Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR.

Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (, also known as ). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of in mixed populations with different proportions. DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in . The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. frequency was determined by two-channel ddPCR. The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of and in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%-70.8%) and 35.1% (95% CI, 29.2%-41%), respectively. The ratio of alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the frequency in the population to be accurately inferred. This new method of detecting alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the frequency by ddPCR using the probes that we designed.