Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCyclerTM

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently-labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.