Genotypisierung von HPA-1 bis HPA-5 mit der allelspezifischen Ligasekettenreaktion nach Amplifikation von genomischer DNA in einer Multiplex-PCR

Abstract

Platelet-specific antibodies are involved in refractoriness to platelet transfusions, neonatal alloimmune thrombocytopenia, and posttransfusion purpura. Binding to different epitopes on platelet glycoproteins, human platelet antigens (HPA), they can cause severe thrombocytopenia. The polymorphic DNA regions of the best known HPA systems HPA-1 to HPA-5 were amplified from genomic DNA in a multiplex polymerase chain reaction (PCR). An automatable ligation-based assay (LCR) using allele-specific oligonucleotide probes was developed for rapid genotyping. The results were correlated with the well established allele-specific restriction enzyme assay (ASRA), and the phenotype was determined by the monoclonal antibody-specific immobilization of platelet antigens (MAIPA). We found a 100% concordance of genotypes between the HPA-LCR in 54 cases of NAIT, PTP and normal controls with the established methods. The gene frequencies calculated from 216 German and 55 Japanese random blood donors were similar to those determined by other time-consuming typing methods in a Caucasian and Japanese population, respectively. Multiplex PCR and the allele-specific HPA ligation assay provide a reliable tool for typing both small series and all platelet donors of a transfusion center in order to identify rare genotypes necessary for the treatment of patients suffering from NAIT, PTP or refractoriness to platelet transfusions.