Genotyping of the human platelet antigen systems 1 through 5 by multiplex polymerase chain reaction and ligation-based typing

Background: Platelet-specific antibodies may be involved in refractoriness to platelet transfusions, disorders such as neonatal alloimmune thrombocytopenia, and posttransfusion purpura. Genotyping for the major human platelet antigen (HPA) systems HPA-1 through HPA-5 is of considerable help in establishing the diagnoses of these diseases. Study Design and Methods: A new genotyping method is described, Alleles of all five systems are amplified in a multiplex polymerase chain reaction. Subsequently, aliquots of the amplification products are thermocycled in the presence of a pair of allele-specific oligonucleotide probes and a heat-stable ligase, After heal denaturation, the probes hybridize adjacent to complementary sequences of the amplification product. In a perfect match, the two probes become covalently joined, Detection of the ligation product is performed with an enzyme-linked immunosorbent assay, Results: Complete concordance of genotypes between the ligation-based typing and established genotyping methods was determined in 54 Austrian (HPA-1, -2, -3, and -5) and 56 Japanese (HPA-4) individuals. Ligation-based genotyping of HPA-1 polymorphism using platelet-derived RNA as starling material gave concordant results in all 15 cases tested. Conclusion: Multiplex polymerase-chain reaction in combination with ligation based typing allows fast typing of large numbers of platelet donors and screening for critical antigens in pregnant women.