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Method for detecting tumor cells or tumor cell debris

A tumor cell and debris technology, applied in the field of detecting tumor cells or tumor cell debris by enhancing the detection signal-to-noise ratio, and detecting tumor cells or tumor cell debris, can solve the problems of not excluding white blood cells, weak CTC detection signal, and low efficiency.

Active Publication Date: 2022-02-25
ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because this method does not exclude the signal interference of white blood cells, the detection signal of CTC is weak, which affects the detection results
[0008] Therefore, it is urgent to develop a new CTC detection method to solve the problems of weak detection signal, low efficiency and low signal-to-noise ratio of CTC during the detection process.

Method used

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  • Method for detecting tumor cells or tumor cell debris
  • Method for detecting tumor cells or tumor cell debris
  • Method for detecting tumor cells or tumor cell debris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1: Comparison of the expression levels of ALPL, ALPP, ALPI and ALPG genes in tumor cells and white blood cells (WBC)

[0122] 1.1 Experimental steps

[0123] (1) Extract total RNA from white blood cells (WBC) and tumor cell lines Lovo, MCF-7, SW480, HCC827, H820, MKN 7, 7721, Caco2, and Bewo (Table 1), and degDNA and reverse the RNA samples recording process to obtain cDNA.

[0124] Table 1 Cell line names and their corresponding tumor types

[0125]

[0126] (2) The cDNA obtained in step (1) was quantitatively amplified by qPCR according to the following system (Table 2 and 3), to detect WBC and tumor cells Lovo, MCF-7, SW480, HCC827, H820, MKN 7, 7721, The average CT value of Caco2, and Bewo, and the obtained average CT value was calculated by 2–ΔΔCT (Livak) method to obtain the relative value of WBC and tumor cell lines MCF-7, SW480, HCC827, H820, MKN 7, 7721, Caco2, and Bewo The relative expression of ALPL, ALPP gene, ALPI gene and ALPG gene in Lovo ce...

Embodiment 2

[0142] Example 2: Activity Test of Tumor Cell Line Alkaline Phosphatase

[0143] 2.1 Experimental steps

[0144] (1) Cell preparation: Digest the tumor cell lines Lovo, Bewo, MKN-7, Caco-2, MCF-7, and 7721 with 0.25% trypsin for 2 minutes, then stop the digestion with 1640 complete medium, and then use 1× Wash the cells with PBS, centrifuge to remove the supernatant, resuspend the cells with 1ml 1×PBS and count with a hemocytometer. 1.5ml EP tubes containing 1000 Lovo cells (Lovo-1000), 1.5ml EP tubes containing 1000 BeWo cells per tube (BeWo-1000), 1000 MKN-7 cells per tube (MKN-7-1000 ), 1.5ml EP tubes containing 1000 Caco-2 cells per tube (Caco-2-1000), 1.5ml EP tubes containing 1000 MCF-7 cells per tube (MCF-7-1000) , 1.5ml EP tube containing 1000 7721 cells (7721-1000) per tube.

[0145] (2) Chemiluminescence detection:

[0146] test group:

[0147] Add alkaline phosphatase chemiluminescence substrate solution (alkaline phosphatase substrate solution A04, purchased f...

Embodiment 3

[0152] Example 3: Effect of TNAP inhibitors on increasing the ratio of tumor cell lines to leukocyte chemiluminescence readouts

[0153] In this embodiment, blood samples are used as samples to be tested for related experiments.

[0154] 3.1 Experimental steps

[0155] (1) WBC cell preparation:

[0156] Take 2ml of blood, lyse with 20ml of 1×red blood cell lysate for 10min, centrifuge at 500g for 5min, remove the supernatant, then wash twice with 1×PBS, centrifuge at 500g for 5min after the first wash, remove the supernatant, and then wash for the second time , Centrifuge again at 300g for 3min, remove the supernatant, resuspend the cells with 1ml 0.5% BSA, obtain WBC cell suspension, and count with a hemocytometer. Add the cell suspension to the 1.5ml EP tube according to the amount of 400,000 white blood cells (WBC-40W) per tube to obtain 1.5ml EP tubes containing 400,000 white blood cells (WBC-40W) per tube.

[0157] (2) Lovo cell preparation:

[0158] Digest Lovo cells w...

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Abstract

The present disclosure relates to a method of detecting tumor cells or tumor cell debris. The method comprises the use of a tissue non-specific alkaline phosphatase (TNAP) inhibitor. By adopting the method disclosed by the invention, the background value of white blood cells can be reduced exponentially, the signal value of the tumor cells is unchanged or slightly reduced, the signal-to-noise ratio is remarkably improved, and the detection sensitivity of the tumor cells is improved.

Description

technical field [0001] The disclosure belongs to the field of biological detection, and in particular relates to a method for detecting tumor cells or tumor cell fragments, in particular to a method for detecting tumor cells or tumor cell fragments by enhancing the detection signal-to-noise ratio. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells shed from solid tumors and enter the blood or lymphatic system. Circulating tumor cells in peripheral blood are very rare, with about 1 to 10 CTCs per milliliter of blood, and CTCs are much lower than other types of cells in blood. Sexuality and heterogeneity increase the difficulty of its detection. Due to their rarity, most techniques require an enrichment step prior to detection of CTCs. This requires that the detection method used can efficiently and accurately detect very few target cells from a large number of non-target cells. CTC detection technology includes CTC enrichment (separation) and...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/42G01N33/573G01N33/569G01N33/574G01N33/543
CPCC12Q1/6886C12Q1/42G01N33/573G01N33/56966G01N33/574G01N33/54326G01N33/54393C12Q2600/158G01N2333/916
Inventor 王鹏飞杨钟林齐盼盼唐东江
Owner ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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