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Isolation and detection method of rare cells at single cell level

A rare cell and single cell technology, applied in the field of rare cell separation and detection, can solve the problems of difficult single cell recovery, non-automation, complex enrichment process, etc., to avoid the loss rate of rare cells, save time and cost, and save The effect of the enrichment step

Active Publication Date: 2020-10-02
HANGZHOU JUNHUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of micromanipulation equipment are non-automated operation and easy loss of cells during operation and transfer of single cells. Inability to accurately transfer to the bottom of the PCR tube makes subsequent molecular reactions difficult
[0007] Therefore, in view of the current complex rare cell enrichment process and difficulty in single cell recovery, a more automated, simple and efficient rare cell separation method at the single cell level is required

Method used

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  • Isolation and detection method of rare cells at single cell level
  • Isolation and detection method of rare cells at single cell level
  • Isolation and detection method of rare cells at single cell level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] (1) Sample preparation:

[0087] 100 non-small cell lung cancer cells H1975 were stained with 3 ul of the dye VybrantDil (Life Tehnologies) for 1 hour. The tumor cells marked by this staining can be identified by fluorescence on the composite filter membrane after filtration. After staining, the cells were spiked into 2ml of blood.

[0088] (2) Pretreatment before filtration (removal of red blood cells)

[0089] The lysed red blood cells were treated with reference to the method in Test Example 1, and similarly, the mixture of H1975 cells and white blood cells was suspended in 1 ml of HBSS, and the cells were counted.

[0090] (3) filter

[0091] Refer to the filtration method of Test Example 9, that is, the filtration pressure is 20mba, the concentration of the sampled cell solution is 500,000 cells / ml, and the total sampled volume is 4 million cells.

[0092] After the filtration, the composite filter membrane was scanned for fluorescence, and the number of positi...

Embodiment 2

[0133] Objective: To prove the feasibility of the method of the present invention in the isolation of lung adenocarcinoma cells.

[0134] according to Figure 8 The process is to isolate and identify circulating tumor cells from peripheral blood of patients, and the specific steps are:

[0135] 1. Take 3ml of blood sample of lung adenocarcinoma known to have the L858R mutation in situ, and lyse the red blood cells (the same step as the red blood cell lysing in the test (1))

[0136] 2. Add 1ml of buffer to the cell pellet in the centrifuge tube, mix by pipetting and transfer to a 1.5ml centrifuge tube.

[0137] 3. Cell counting, calculate the total amount of cells in the cell suspension (about 4 million).

[0138] 4. Add cell staining reagents according to the number of cells (add 10ul CD45 and 10ulEpCAM to 5 million white blood cells, increase the amount of antibodies proportionally if the number of cells is greater than 5 million, and add antibodies to the amount of 500 ce...

Embodiment 3

[0150] Objective: To prove the feasibility of the method of the present invention in gastric cancer.

[0151] The blood sample was taken from a gastric cancer patient, and the steps 1-12 were the same as those in Example 2. After amplification, use Sanger to detect gene mutations. (eg EGFR, KRAS, TP53, etc.), A750T mutation was detected.

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Abstract

The invention relates to a method for separating unicellular level of rare cells in a body fluid sample. The body fluid sample is uniformly added to a composite filter membrane with a cell suspensionform, the cell suspension comprises leucocyte as most cells and pathological cell as the rear cells, the composite filter membrane comprises more than 1000 of independent and addressable sub-filter membranes of an array, each sub-filter membrane contains micropores with a size being 3 micrometers to 10 micrometers, so that the respective intercepted leukocyte number by more than 50% of a sub-filter membrane does not exceed 10, and a fluorescent labeled pathological cell marker used for karyote is labeled; the intercepted karyote is subjected to fluorescence imaging, so that the position of thesub-filter membrane of the pathological cells having specific fluorescence characteristic is determined and intercepted. According to the invention, the rare cells are separated by the unicellular level in a rapid and simple mode with high recovery rate.

Description

technical field [0001] The invention belongs to the field of separation and detection of rare cells, and in particular relates to a method for separating and detecting rare cells from human body fluid. Background technique [0002] Circulating tumor cells (CTCs) in human peripheral blood refer to tumor cells that spread from tumor lesions into the peripheral blood circulation, and can develop into tumor metastatic lesions under certain conditions. CTCs reflect the molecular characteristics of tumor lesions and are the direct source of tumor hematogenous metastasis, so CTC detection has attracted more and more attention. The content of CTC in peripheral blood is very small, and every 10ml of blood may only contain a few to dozens of circulating tumor cells, but there are as many as 100 million white blood cells and 50 billion red blood cells, so they can be quickly and efficiently separated from peripheral blood Circulating tumor cells are the prerequisite for subsequent cou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12Q1/6886
CPCC12N5/0693C12N2509/10C12Q1/6886C12Q2600/156
Inventor 阎灼辉钱祺
Owner HANGZHOU JUNHUI BIOTECHNOLOGY CO LTD
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