Genotyping Partial HPA For Chinese Han Ethnic Group And Establishment Of B Lymphoblastoid Cell Lines As A Source Of Refrence DNA For HPA-15

Objective Aimed to establish a protocol, named simultaneous PCR-SSP, to simultaneously genotype HPA-1 through 7 and HPA-15, then to investigate the gene frequencies of unrelated blood donors of Han ethnic group in Fujian province using the simultaneous PCR-SSP method, and then to establish B lymphoblastoid cell lines (BLCLs) as a source of reference DNA for HPA-15.Methods (1) The DNA sequences of HPAs were obtained, and allele-specific primers of HPA and internal control primers of HGHG were designed using the software of Primer Premier 5 and BLAST. Gradient temperatures were used to determine the optimum Tm of HPA systems and HGHG, and reaction conditions were regulated using the touch down protocol. (2) In order to identify the accuracy of simultaneous PCR-SSP, the reference samples were genotyped. (3) Primer mixtures were prepared and dispensed. The gene frequencies of 160 unrelated blood donors of Han ethnic group in Fujian province were investigated using the simultaneous PCR-SSP assay and the reproducibility of the method was determined. (4) B95-8 cell line was cultured with low concentration of calf serum for about two weeks when Epstein-Barr virus could be harvested. (5) After selecting blood samples of three genotypes of HPA-15 using PCR-SSP protocol, the B lymphocytes of these samples were transformed with Epstein-Barr virus to establish B lymphoblastoid cell lines (BLCLs). (6) The genomic DNA of BLCLs was extracted and genotyped for HPA-15 using PCR-SSP and Nest PCR methods. To test the stability of BLCLs, the 3rd, 10th and 15th subcultivation of BLCLs were genotyped for HPA-15.Results (1) The established simultaneous PCR-SSP method was simple, rapid, and has a percentage of 100% for accuracy and reproducibility, respectively. (2) The gene frequencies of HPA-3a and HPA-15a in blood donors of Han ethnic group in Fujian province were 0.5406 and 0.5531, respectively, and HPA-3 and HPA-15 had the greatest heterozygosity with a percentage of 53.13% and 45.63% for HPA-3a/3b and HPA-15a/15b, respectively. It was noted that the gene frequencies of HPA-4a and HPA-7a were both more than 0.9999, actually, the observed frequencies of the two genes were both 1.0000. For the remaining 4 HPAs, the predominance of a/a homozygosity was noted for HPA-1, -2, -5, and -6, with a frequency more than 0.9500 for gene a,that is, the frequencies of HPA -1a, -2a, -5a, and -6a were 0.9938, 0.9688, 0.9813, 0.9625, respectively. (3) Seven BLCLs were established with a mean time of eight weeks, and four BLCLs were chosen to be cryoprotected. Our results indicated that the HPA-15 genotypes of BLCLs were in concordance with the genotypes of blood samples. (4) The HPA-15 genotypes of 3rd, 10th and 15th subcultivation of BLCLs were detected and the results indicated that the HPA-15 genotypes of four BLCLs were stable and reliable. (5) The establishment of BLCLs for HPA-15 is convenient for quality control of HPA-15 genotyping. After thawing, it only took about one week to culture the BLCLs for DNA extraction and the genotypes of BLCLs were reliable.Conclusions The simultaneous PCR-SSP method could be established when fine primers were designed and cycling parameters, reaction conditions were optimized. The HPA gene frequencies of unrelated blood donors of Han ethnic group in Fujian province were in concordance with the frequencies of the Korean, Vietnamese and Taiwanese and showed significantly different from the European, African,American and Australian. The establishment of BLCLs as a source of reference DNA for HPA-15 could exactly be done.