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

Abstract

Alloimmunization against human platelet alloantigens (HPA) 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.

New polymorphisms of human CD80 and CD86: lack of association with rheumatoid arthritis and systemic lupus erythematosus

“…Genotyping of CD80 downstream promoter region using PCR-PHFA PCR-PHFA has been successfully applied to the allele typing for HLA-DPB1, 45 human platelet antigens (HPAs), 48 and Fc␥RIIIB, 24 and we have recently established the allele typing method of human TNFA 5′-flanking region using PCR-PHFA. 49 The downstream promoter regions of the genomic DNA from three samples possessing (−79C/C, −7T/T, +5C/C), (−79G/G, −7T/T, +5C/C) and (−79C/C, −7C/C, +5A/A) genotypes, respectively, were amplified using biotin-labeled forward primer and DNP-labeled reverse primer and were used as the standard DNAs.…”

Neonatal alloimmune thrombocytopenia caused by an antibody specific for a newly identified allele of human platelet antigen‐7

“…HPA‐4b–incompatible NAIT represents the majority (72%) of HPA‐related NAIT cases in Japan, 24 and cases due to HPA‐2b, HPA‐3a, HPA‐5b, and HPA‐6b have also been reported 22 . Furthermore, before the present study, the low‐frequency HPA‐7 to HPA‐13 antigens were found to be monomorphic in Asian (including Japanese) populations 2,4,25 …”

Determination of Granulocyte–Specific Antigens on Neutrophil FcÁ Receptor IIIb by PCR–Preferential Homoduplex Formation Assay, and Gene Frequencies in the Japanese Population

“…The double–labeled standard DNA was captured on a streptavidin–coated microtiter plate and detected using an alkaline–phosphatase–conjugated anti–DNP antibody with a chromogenic substrate, as described previously [18]. …”

“…Earlier, we developed a new DNA typing method, the PCR–preferential homoduplex formation assay (PHFA) [17], and applied it to the typing of human platelet alloantigens (HPA)–1, –2, –3, –4, –5, and –6 systems [18]. Now, we have applied this method to DNA typing of neutrophil–specific antigens, namely, NA1, NA2, and SH, located on the neutrophil FcγRIIIb, and determined the FcγRIIIB gene frequencies in the Japanese population.…”