Representative flow cytometric analysis for detecting antineutrophil antibodies and classification of patients.

All histograms showed the analysis of neutrophils, which were gated electronically in medium to high forward and orthogonal light scatter, and the green fluorescence of this population was collected. Data acquisition and analysis were performed with CellQuest software (Becton Dickinson Immunocytometry Systems) or ORTHO ImmunoCount II software (Ortho Diagnostic Systems). Various concentrations of TAG1 bind to HNA-1a–homozygous neutrophils and those of TAG2 to HNA-1b–homozygous neutrophils (A). The relative fluorescence intensity (RFI) of each concentration of TAG1 or TAG2 is presented (B). The representative flow cytometric findings of weakly positive and strongly positive sera were shown against 2 different HNA-1a–homozygous donors (C). (D) The RFI of negative antibodies was 1.5 or under, whereas that of weakly positive antibodies ranged from 1.6 to 2.9 and that of strongly positive antibodies was 3.0 and above. According to the results of RFI, 41 patients were classified into 3 groups: 12 antibody negative (○), group A), 13 antibody-weakly positive (group B), and 16 antibody-strongly positive (group C). Antibodies specificity in groups B and C are presented as follows: anti–HNA-1a, (●); anti–HNA-1b, (⊕); FcγRIIIb, (⊗); and inconclusive, (⊙).