METHOD FOR DETECTION OF NEUTROPHIL EXTRACELLULAR TRAPS IN SUPRAVITAL STAINED BLOOD PREPARATION

摘要

FIELD: medicine.;SUBSTANCE: invention relates to medicine, namely to immunology, pathophysiology and experimental medicine, and can be used to detect morphological equivalents of the stages of formation of neutrophil extracellular traps (NETs). A suspension of neutrophils isolated on a double density gradient of ficoll-verografin solution after stimulation with a mixture of Lactobacillus reutri, L. acidophilius, L. rhamnosis and Bifidumbacterium longum is supravitally stained using propidium iodide intercalating dye and incubated under a cover slip in the dark for 5 minutes at 37°C with monoclonal antibodies to the neutrophil-specific antigen CD15, marked with FITC fluorescent dye. The results are visualized using fluorescence microscopy using an excitation filter with a wavelength range of 450-480 nm and an emission filter in the wavelength range of 515-700 nm. The morphological equivalents of cells that are in different degrees of activation in the process of a neoforming reaction are calculated: intact bright green cells without a stained nucleus, weakly activated cells with bright green surface structures and a weakly stained nucleus, activated cells with bright green and red surface structures, orange core, hyperactivated cells - bright green superficial structures and an enlarged red-orange nucleus reaching the border of the cytolemma, hyperactivated cells with initial signs of netosis - early netosis - with bright green surface structures and an enlarged red-orange nucleus with visualized output nuclear matter in at least one location. Simultaneously with cellular equivalents, two variants of netosis are counted: filamentous netosis - red-orange structured filamentous NET, exceeding the cell size by more than 2 times, cloud-like netosis - unstructured homogeneous NET of red-orange color, located around the source cell, exceeding the cell size by 1 ,5-2 times. Separately, the number of microorganisms located directly in neutrophil traps is counted in terms of one network, followed by the calculation of the percentage of different groups of morphological elements - the morphological profile of netosis.;EFFECT: method provides not only the possibility of identifying viable (intact) neutrophils, but also determining the morphological equivalents of cells that are in varying degrees of activation in the process of non-forming reaction, early netosis and the possible detection of specific antigens of leukocyte differentiation expressed on their surface with simultaneous visualization of NET due to two-color staining preparation - bright green structures (CD15-FITC) and red-orange DNA strands stained with propidium iodide, allowing to simultaneously determine neutrophils by their immunophenotyping with visualization of leukocyte differentiation antigens (CD15) expressed on the surface, assess their viability and visualize various types of neutrophil traps.;1 cl, 3 ex, 8 dwg