We developed an allele-specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy-three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA-2, -3 and -4 in parallel without the use of platelets, platelet-specific alloantibodies or restriction enzymes.

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We identified a new third allele of HPA-7, which is characterized by a 1297C>T mutation in the GPIIIa gene. This 1297C>T allele was found in 0.15% of the Japanese population. An antibody against this antigen could be the cause of severe NAIT.

Recently, the polymorphism of a new human platelet antigen, Ca/Tu, was shown to be derived from a G-A nucleotide substitution at base 1564 of GPIIIa cDNA, which leads to a single amino acid difference, Arg/Gln at amino acid 489 of GPIIIa. We developed a PCR-RFLP method to determine the genotypes of Ca/Tu and their frequencies in a Japanese population. Fifteen Ca/Tu(a) donors comprising 1 Ca/Tu(a/a) homozygous donor and 14 Ca/Tu(a/b) heterozygous donors were found among the 314 random donors analyzed. The frequencies of Ca/Tu genes were 0.025 (Ca/Tu(a)) and 0.975 (Ca/Tu(b)). The present study showed that the frequency of Ca/Tu[l individuals in the Japanese (15/314) was approximately 7-fold higher than in the Finnish population (1/150) previously reported by Kekomäki et al. Therefore, attention must be given to the involvement of the Ca/Tu alloantigen in neonatal alloimmune thrombocytopenia and the refractoriness of platelet transfusion.

Recently, the polymorphism of a new human platelet antigen, Ca/Tu, was shown to be derived from a G-A nucleotide substitution at base 1564 of GPIIIa cDNA, which leads to a single amino acid difference, Arg/Gln at amino acid 489 of GPIIIa. We developed a PCR-RFLP method to determine the genotypes of Ca/Tu and their frequencies in a Japanese population. Fifteen Ca/Tua donors comprising 1 Ca/Tu(a/a) homozygous donor and 14 Ca/Tu(a/b) heterozygous donors were found among the 314 random donors analyzed. The frequencies of Ca/Tu genes were 0.025 (Ca/Tua) and 0.975 (Ca/Tu(b)). The present study showed that the frequency of Ca/Tua individuals in the Japanese (15/314) was approximately 7-fold higher than in the Finnish population (1/150) previously reported by Kekomäki et al. Therefore, attention must be given to the involvement of the Ca/Tu alloantigen in neonatal alloimmune thrombocytopenia and the refractoriness of platelet transfusion.

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive.

We developed an allele-specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy-three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA-2, -3 and -4 in parallel without the use of platelets, platelet-specific alloantibodies or restriction enzymes.