METHOD FOR DETECTING TUMOUR CELLS CIRCULATING IN BLOOD BY MULTIPARAMETER FLOW CYTOMETRY Russian patent published in 2024 - IPC G01N33/574 G01N33/49 G01N33/533 

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to laboratory diagnostics, and can be used for detection of tumour cells circulating in blood by multiparameter flow cytometry. Tumour cells are recovered from 7.5 ml of EDTA-stabilized peripheral venous blood and stained with fluorochromic antibodies. Cell detection is carried out as follows: at the first stage, Fc receptor blockers are added to the sample – inactivated 10% blood serum of AB, IV group; isolated cells are added with a cocktail of monoclonal antibodies to panepithelial antigens – EpCAM CD326, general leukocyte antigen CD45 and marker CD133, incubated for 20 minutes at a temperature of +4 °C, then cells are washed in 5 ml of phosphate-salt buffer at 1,000 rpm for 5 minutes; cell membrane permeabilization stage is carried out and cytokeratin antibodies are added, CAM5.2 is incubated for 20 minutes at a temperature of +4 °C, then cells are washed in 5 ml of phosphate-salt buffer at 1,000 rpm for 5 minutes; then nuclear dye SYTO16 – 2 mcl is added and incubated in the dark at room temperature for 5 minutes; then adding 300 mcl of phosphate-buffered saline and the whole volume of the sample is analysed on a double-laser flow cytometer, after which the flow cytometry data are processed using a program for processing native flow cytometry files on a personal computer; additionally, a fluorescence-minus-one control is used, successively excluding one antibody from the stained sample of the analysed sample. Presence of living CAM5.2+CD326+CD133+CD45-, CAM5.2-CD326+CD133+CD45-, CAM5.2+CD326-CD133-CD45- and CAM5.2+CD326+CD133-CD45- tumour cells in the sample is detected and their number is counted.

EFFECT: method enables detection of tumour cells, circulating in blood, as additional biomarkers, in order to predict the clinical course of the disease and assess the effectiveness of therapy of malignant tumours of an epithelial nature, by detecting tumour cells circulating in the blood by multiparameter flow cytometry.

1 cl, 2 dwg, 2 ex