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

Authors:
Stefan Roitsch
Stefan Roitsch
RWTH Aachen University
Germany
Markus F Neurath
Markus F Neurath
University of Erlangen-Nuremberg
Erlangen | Germany
Moritz Leppkes
Moritz Leppkes
*Medical Clinic 1 and Medical Clinic 3
Gurgaon | India

Autoimmunity 2018 Sep;51(6):288-296

a Department of Internal Medicine 1 - Gastroenterology, Pneumology and Endocrinology , Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Universitätsklinikum Erlangen , Erlangen , Germany.

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.

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Source
http://dx.doi.org/10.1080/08916934.2018.1527317DOI Listing
September 2018

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References

(Supplied by CrossRef)

jona2 et al.
2009

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