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Novel circulating tumor cell identification technology

A new type of tumor cell technology, applied in the field of medical diagnosis, can solve the problems of false positives, imperfect detection methods, and difficulty in accurate quantification by fluorescence microscopy

Pending Publication Date: 2020-10-23
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because normal epidermal cells in the blood also express a certain degree of conventional marker proteins such as cytokeratin (CK18 or 19) and vimentin, which are also present in normal tissue cells, but the content is significantly higher in tumor cells , and fluorescence microscopy is difficult to accurately quantify, and the imperfect detection method also leads to false positives. Therefore, the fluorescence detection of keratin and other related protein markers still cannot be used as the main basis for the detection of CTC

Method used

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  • Novel circulating tumor cell identification technology
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Examples

Experimental program
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Effect test

Embodiment 1

[0051] (1) Collection, transportation and storage of peripheral blood, sample pretreatment:

[0052] According to the standard sampling procedure, take 3-5 ml with BD EDTA anticoagulant tube, press the angle of 90 degrees, shake gently up and down 8 times, and transport the blood sample with a low-temperature ice pack (8°C). If left at room temperature, it needs to be used for experimental operations within 2 hours, or it can be stored in a 4-degree refrigerator for about a day.

[0053] (2) Enrichment and separation of CTCs: Enrichment and separation were carried out with a CTC capture device, and separated on a microporous membrane.

[0054] (3) Cell immobilization on microporous membrane: cell morphology identification and immunofluorescence detection of CTC;

[0055] CTCs need to be fixed after being enriched on the membrane. The fixative: 90% alcohol, 5% of 1x PBS and 5% acetic acid are prepared immediately before use. Add 1ml of fixative to the cell strainer, fix for at...

Embodiment 2

[0065] (1) Collection, transportation and storage of peripheral blood, sample pretreatment:

[0066] According to the standard sampling procedure, take 3-10 ml of BD EDTA anticoagulant tube, press the angle of 90 degrees, shake gently up and down 8 times, and transport the blood sample with a low-temperature ice pack (4°C). If left at room temperature, it needs to be used for experimental operations within 2 hours, or it can be stored in a 4-degree refrigerator for about one day.

[0067] (2) Enrichment and separation of CTCs: Enrichment and separation were carried out with a CTC capture device, and separated on a microporous membrane.

[0068] (3) Cell immobilization on microporous membrane: cell morphology identification and immunofluorescence detection of CTC;

[0069] CTCs need to be fixed after being enriched on the membrane. The fixation solution: 90% alcohol, 5% of 1x PBS and 5% acetic acid, prepared immediately before use, can be added to the cell strainer with 1ml fi...

Embodiment 3

[0079] (1) Collection, transportation and storage of peripheral blood, sample pretreatment:

[0080] According to the standard sampling procedure, take 3-5 ml with BD EDTA anticoagulant tube, press the angle of 90 degrees, shake gently up and down 8 times, and transport the blood sample with a low-temperature ice pack (6°C). If left at room temperature, it needs to be used for experimental operations within 2 hours, or it can be stored in a 4-degree refrigerator for about one day.

[0081] (2) Enrichment and separation of CTCs: Enrichment and separation were carried out with a CTC capture device, and separated on a microporous membrane.

[0082] (3) Cell immobilization on microporous membrane: cell morphology identification and immunofluorescence detection of CTC;

[0083] CTCs need to be fixed after being enriched on the membrane. The fixation solution: 90% alcohol, 5% of 1x PBS and 5% acetic acid, prepared immediately before use, can be added to the cell strainer with 1ml f...

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Abstract

The invention provides an identification technology of a novel circulating tumor cell (CTC). The identification technology is used for identifying the CTC. The method is characterized in that body fluid (such as peripheral blood) of a tumor patient is subjected to standard sampling, transportation and storage, sample pretreatment, CTC enrichment and separation on a microfiltration film, cell immobilization, cell silver staining and immunofluorescence in-situ detection, whether the cells are tumor cells or not is determined according to the karyoplasm proportion, the irregular nucleolus characteristics and the number, and finally the organ tissue source of CTC in blood is determined through fluorescence immunoassay of tissue and organ specific protein markers. According to the method, CTC is subjected to in-situ microscopic examination, tumor cells are detected, organ tissue sources of circulating tumor cells are determined, a long-term problem that CTC morphological identification lacks a believable method is solved, and establishment of a unified standard for CTC detection is facilitated. The method has advantages of being easy to operate, good in repeatability, high in stabilityand the like, and can be used for early screening of tumor high-risk groups, curative effect evaluation of cancer patients and recurrence monitoring.

Description

technical field [0001] The invention belongs to the technical field of medical diagnosis, and in particular relates to an identification technology of a novel circulating tumor cell (Circulating Tumor Cell, CTC). Background technique [0002] Tumors can be divided into two categories: solid tumors and non-solid tumors. The former are tumors formed by the differentiation of epithelial or endothelial cells, while the latter are formed by cells of the hematopoietic system, such as lymphocyte carcinoma, which do not form tumors. Tumor cells detach from the primary tumor or metastases of solid tumors and enter the human circulatory system to form circulating tumor cells (CTCs), which exist in peripheral blood. [0003] CTC exists in the form of free single cells, or in the form of cell clusters (Circulating Tumor Microemboli, CTM). These cells are the basis of tumor metastasis. The study of CTC helps to reveal the mechanism of malignant tumor occurrence and metastasis. CTC is al...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/58G01N33/533G01N33/574G01N33/68
CPCG01N21/6486G01N33/582G01N33/533G01N33/57484G01N33/57438G01N33/68
Inventor 吴少波陈利民
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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