Techniques for measuring NETosis: A critical literature review and outlook for standardization
Constant Gillot1, Hachem Bouarroudj1, Jonathan Decarpentrie1
1Clinical Pharmacology and Toxicology Research Unit, Namur Research Institute for Life Science, University of Namur, 5000 Namur, Belgium.
Background:
Neutrophil extracellular traps (NETs) are key components of innate immunity, but NETosis research is limited by the absence of standardized biomarkers and analytical protocols. The rapid expansion of NET-related studies emphasizes the need for a critical evaluation of current measurement approaches.
Objective:
To systematically map and assess methodological strategies used to detect and quantify NETosis, highlighting analytical diversity, limitations, and the need for standardized, validated techniques to improve reproducibility and support clinical translation.
Methods:
A Medline search (2004-June 2025) identified 162 studies reporting NETs detection techniques in vitro, ex vivo, or in vivo. Extracted data included biomarkers (cfDNA, MPO, NE, citrullinated histones, PAD4, ROS) and detection methods (fluorometry, microscopy, ELISA, qPCR, flow cytometry, western blot, proteomics). A narrative synthesis identified methodological trends, variability, and gaps.
Results:
Marked heterogeneity was observed in biomarker choice and analytical methods. cfDNA was the most common marker (106 studies), followed by MPO (58), histones (52), and NE (43). Frequently used techniques included fluorometry (38.7%), microscopy (34.9%), and ELISA (13.2%). Notably, 85-90% of protocols relied on non-validated, home-made assays. Imaging offered structural specificity, whereas high-throughput assays such as fluorometry and ELISA suffered from limited specificity and inter-laboratory variability. Secondary biomarkers (PAD4, ROS, MMP-9) were inconsistently assessed, and no harmonized analytical framework exists. No single biomarker or assay currently enables reliable, standardized NETosis quantification.
Conclusions:
NETosis research remains highly fragmented. Standardized, multimodal protocols combining structural (cfDNA, H3Cit), enzymatic (MPO, NE), and regulatory (PAD4, ROS) markers, together with validated assays, are required to enhance reproducibility and support future clinical implementation.
