Genotyping of human platelet antigen-1 by gene amplification and labelling in one system and automated fluorescence correlation spectroscopy.

Genotyping of human platelet antigen-1 (HPA-1) is required for the diagnosis and appropriate therapy of alloimmunization. Recently, the HPA-1 polymorphism has been identified as an inherited risk factor for thrombosis. Most currently used methods for HPA-1 genotyping have the disadvantage of time-consuming post-polymerase chain reaction (PCR) processes such as ligation (oligonucleotide ligation assay), restriction enzyme digestion (allele-specific restriction enzyme analysis) and electrophoresis (single-strand conformation polymorphism). We present a novel method for HPA-1 genotyping based on a homogeneous PCR strategy (GALIOS, gene amplification and labelling in one system) combined with automated fluorescence correlation spectroscopy (FCS). The PCR uses one pair of gene-specific amplification primers and two allele-specific, semi-nested labelling primers. The allele-specific labelling primers differ in a single nucleotide (T for HPA-1a/1a, C for HPA-1b/1b) and are coupled to different fluorescent dyes. The quantities of generated fluorescent PCR products are analysed by FCS at 543 nm and 633 nm excitation wavelength respectively. The genotypes determined using this method were in 100% concordance with the results obtained by allele-specific restriction analysis (n = 380 samples). The assay was validated for specificity, reliability and the dynamic range. This innovative method of rapid HPA-1 genotyping offers a specific and robust system, which is applicable for routine HPA-1 genotyping.