Immunofluorescent staining and interpretation method for circulating tumor cell

Abstract

The invention discloses an immunofluorescent staining and interpretation method for circulating tumor cells. The method comprises the following steps: S10, performing centrifugal treatment on a blood sample diluted with a phosphoric acid buffer solution, and removing erythrocyte and leukocyte in the blood sample; S20, moistening the treated blood sample with the phosphoric acid buffer solution, adding a stationary liquid for immobilizing cells in the blood sample, adding a permeabilization liquid for permeabilization, and further adding a confining liquid for sufficient confining; and S30, dripping dye liquids of anti-pan-CK-FITC, anti-CD45-AF59 and anti-Vimentin-FITC into the treated blood sample at a dark place, performing light shielded incubation, removing the dye liquids, adding a nucleic acid fluorescent dye to continue incubation. The interpretation method comprises steps of scanning observation and result interpretation. Compared with the prior art, the dyeing method disclosed by the invention has the advantages of being simple and convenient to operate, easy to popularize and the like, and the interpretation method has the advantages of being accurate interpretation and the like.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis and relates to the identification of tumor cells, in particular to an immunofluorescence staining and interpretation method for circulating tumor cells. Background technique [0002] According to the "World Cancer Report 2014" released by the World Health Organization, tumors have become the leading cause of death in the world, and malignant tumors are a major public health problem that seriously threatens human health and social and economic development. According to epidemiological survey results, the incidence and mortality of malignant tumors such as lung cancer, breast cancer, pancreatic cancer, liver cancer, and colorectal cancer rank among the top. In 2012, there were 14 million new cancer cases and 8.2 million people in the world. die. As early as 2006, the World Health Organization proposed the three golden principles of "early detection, early diagnosis, and early treatment" ...

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Problems solved by technology

RT-PCR detection can accurately obtain cancer mutation information, but it cannot count CTCs, and it is impossible to evaluate the therapeutic effect by observing and comparing the changes in the number of CTCs before and after treatment clinically, so its significance is limited

Fluorescent in situ hybridization uses fluorescein-labeled probes to react with nucleic acids to achieve the purpose of detection. It has the characteristics of fast response and high sensitivity, but it cannot achieve 100% hybridization, especially when using shorter cDNA probes Significant drop in efficiency

Chinese patent document CN104007257B reported the use of a new type of FISH technology, combining it with biological humoral cell surface markers, using two FISH specific probes for antibodies to humoral cell surface markers and non-humoral rare nucleated cells The method of treating the obtained cells with reagents can improve the accuracy; however, due to the use of double staining, the method increases the staining steps and designs different probes for different samples, which increases the difficulty of work and the complexity of operation, and also increases the complexity of clinical laboratory personnel. difficulty of mastering

[0006] In summary, although the existing CTC identification methods have their own advantages, there are still some problems, such as easy to produce missed detection, complicated operation, high cost, difficult to popularize, etc. Therefore, it is urgent to establish a method that can quickly and Reliable, accurate, and easy-to-promote CTC detection technology as a routine screening method for circulating tumor cells

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