Determination of human neutrophil antigen-1, -3, -4 and -5 allele frequencies in English Caucasoid blood donors using a multiplex fluorescent DNA-based assay

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Background and Objectives

A number of DNA-based methods to genotype the alleles coding for HNA have been described, but all require the separate amplification and analysis of each allele. The aim was to develop a DNA-based method for simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles.

Materials and Methods

An allele-specific primer extension method was used in combination with magnetic beads from Luminex technology. PCR–sequence-specific primers (SSP) was used to resolve the presence of the HNA-1b allele in samples assigned by the Luminex bead assay as HNA-1a/-1b/-1c or HNA-1b/-1c. HNA allele frequencies were determined in a panel of 140 randomly selected English Caucasoid blood donors.


HNA allelic types were compared with historical results, and 100% concordance was found. Only eight of the 97 samples used in the validation required additional testing by PCR–SSP. Allele frequencies were determined in the blood donor population as follows: 0·318 for HNA-1a, 0·668 for HNA-1b, 0·014 for HNA-1c, 0·768 for HNA-3a, 0·232 for HNA-3b, 0·882 for HNA-4a, 0·118 for HNA-4b, 0·736 for HNA-5a and 0·264 for HNA-5b.


A multiplex Luminex bead assay for the simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles is described that enables rapid typing of donors to support HNA alloimmunized patients who require HNA-compatible blood products.

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