METHOD FOR DETERMINING CIRCULATING TUMOR CELLS IN PATIENTS WITH BREAST CANCER Russian patent published in 2022 - IPC G01N33/533 G01N33/574 A61B5/00 

FIELD: medicine diagnostic methods.

SUBSTANCE: invention relates to diagnostic methods in medicine and can be used in oncology to determine circulating tumor cells in patients in order to determine the sensitivity of circulating tumor cells to chemotherapy and predict the likelihood of hematogenous metastases in breast cancer. The expression on nucleated cells of the common leukocyte marker CD45, epithelial markers CD326 (EpCAM) and pan-cytokeratin AE1/AE3 and erythrocyte marker CD235a (glycophorin A) is determined, for this a sample of EDTA-stabilized venous blood in a volume of 9 ml is taken in the morning on an empty stomach, the first ml blood samples are not taken, then, the population of circulating tumor cells (CTCs) is enriched using the negative selection method, polystyrene particles with adsorbed antibodies to CD45, CD66b and CD235a (glycophorin A) are added to the blood sample at a concentration of 50 mcl per 1 ml of blood and incubated at room temperature for 20 minutes, then the sample is diluted 1:1 with phosphate buffered saline, applied to a density gradient with p equal to 1.077 at a ratio of 1 part of the gradient and 2 parts of the diluted sample, then the samples are centrifuged at 1200g for 20 minutes, then 2/3 of the upper fraction is removed and the contents are collected rings at the gradient-plasma phase boundary, then the resulting cell suspension is washed in 3 ml of phosphate-buffered saline containing 5% fetal bovine serum by centrifugation at 300g for 10 minutes and twice at 110g for 10 minutes, the supernatant is removed, to the cell sediment add 150 µl of buffer, then the sample is divided into two parts: an unstained control sample in a volume of 50 μl and a colored sample in a volume of 100 mcl, 25 mcl of PerFix-nc Buffer 1 fixing buffer are added to both samples, mixed on a vortex and incubated for 15 minutes at 18-25°C, then 300 mcl of permeabilizing buffer PerFix-nc Buffer 2 is added, mixed on a vortex, antibodies anti-CD45-APC-Cy7 (clone HI30, mouse IgG1), anti-CD326 (EpCAM)-AF488 (clone 9C4 , mouse IgG2b), anti-pan-cytokeratin-AP647 (clone AE1/AE3, mouse IgG1) and anti-CD235a (glycophorin A)-PerCP-Cy5.5 (clone HI264, mouse IgG2a) are immediately added at the rate of 5 mcl of antibodies per 10⁶ cells, then incubated in the dark for 20-30 minutes at 18-25°C, then 3 ml of PerFix-nc Buffer 3 are added to the samples and centrifuged at 300g for 6 minutes, the supernatant is removed, then 100 mcl of buffer are added to the samples , after which the entire volume of the sample obtained is analyzed on a flow cytometer, the signals of fluorescent labels are simultaneously detected: AF488, RegCP-Cy5.5, AF647, APC-Cy7, and the concentration of circulating tumor cells per 1 ml of whole blood is calculated using the following formula.

EFFECT: application of the invention provides an increase in the information content and sensitivity of the method.

1 cl, 2 ex, 2 dwg, 2 tbl