Large-scale DNA typing for human platelet alloantigens by PCR-PHFA (preferential homoduplex formation assay)

Alloimmunization against human platelet alloantigens (TTPA) is known to be involved in disorders such as neonatal alloimmune thrombocytopenic purpura, posttransfusion purpura, and refractoriness to platelet transfusion therapy. HPA typing is essential in diagnosis and management of patients. Therefore a reliable and speedy method is necessary for HPA typing. We have successfully applied a new DNA typing method, PCR-preferential homoduplex formation assay (PHFA) method, to typing for the HPA-1, -2, -3, -4, -5 and -6 systems. This method is based on DNA strand competition during hybridization under a precisely controlled temperature gradient between a double-labelled amplicon (standard DNA), prepared from biotin- and DNP-labelled primers, and an unlabelled amplicon (sample DNA). The results obtained by PCR-PHFA typing were in good agreement with the allotypes determined by serological typing and by other DNA typing methods. The PCR-PHFA method can be easily automated, is suitable for typing both small and large numbers of samples, and thus is applicable to routine HPA typing.