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Human neutrophil antigen (HNA)-typed granulocyte panels are widely used to screen for the presence of HNA antibodies and to determine antibody specificity. Many laboratories screen donors for HNA genotypes using low-throughput methods such as allele-specific polymerase chain reaction (PCR), PCR-restriction fragment-length polymorphism, and multiplex PCR. In the present study, we used a high-resolution melting (HRM) analysis to determine HNA genotypes. For the HRM analysis, purified genomic DNA samples were amplified via PCR with HNA-specific primers. Nucleotide substitutions in genes encoding HNAs were differentiated on the basis of the HRM curves, and the results of HRM and DNA sequencing analyses were determined to be in complete agreement. The gene frequency of HNA-1a, -1b, -1c, -3a, -3b, -4a, -4b, -5a, and -5b in the Japanese population was consistent with the previous reports. Our results suggest that HRM analysis can be used for genotyping HNA antigens determined by single nucleotide substitutions.

Background and Objectives The Fcgamma receptor IIIb (Fc γ RIIIb) carries the human neutrophil antigen ( HNA)-1 system. The three FCGR3B alleles encode four antigens: one allele encodes HNA-1a, and two alleles encode two antigens each, HNA-1b and HNA-1d and HNA-1b and HNA-1c, respectively. Several other non-synonymous substitutions have also been reported. Little is known about the properties of HNA-1d antibodies because HNA-1d was recently discovered, and HNA-1d antibodies have been reported only in a couple of cases. These findings raise the possibility that the polymorphisms in the HNA-1 system and spectrum of the specificity of resulting antibodies are more complex than previously thought. We address this possibility in this work. Materials and Methods Several HNA-1 antibodies, which had been classified as HNA-1a or HNA-1b before HNA-1d was discovered, were re-evaluated for their reactivity to different recombinant variants of Fc γ RIIIb. Results The HNA-1a antibody, but not the reference HNA-1a antibody, cross-reacted to HNA-1d. Further, nucleotide replacement in FCGR3B alleles *02 at 193G→A ( AA position 54 E→K) resulted in the disappearance of HNA-1d, but not HNA-1b, antigenicity. Conclusion The HNA-1 system may be more complex than previously understood. [ABSTRACT FROM AUTHOR]

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive. [ABSTRACT FROM AUTHOR]