Anal Biochem. 2025 Dec 26:116037. doi: 10.1016/j.ab.2025.116037. Online ahead of print.
ABSTRACT
BACKGROUND: Neutrophil extracellular traps (NETs) could help prevent and treat thrombosis-related diseases. Detecting NETs primarily relies on plasma; however, the impact of anticoagulants and plasma centrifugation on these results remains unclear.
OBJECTIVES: This study aimed to identify the most effective anticoagulant and centrifugation methods for detecting NETs in plasma.
METHODS: Plasma samples were collected from sepsis rats using sodium citrate and EDTA as anticoagulants. These samples were subjected to various centrifugal forces, durations, and temperatures to determine the optimal method for NETs analysis. Similarly, plasma samples from infected patients were collected and analyzed using different centrifugation techniques to identify the optimal method. Gene expression analysis in endothelial cells stimulated by these plasma samples was conducted to compare the endothelial responses to NETs levels.
RESULTS: In rats, EDTA-plasma exhibited increased levels of myeloperoxidase (MPO)-DNA, while dsDNA levels remained unchanged, with both concentrations being higher at 400 g. Similarly, EDTA and a centrifugal force of 400 g increased MPO-DNA levels without affecting dsDNA levels in patients. Plasma isolated using a centrifugal force of 400 g enhanced adhesion, inflammation, and chemotaxis gene expression in endothelial cells, demonstrating that low centrifugal force is effective for NETs detection.
CONCLUSION: For an accurate NETs assay, it is recommended to use EDTA anticoagulant and prepare plasma using a low centrifugal force, such as 400g.
PMID:41456846 | DOI:10.1016/j.ab.2025.116037