High-throughput multiplex single-nucleotide polymorphism analysis for red cell and platelet antigen genotypes

BACKGROUND: Transfusion recipients who become alloimmunized to red cell or platelet (PLT) antigens require antigen-negative blood to limit adverse transfusion reactions. Blood collection facilities use regulated and unregulated antibodies to phenotype blood, the cost of which can be prohibitive depending on the antisera and demand. An alternative strategy is to screen blood for these antigens with genomic DNA and the associated single-nucleotide polymorphisms (SNPs). STUDY DESIGN AND METHODS: A multiplex polymerase chain reaction (PCR)-oligonucleotide extension assay was developed with genomic DNA and a SNP genotyping platform (GenomeLab SNPstream, Beckman Coulter) to identify SNPs related to D, C/c, E, S/s, K/k, Kp(a/b), Fy(a/b), FY0 (-33 promoter silencing polymorphism), Jk(a/b), Di(a/b), and human PLT antigen (HPA)-1a/1b. A total of 372 samples were analyzed for 12 SNPs. The genotypes were compared to the blood group and PLT antigen phenotypes. RESULTS: Individual sample results varied from 98 to 100 percent for 11 of 12 SNPs. D was correctly identified in 292 of 296 (98.6%) D+ donors. The RHCE exon 5 E/e SNP analysis had the lowest concordance (89.5%). Thirty-three R1R1 and 1 r"r were correctly identified. PCR-restriction fragment length polymorphism (RFLP) on selected samples confirmed the presence of the FY0 silencing polymorphism in nine donors. Homozygous HPA-1b/1b was identified in four donors, which was confirmed by PCR-RFLP (n = 4) and anti-HPA-1a serology (n = 2). The two HPA-1a-negative donors were recruited into the plateletpheresis program. CONCLUSION: The platform has the capacity to genotype thousands of samples per day. The suite of SNPs provides genotype data for all blood donors within 36 hours of the start of testing.