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Hematoxylin and eosin staining kit for circulating tumor cells (CTC)

A staining reagent and tumor cell technology, applied in the field of cell staining, can solve the problems of high false positive rate, low detection efficiency, and inability to exclude blood leukocyte interference, and achieve the effect of good effect and less blood leukocyte interference.

Active Publication Date: 2015-09-23
NINGBO MEIJING MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The publication number is CN102980793, which discloses a circulating tumor cell staining kit, which includes fixative (paraformaldehyde + bovine serum albumin), isotonic solution (sodium chloride solution or glucose solution), eosin Dyeing solution, methylene blue + azure dyeing solution, eosin dyeing solution can be combined with alkaline cytoplasm, and methylene blue + azure can be combined with cell nucleus to achieve double staining effect, but the staining kit needs to be specific for circulating tumors. Cells can only be stained after separation, the detection process is complicated in cell separation steps, and the detection efficiency is not high
The publication number is CN 103472227, which discloses a circulating tumor cell detection kit, which includes immunomagnetic microspheres, a magnetic separation device (magnetic frame) and an analysis and counting device; the application of the kit includes the counting of circulating tumor cells, Quantitative, molecular and gene-level analysis, but when the antibody of the immunomagnetic microspheres in the kit enriches and captures CTC cells, blood leukocytes are also enriched and captured at the same time, so the interference of blood leukocytes cannot be ruled out. Positive rate is higher

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0010] A staining kit for circulating tumor cells, including a fixative (acetone), immunomagnetic microspheres and staining reagents, and a blocking solution: containing 0.1% to 1% high-carbon alcohol in mass percent concentration, and a mass percent concentration of 0.5% to 3% glycerin solution, anti-fluorescence quenching agent: a glycerin solution containing 0.1% to 1% p-phenylenediamine in mass percent, wherein the surface of the immunomagnetic microspheres is coated with anti-EpCAM and human epidermal growth Combined antibody of factor receptor (Her2), the staining reagent is a composite fluorescent staining reagent containing: FITC-CK19, Cy3-CD45 and DAPI.

Embodiment 2

[0012] It is basically the same as Example 1, except that the high-carbon alcohol in the blocking solution is replaced by fatty acid or high-carbon fatty amine or fatty amide or ethylene oxide or polyethylene glycol, and glycerin is replaced by polysaccharides such as galactose, arabinose and maltose , FITC-CK19 fluorescent staining reagent was replaced by FITC-Her2 fluorescent staining reagent.

Embodiment 3

[0014] Staining process of circulating tumor cells using the staining kit in Example 1: 2.5 mL of blood was added to a 15 ml centrifuge tube, and then 2.2 mL of blood pretreatment reagent (0.1-1% surfactant and 0.5- 3% protein protectant), GF=800g, Temp=25°C and centrifuge for 10 min; carefully take out the centrifuge tube, use a pipette to absorb the supernatant (keep about 1 mL of the supernatant above the red blood cells), and put it in the centrifuge tube Then add 1 mL of blood pretreatment reagent; mix the centrifuge tube thoroughly; then add 100 μL of immunomagnetic microtube with combined antibody against EpCAM and Her2, incubate at room temperature for 30 min, mix well every 10 min, and place on magnetic On the device, perform magnetic separation. After magnetic separation, add phosphate buffer solution (pH = 7.4, 0.1mol / L) to wash for 1 to 3 times. After washing, take 10-20 μL (about 200) cell drops with a pipette gun Add it to the glass slide, carefully mark the posi...

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Abstract

The invention discloses a hematoxylin and eosin staining kit for circulating tumor cells (CTC). The kit comprises stationary liquid, immunomagnetic microspheres, a staining reagent as well as confining liquid, wherein the confining liquid comprises an ion detergent and a protein protectant; the surfaces of immunomagnetic microspheres are carried with a combined antibody for resisting EpCAM and Her2; the staining reagent is a compound fluorescence staining reagent containing FITC-CK19 or FITC-Her2, Cy3-CD45 and DAPI. The hematoxylin and eosin staining kit for circulating tumor cells (CTC) has the advantages that the interference of blood leucocytes is low, the CTC cells are specifically enriched and captured, and the staining effect on CTC cells is good.

Description

technical field [0001] The invention relates to cell staining, in particular to a staining kit for circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells that enter the peripheral blood of the human body. Circulating tumor cells with high vitality and high metastatic potential can survive in the blood circulation system and proliferate in a suitable environment, leading to tumor recurrence and metastasis. The detection of circulating tumor cells can be effectively used in early diagnosis in vitro, rapid evaluation of chemotherapy drugs, individualized treatment including clinical drug screening, detection of drug resistance, monitoring of tumor recurrence and development of new tumor drugs, etc. Circulating tumor cell detection includes blood processing, cell capture, cell staining and qualitative and quantitative detection of circulating tumor cells. The publication number is CN102980793, which discloses a circulating tu...

Claims

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Application Information

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IPC IPC(8): G01N1/30
Inventor 张晓晶沈挺乐飚
Owner NINGBO MEIJING MEDICAL TECH
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