Single PCR Multiplex SNaPshot Reaction for Detection of Eleven Blood Group Nucleotide Polymorphisms Optimization, Validation, and One Year of Routine Clinical Use

Hemagglutination-based assays have several clinical shortcomings. To overcome this difficulty, we have developed a multiplex-PCR SNaPshot assay adapted to the Southern French population, which includes individuals from sub-Saharan Africa and the Comoros archipelago. Single nucleotide polymorphisms (SNPs) associated with clinically relevant blood antigens as well as with null phenotypes were profiled (i.e., K/k, Fy(a)/Fy(b)/Fy(bw)/Fy(null), S/s/U-/U+(var), Jk(a)/Jk(b), Do(a)/Do(b), Yt(a)/Yt(b), and Co-a/Co-b). A single multiplex-PCR reaction was used to amplify nine gene regions encompassing 11 SNPs. Identification was obtained by incorporation of the complementary dye-conjugated single base at the 3' end of each probe primer annealed proximal to the target SNP. After optimization, the SNaPshot assay was validated with 265 known allele or phenotype pairs. Results were found fully concordant with those of hemagglutination, allele-specific PCR, and/or sequencing. The assay was then evaluated on 227 blood samples in a clinical context. A total of 203 derived-phenotypes were generated, including 82 atypical phenotypes [i.e., Fy(b+(w)) (n = 32); K+ (n = 22); Co(b+) (n = 8); Yt(b+) (n = 18); S-s+U+(var) (n = 2), 105 null phenotypes, i.e., Fy(a-b-) (n = 97); S-s-U- (n = 6); S-s-U+(var) (n = 2)] and sixteen Fy-positive samples carried a FY*Fy allele. The findings show that this assay can provide a low-cost and fast genotyping tool well adapted to local ethnically mixed populations. (J Mol Diagn 2010, 12:453-460; DOI: 10.2353/jmoldx.2010.090222)