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Kit for identifying circulating tumor cells by combining TCPP with CEP probe and application

A technology of tumor cells and kits, which is applied in the fields of oncology and medical testing, can solve the problems of missed screening and low coincidence rate of positive detection, and achieve the effects of improving accuracy, reducing costs, and saving costs

Inactive Publication Date: 2020-03-27
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problems of low detection positive coincidence rate and missed screening in the immunofluorescence labeling method and in situ hybridization method in the existing circulating tumor cell detection methods

Method used

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  • Kit for identifying circulating tumor cells by combining TCPP with CEP probe and application
  • Kit for identifying circulating tumor cells by combining TCPP with CEP probe and application
  • Kit for identifying circulating tumor cells by combining TCPP with CEP probe and application

Examples

Experimental program
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Effect test

Embodiment 1

[0049] This embodiment provides a kit for identifying circulating tumor cells using TCPP combined with CEP probes. The components include: TCPP stock solution (concentration is 1 mg / mL), 100 mM pH value of 6.1 MES buffer, cell washing solution (PBST ), 5% paraformaldehyde cell fixation solution, tissue fixation solution (PBS solution containing 50% ethanol by volume fraction, which contains 1% polyethylene glycol by mass fraction), CEP8 probe solution, cell permeation solution (PBS solution containing 0.5% Triton X-100 by volume fraction), DAPI staining solution, PBS solution.

[0050] 1. Enrichment of circulating tumor cell CTCs

[0051] 1. Collect 6 mL of peripheral blood into ACD blood collection tubes. Add 10 μL of H460 lung cancer cell culture suspension to it. The sample source is peripheral blood, but not limited to peripheral blood, and also includes pleural and ascites fluid or cerebrospinal fluid, etc.

[0052] 2. The sample was centrifuged at 400g for 15min, and ...

Embodiment 2

[0091] This embodiment provides a kit for identifying circulating tumor cells using TCPP combined with CEP probes. The components include: TCPP stock solution (concentration is 1 mg / mL), 100 mM pH value of 6.1 MES buffer, cell washing solution (PBST ), 5% paraformaldehyde cell fixation solution, tissue fixation solution (PBS solution containing 50% ethanol by volume fraction, which contains 1% polyethylene glycol by mass fraction), CEP8 probe solution, cell permeation solution (PBS solution containing 0.5% Triton X-100 by volume fraction), DAPI staining solution, PBS solution.

[0092] Detection of peripheral blood of healthy people.

[0093] 1. Enrichment of circulating tumor cell CTCs

[0094] 1. Collect 6mL of peripheral blood from healthy people in ACD blood collection tubes.

[0095] 2. The sample was centrifuged at 400g for 15min, and the supernatant was discarded. Make up 6mL with PBST cell washing solution, invert up and down several times, and mix well. Wherein th...

Embodiment 3

[0133] Detect the enrichment rate of tumor cells.

[0134] 1. Enrichment of circulating tumor cell CTCs

[0135] 1. Collect 6 mL of peripheral blood from healthy people into ACD blood collection tubes, and add 25 H460 tumor cell lines into it.

[0136]2. The above samples were centrifuged at 400g for 15min, and the supernatant was discarded. Make up 6mL with PBST cell washing solution, invert up and down several times, and mix well. Wherein the PBST cell washing solution includes a PBS solution with a volume fraction of 0.02% Tween 20.

[0137] 3. Slowly superimpose the blood sample on the 3mL lymphocyte separation medium Ficoll to ensure that the liquid surface is layered and clear. Centrifuge at 350 g for 6 min.

[0138] 4. After centrifugation, it is divided into three layers, the upper layer is the light yellow plasma layer, the middle layer is the buffy coat layer (CTCs and white blood cells are mainly in this layer), and the lower layer is the red blood cell layer. ...

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Abstract

The invention belongs to the field of oncology and medical examination, and relates to a kit for identifying circulating tumor cells by combining TCPP with a CEP probe and application. The kit comprises a TCPP stock solution, a subacid buffer solution, a cell cleaning solution, a cell fixing solution, a tissue fixing solution, a CEP probe solution, a cell membrane rupturing solution, a DAPI staining solution and PBS. The kit is applied to non-disease diagnosis and treatment purposes, and the method for identifying the circulating tumor cells by the kit comprises a solid-phase staining method or a liquid-phase staining method. The method does not depend on antigen expression on the surfaces of tumor cells as a detection means. False negative caused by antigen loss of tumor cells in the EMTprocess due to some existing antigen-dependent detection means is avoided, various epithelial tumors can be identified, and the defect that no unified tumor marker is provided for identifying circulating tumor cells due to heterogeneity of different types of tumor cells is avoided.

Description

technical field [0001] The invention relates to the fields of oncology and medical testing, in particular to a kit and application for detecting and identifying circulating tumor cells from human isolated samples by using TCPP combined with CEP probes. Background technique [0002] With the continuous development of medical technology, more and more malignant tumors can be effectively controlled or even cured. However, there are still a large number of cancer patients with poor prognosis, which puts forward higher requirements for early diagnosis and prognosis monitoring of malignant tumors. Due to factors such as insidious onset, no obvious symptoms in the early stage, and easy recurrence and metastasis, detection of circulating tumor cells (CTCs) has attracted more and more attention as a new method of tumor diagnosis and monitoring in recent years. It has been confirmed that the dynamic changes of circulating tumor cell counts are related to the curative effect, recurren...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N33/58G01N21/64G01N1/30
CPCG01N1/30G01N21/6458G01N33/5005G01N33/582
Inventor 韦玉军陆宝石刘文干
Owner 安徽安龙基因科技有限公司
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