Detection of platelet specific antigens by flow cytometry and genotyping method

Wee, Jai Ho; Jeong, Ki Cheol; Kim, Jung Man

Abstract

BACKGROUND
To identify the human platelet antigens (HPA) associated with neonatal alloimmune thrombocytopenia (NATP), posttransfusion purpura (PTP), and platelet refractoriness, polymerase chain reaction-sequence specific primer (PCR-SSP) method and immunofluorescent method by flow cytometry were used. The frequencies of the genonotypes of HPA systems by PCR-SSP method and those of phenotypes by flow cytometry were determined. Then both types were compared each other and each types were compared with those of established reports. METHOD: Platelet suspensions were prepared from peripheral blood specimens of 200 blood donors and DNA specimens were extracted from those of 160 donors among them. Phenotypes of 200 specimens and genotypes of 160 ones were tested by flow cytometry and PCR-SSP method, respectively. RESLUTS: Frequencies of penotypes of HPA-1a, -3a, -4a, -4b and NaKa were 100.0%, 88.0, 100.0%, 0.5% and 94.0%, respectively. HPA-5 system could not be identified due to a few antigenic sites of HPA-5 systems. The genotype fequencies are of HPA-2 were a+b- 63.75%, a+b+ 35.00%, a-b+ 1.25%; HPA-3, a+b- 38.12%, a+b+ 48.13%, a-b+ 13.75%; HPA-4, a+b- 100.00%, a+b+ 0.00%, a-b+ 0.00%; HPA-5, a+b- 98.12%, a+b+ 1.88%, a-b+ 0.00%. The frequencies of HPA-4 and -5 were almost same as those of other reports but the frequencies of HPA-2 and -3 were somewhat different from others. Concordant rate between phenotype and genotype of HPA-3a,-4a and -4b were 95.6%, 100% and 99.4%, respectively.
CONCLUSION
Phenotyping method by flow cytometry was rapid and objective for identifying HPA systems except HPA-5 system which has a few antigenic sites on platelet membrane. Especially it will be useful method for screening HPA-4b as possible cause of NATP in Koreans. But like other serologic methods, phenotyping by flow cytometry also require the highly qualified antiserum and appropriate amount of platelet. PCR-SSP method was also rapid and simple to test of genotypes of HPA-2~5 systems. Because PCR-SSP method is thought to be one of the most simple and economic genotyping methods to overcome the shortages of serologic methods, it is suggested to be the efficient screening method of HPA systems substituting the serologic methods in the cases which HPA sytems can not be identified by flow cytometry.