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

Abstract

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.

Keywords:

• ANCA
• NET
• autoimmunity
• neutrophils
• ANCA-associated vasculitis
• immunofluorescence
• microbeads
• DNase-1
• ulcerative Colitis

Disclosure statement

M.L. received a research scholarship from MSD Sharp & Dohme GmbH, Germany. The remaining authors have no conflict of interest to declare.

Notes on contributors

S.R., M.L. and S.G. performed experiments, S.R. drafted the manuscript, M.L. designed the study. All authors read and approved the manuscript.