Detection of neutrophils and neutrophil extracellular traps in biological fluids by double staining assay

Generalov S.I., Ishchenko O.V., Konevalova N.Y., Frolova A.V., Zherulik S.V., Sushkova S.A., Pavlov Y.A.

Vitebsk State Medical University

Netosis is a recently discovered pathway of neutrophil death, which is followed by neutrophil extracellular traps formation. These structures play an important role in many pathological processes. The main goal of this study is to create a method for detection of neutrophil extracellular traps in biological fluids by identification neutrophil-specific enzyme - naphthol-AS-D-chloroacetate esterase and extracellular DNA using bright field microscopy. The developed method is based on the azo coupling reaction of nitried fuchsin with α-naphthol. The cultures of neutrophils obtained from the venous blood of healthy donors, ascitic fluid and sputum were used as a material for the study. The number of segmented and desegmented neutrophils, neutrophil extracellular traps and cytoplasts was determined. This method was compared with the previously developed test for the determination of neutrophil extracellular traps with Fast Corinth V Salt dye.