Background/Purpose: Neutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple rheumatologic diseases including rheumatoid arthritis, lupus, and vasculitis. Unfortunately, methods for quantifying NETs have limitations involving cost, speed, bias, and/or ease of use, which constrain the study of NETs in disease. The purpose of this project is to advance the methodology for NET quantification in order to maximize the ability of investigators to further elucidate the role of NETs in health and disease.

Methods: We created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area for many fluorescent microscope images at once providing data on a per cell, per image, and per sample basis. To test DANA, 2 different individuals determined the frequency of NET-like structures for fluorescent microscope images of Sytox-stained human neutrophils by eye followed by running DANA on those images. DANA was then used to analyze images of neutrophils from rheumatoid arthritis subjects and control subjects. To test the ease of implementing DANA and applicability to other species and DNA stains, two individuals with no programming background installed DANA and repeated the above experiments using images of DAPI-stained unstimulated and stimulated murine bone marrow derived neutrophils.

Results: DANA quantified a similar frequency of NET-like structures to the frequency determined by eye, and in a fraction of the time, for both human Sytox-stained neutrophil images and murine DAPI-stained neutrophil images. Also, as expected, DANA detected increased DNA area and frequency of NET-like structures in rheumatoid arthritis subjects compared to controls and in stimulated murine neutrophils compared to unstimulated.

Conclusion: DANA provides a means to quantify DNA decondensation and the frequency of NET-like structures in a reliable, simple, high-throughput, and cost-effective manner making it ideal to assess NETosis in a variety of conditions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-area-and-netosis-analysis-dana-a-high-throughput-method-to-quantify-neutrophil-extracellular-traps-in-fluorescent-microscope-images/