Detection by flow cytometry of anti-neutrophil cytoplasmic antibodies in a novel approach based on neutrophil extracellular traps

Background:Anti-neutrophil-cytoplasmic antibodies (ANCA) are auto-antibodies directed against components of neutrophil granulocytes and may be found in various inflammatory conditions, like small-vessel vasculitis or ulcerative colitis (UC). Routine ANCA screening is performed on ethanol-fixed neutrophils using indirect immunofluorescence technique. Yet, how neutrophil granule proteins become available to immunologic presentation is a matter of debate. In recent years, various studies have shown that neutrophils are able to extrude their chromatin decorated with granular proteins as neutrophil extracelullar traps (NETs).Aim: We hypothesized that (I) ANCA immunoreactivity may be found on NETs and (II) NETs may serve as a useful tool in a novel approach for ANCA detection.Methods: Sera from patients suffering from either ANCA-associated vasculitis (n=10), UC (n=30) or sera from patients without diagnosed ANCA-associated diseases (n=20), respectively, were subjected to indirect immunofluorescence and a newly developed method to detect ANCA by flow cytometry employing microbead technology.Results: ANCA-related immunofluorescence was readily detectable on ethanol-fixed NETs, establishing NETs as a structure carrying ANCA target antigens. Moreover, we observed that neutrophils form NETs in response to microbeads and stick to the surface of these beads. Using these NET-coated microbeads in flow cytometry, we were capable of reliably detecting p-ANCA, c-ANCA, and a-ANCA in tested patient sera. UC-related complex DNase-1-sensitive ANCA (NET-ANCA) antigens were also detected on NET-coated microbeads.Conclusion: NET-coated microbeads may be commercially developed as a novel tool for automated ANCA screening assays using flow cytometry.