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Enrichment and analysis method for circulating tumor cells

A tumor cell and analysis method technology, applied in the direction of tumor/cancer cell, sterilization method, support/fixation method of microorganisms, etc., can solve the problems of impure tumor cells, loss of tumor cells, low separation efficiency, etc. Enrichment effect, improve removal effect, improve recovery effect

Active Publication Date: 2014-12-03
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Density gradient centrifugation has the advantages of being simple, cheap, and fast, but the isolated tumor cells are not pure, the separation efficiency is not high, and a considerable part of the tumor cells are lost, which still has great limitations in practical application.

Method used

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  • Enrichment and analysis method for circulating tumor cells
  • Enrichment and analysis method for circulating tumor cells
  • Enrichment and analysis method for circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Used for red blood cell cracking liquid, with the following composition:

[0041] Table 1 Red blood cell cracking solution

[0042] test group Ammonium chloride Potassium potassium bicarbonate Tethamine tetharium tetrazide formaldehyde Group1 0.1M 0.001M 0.001M 0.01% Group2 0.3M 0.018M 0.0036M 0.1% Group3 0.31M 0.021M 0.0042M 0.1% Group4 0.32M 0.022M 0.0045M 0.1% Group5 0.5M 0.2M 0.05M 0.2%

[0043]The distilled water can be added to 1L, and the pH can be 7.0 to 8.0, and the examples are adjusted to 7.4 in this embodiment.

[0044] 2. Immune micro beads mixed fluid

[0045] Immune micro beads are consolidated by covalent or non-covalent prices on the surface of the antibody and micro beads. The diameter of the micro beads is from 10 nanometer-100 microns.One or more ingredients of polymethyl acrocylite, and tetraxide.When immune micro beads contain polystyrene and tetralet oxide, the micro beads are magnetic beads and...

Embodiment 2

[0056] The specific enrichment method includes:

[0057] 1) Sample processing

[0058] Blood samples and red blood cells are mixed with a 1: 3 ratio to crack red blood cells.

[0059] 2) Collect cells

[0060] The pre -processing sample 600g centrifugal 5min.Go to the Qing Dynasty, add the phosphate buffer (PBS, pH7.4) heavy suspension, 600G centrifugal 5min, add PBS heavy suspension cells.

[0061] 3) Remove white blood cells

[0062] Add an immunore bead mixture to the cell suspension and react on the mixture for 30 minutes.Place the centrifugal tube on the magnetic frame, stop for 5 minutes, and transfer the liquid to a new centrifugal tube to obtain the cell suspension.

[0063] Example 3 An Analysis and Appraisal Method of Cycling Tumor cells

[0064] 1. solution reagent

Embodiment 3

[0065] 1) Reaction buffer: PBS with 0.1%BSA, 2mm EDTA, pH7.4.Provide suitable solution conditions for immune binding reactions on immune magnetic beads and cell surface antigens.

[0066] 2) Fixed liquid: 13%neutral Holmarin.

[0067] 3) Close solution: PBS containing 10%fetus serum, pH7.4.Anti -specific antibody binding sites on closed cells.

[0068] 4) Cell marking solution

[0069] Formula: Contains 0.1mg / ml Fitc to mark CD45 antibodies, 0.1mg / ml PE tags CK antibodies, 15ug / mLDAPI.

[0070] Function: Mark different colors of fluorescentin to white blood cells and tumor cells for cell distinctions.

[0071] 2. Analysis and identification method

[0072] 1) Cell marking

[0073] Add fixed solution to the cell suspension and react for 10min.600g centrifugal 5min, remove the Qingqing.Add the closed liquid cloak cell and react for 10min.600g centrifugal 5min, remove the Qingqing.Add the cell marking solution to the cells and react for 30min.600g centrifugal 5min, remove the Qingqin...

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Abstract

The invention provides an enrichment and analysis method for circulating tumor cells. The method mainly comprises the following steps: subjecting a biological fluid sample to red blood cell lysis treatment; adding immune beads specifically bonding with white cells, red cells and / or platelets for incubation; and filtering cell suspension by using a filter membrane and enriching circulating tumor cells. According to the invention, through integration and optimization of CTCs enrichment methods such as red blood cell lysis treatment, an immune bead method and membrane filtration, the enrichment effect of the circulating tumor cells is greatly improved; with the method, the removal rate of leukocytes is greater than 99.9%, and the enrichment rate of tumor cells is greater than 80%. Meanwhile, the invention also provides a kit using the enrichment method for the circulating tumor cells and discloses a device for enrichment of the circulating tumor cells. The invention also provides an analysis method for the enriched circulating tumor cells. With the analysis method, analysis of dynamic changes like the amount of tumor cells, gene mutation and gene expression profiles can be realized.

Description

Technical field [0001] The present invention is a molecular biology field, which involves medicine and biotechnology, and specifically involves the enrichment and analysis methods of a circular tumor cell. Background technique [0002] It is currently believed that the tumor microe metastases originated in tumor cells invading the cycle. Therefore, cyclic tumor cells (CTCS) may be a sign of tumor metastasis.In recent years, with the development of molecular technology, it is possible to separate and count the peripheral blood CTCS.In the early stages of the disease, CTCS may help the identification of good and malignant tumors, and the prediction and judgment of metastasis risks.During the progress of the disease, CTCS may provide important prognosis information and help doctors monitor the treatment effect.Moreover, CTCS may reflect tumor characteristics, can instruct the formulation of treatment plans, and can be used as target targets. [0003] The existing CTCS enrichment met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12M1/12G01N33/569
Inventor 许嘉森刘志明吴诗扬刘苏燕林丽
Owner SUREXAM BIO TECH
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