Genotyping of human neutrophil antigens 1, 3, 4 and 5 using a novel multiplex polymerase chain reaction.

Abstract

Our study aimed to establish a novel multiplex polymerase chain reaction (PCR) for rapid simultaneous detection of all relevant human neutrophil antigen (HNA)-1, -3, -4 and -5 alleles. Granulocyte-reactive antibodies are involved in several diseases, such as neonatal alloimmune neutropenia, autoimmune neutropenia and transfusion-related acute lung injury. A panel of well-defined test granulocytes is required for diagnostic antibody detection and prospective blood donor screening. Several genotyping methods for the detection of HNA alleles have been described, but most approaches require separate amplification of each HNA allele or at least a separate amplification of the HNA-1 alleles. The new method is based on simultaneous detection in one reaction tube, where each HNA-1 allele is amplified by two allele-specific primers, one primer of which is labelled with a fluorescent dye (HEX, FAM). Allelic polymorphisms for HNA-3, -4 and -5 were amplified with one common unlabelled primer and two fluorescence-labelled (HEX, FAM) allele-specific primers. DNA fragments of HNA alleles are analysed on a Genetic Analyser 3130xl by amplicon size and fluorescent dye. A total of 110 blood donors with known genotypes were studied. In the 110 DNA samples studied, all HNA-1, -3, -4 and -5 alleles could be detected precisely. All results matched perfectly with those from reference typing by PCR-sequence-specific primer. Amplification performed well even at low DNA concentrations (10ng μL-1 ). Our method enables fast and easy genotyping of all relevant HNA-alleles in one PCR reaction. Results are easy to analyse due to different amplicon sizes and fluorescent dyes. Furthermore, the method is suitable for high sample throughput.