HNA-1, -3, -4, and -5 genotyping using multiplex PCR among southern Thais: Developing continual HNA-1 null detection

Abstract

Background: Antibodies against human neutrophil antigens (HNAs) are involved in various clinical conditions including transfusion-related acute lung injury and auto/alloimmune neutropenia. We aimed to determine HNA-1, -3, -4, and -5 frequencies among southern Thais using multiplex PCR and to develop HNA-1 null detection. Methods: Samples obtained from 427 southern Thai blood donors were genotyped HNA-1, -3, -4, and -5 using multiplex PCR and compared their allele frequencies with those previously reported in Thai populations. HNA-1 null samples were tested by newly developed PCR-SSP and PCR-RFLP and confirmed by DNA sequencing. Results: The frequencies of FCGR3B*01, FCGR3B*02, FCGR3B*03, SLC44A2*01, SLC44A2*02, ITGAL*01, and ITGAL*02 among southern Thais differed from other Thai populations, except ITGAM*01 and ITGAM*02 frequencies. Two samples without specific fragments of FCGR3B*01, FCGR3B*02, and FCGR3B*03 tested by multiplex PCR were confirmed by PCR-RFLP to identify FCGR3B deficiency (HNA-1 null). Moreover, to reduce test steps, the newly developed PCR-SSP for FCGR3B deficiency was validated and tested in all samples and the results were in agreement with DNA sequencing. Conclusions: This was the first study to report HNA-1, -3, -4, and -5 frequencies among southern Thais. The indeterminate results of multiplex PCR for HNA-1 genotyping led to establish HNA-1 null detection using PCR-SSP, which is simple, convenient and cost-effective and can be used to identify FCGR3B deficiency.