption-elution test, salivary ABH inhibition test, and anti-A1 (lectin) saline method could be used. However, these serological methods are laboring and obscure. Therefore, reliable and affordable method to assess the ABO subgroups is of particular interest. Methods: To solve this problem, the multiplex SNaPshot-based assays were designed to determine rare A and B subgroups. Primers used as probes for determination of rare ABO blood groups known in Taiwanese population were designed. Many ABO subtype samples were used to validate the accuracy and reproducibility of our SNaPshot panel. Results: A panel of primer probes were successfully designed in determining 8 SNP sites (261, 539, 838, 820, 745, 664, IVS6 +5, and 829 in exon 6 and 7) for A phenotype and 6 SNP sites (261, 796, IVS3 +5, 247, 523, and 502 in exon 2, 6 and 7 and intron 3) for B phenotype. SNaPshot analysis for defining blood group A alleles (A ₁ , A ₂ , A ₃ , A _m and A _el ) and blood group B alleles (B ₁ , B ₃ , B _w and B _el ) was therefore available. Conclusion: SNaPshot analysis could be used in reference laboratories for typing known rare subgroups of A and B without DNA cloning and traditional sequencing. Moreover, this method would help to construct databases of genotyped blood donors, and it potentially plays a role in determining fetal–maternal ABO incompatibility.