[A novel method for detection of anti-neutrophil cytoplasmic antibody (ANCA) using flow cytometry]

It has been reported that anti-neutrophil cytoplasmic antibody (ANCA) is a specific serological marker for systemic necrotizing vasculitis, such as Wegener's granulomatosis, microscopic polyarteritis and idiopathic crescentic glomerulonephritis. Although the standard method for ANCA detection enplays an indirect immunofluorescence technique (IIF), the method is not objective. We attempted to detect ANCA by a more objective method using flow cytometry.

Thirty ANCA positive sera (cytoplasmic in 4, perinuclear in 26) and 20 ANCA negative sera including 2 sera with anti-nuclear antibody (ANA) detected by standard IIF were used for this study. For flow cytometric analysis, heparinized blood was taken from normal healthy volunteers. Neutrophils in the heparinized blood was incubated with human recombinant tumor necrosis factor alpha (rTNF alpha) in order to facilitate reaction between the antigens of ANCA and ANCA. Primed neutrophils were incubated with sera. After washing with phosphate buffered saline, immunofluorescence-labeled antibody against human IgG was added and incubated. The mean intensity of fluorescence (MIF) of the neutrophils was measured by flow cytometry (FACS, Becton-Dickinson CO., U.S.A.) for each sample. ANCA was considered positive by flow cytometric analysis if MIF of the sample was over the mean +2SD for normal human sera.

By flow cytometric analysis, ANCA was detected in all ANCA positive samples by IIF and in 3 ANCA negative samples by IIF, of which 2 had ANA. However, it was possible to discriminate serum with positive ANCA from serum with positive ANA by flow cytograms because ANA reacted with the nuclei of both lymphocytes and neutrophils showing two peaks on the histogram.(ABSTRACT TRUNCATED AT 250 WORDS)