Positive circulating tumor cell enrichment method

Abstract

The invention relates to a positive circulating tumor cell enrichment method. The positive circulating tumor cell enrichment method comprises the following steps that S1, a buffer solution and a tumorpatient blood sample are sequentially added in a cell separation solution, and first-time centrifugal treatment is performed; S2, the tumor patient blood sample obtained after the first-time centrifugal treatment is subjected to second-time centrifugal treatment, supernate is removed, red blood cell lysate is added, and then low-temperature splitting decomposition is performed; S3, the tumor patient blood sample obtained in the step S2 is subjected to third-time centrifugal treatment, supernate is removed, incubation liquid is added, then immunomagnetic beads are added, incubation is performed after even mixing, and the immunomagnetic beads are coupled with epithelial cell adhesion molecules; S4, the incubation liquid is removed after incubation, and elution is performed to obtain a target; S5, the target obtained in the step S4 is transferred to a complete culture medium, and continuous culture is performed to obtain circulating tumor cells. The positive circulating tumor cell enrichment method has the advantages of being accurate in capture, capable of keeping cell viability to the most degree, low in cost and efficient.

Description

technical field

[0001] The invention relates to in vitro diagnostic technology, in particular to a positive enrichment method for circulating tumor cells. Background technique

[0002] Circulating Tumor Cells (CTC, Circulating Tumor Cell) is a general term for various tumor cells existing in peripheral blood. Due to spontaneous or therapeutic operations, they fall off from solid tumor lesions (primary tumors, metastases), and most of the CTCs enter the peripheral blood. After apoptosis or phagocytosis, a few can escape and anchor to develop into metastases, increasing the risk of death in patients with malignant tumors.

[0003] It is well known that tumor metastasis is the most important cause of death in cancer patients. According to statistics, 90% of tumor-related deaths are due to tumor metastasis caused by tumor cell invasion and shedding into the blood. Therefore, CTC has always attracted great interest of many oncology experts. Circulating tumor cells (CTCs) that ...

Examples

Embodiment 1

[0058] In order to verify the capture ability of this enrichment method for different cell lines, the MCF-7 and NCI-H226 cell lines pre-labeled with Cell Tracker GreenCMFDA were respectively selected, and after accurate counting, they were mixed into the peripheral blood of healthy volunteers and carried out according to this method. Enrichment, statistical recovery, the specific operation method is as follows:

[0059] 1. PBMC blood separation: Before the experiment, take out the sample density separation solution and equilibrate to room temperature, add 3mL sample density separation solution to the lymphatic separation tube in advance, then add 3mL PBS and 3mL peripheral blood of healthy volunteers in order, and add accurately counted Cell Tracker Green CMFDA pre-labeled MCF-7 and NCI-H226 cells, 2000rpm, centrifuge for 20min; use a Pasteur pipette to extend into the buffy coat (mononuclear cell enrichment area) to absorb, transfer to a new centrifuge tube, discard The red b...

Embodiment 2

[0072] In order to verify the possibility of using this method to isolate and obtain target cells in vitro, the peripheral blood samples of lung cancer patients were used to separate circulating tumor cells, and relevant technical means were used to obtain stable proliferating cell lines. The specific operation methods are as follows:

[0073]1. PBMC blood separation: Before the experiment, take out the sample density separation solution and equilibrate to room temperature, add 3mL sample density separation solution to the lymphatic separation tube in advance, then add 3mL PBS and 3mL peripheral blood of lung cancer patients in sequence, centrifuge at 2000rpm for 20min; use Insert the Pasteur pipette into the buffy coat (the mononuclear cell-enriched area) to absorb and transfer to a new centrifuge tube, and discard the red blood cell layer at the bottom of the lymphatic separation tube. Centrifuge at 300g for 10min, discard the supernatant;

[0074] 2. According to 1 volume o...

technical field

[0001] The invention relates to in vitro diagnostic technology, in particular to a positive enrichment method for circulating tumor cells. Background technique

[0002] Circulating Tumor Cells (CTC, Circulating Tumor Cell) is a general term for various tumor cells existing in peripheral blood. Due to spontaneous or therapeutic operations, they fall off from solid tumor lesions (primary tumors, metastases), and most of the CTCs enter the peripheral blood. After apoptosis or phagocytosis, a few can escape and anchor to develop into metastases, increasing the risk of death in patients with malignant tumors.

[0003] It is well known that tumor metastasis is the most important cause of death in cancer patients. According to statistics, 90% of tumor-related deaths are due to tumor metastasis caused by tumor cell invasion and shedding into the blood. Therefore, CTC has always attracted great interest of many oncology experts. Circulating tumor cells (CTCs) that ...

Examples

Embodiment 1

[0058] In order to verify the capture ability of this enrichment method for different cell lines, the MCF-7 and NCI-H226 cell lines pre-labeled with Cell Tracker GreenCMFDA were respectively selected, and after accurate counting, they were mixed into the peripheral blood of healthy volunteers and carried out according to this method. Enrichment, statistical recovery, the specific operation method is as follows:

[0059] 1. PBMC blood separation: Before the experiment, take out the sample density separation solution and equilibrate to room temperature, add 3mL sample density separation solution to the lymphatic separation tube in advance, then add 3mL PBS and 3mL peripheral blood of healthy volunteers in order, and add accurately counted Cell Tracker Green CMFDA pre-labeled MCF-7 and NCI-H226 cells, 2000rpm, centrifuge for 20min; use a Pasteur pipette to extend into the buffy coat (mononuclear cell enrichment area) to absorb, transfer to a new centrifuge tube, discard The red b...

Embodiment 2

[0072] In order to verify the possibility of using this method to isolate and obtain target cells in vitro, the peripheral blood samples of lung cancer patients were used to separate circulating tumor cells, and relevant technical means were used to obtain stable proliferating cell lines. The specific operation methods are as follows:

[0073]1. PBMC blood separation: Before the experiment, take out the sample density separation solution and equilibrate to room temperature, add 3mL sample density separation solution to the lymphatic separation tube in advance, then add 3mL PBS and 3mL peripheral blood of lung cancer patients in sequence, centrifuge at 2000rpm for 20min; use Insert the Pasteur pipette into the buffy coat (the mononuclear cell-enriched area) to absorb and transfer to a new centrifuge tube, and discard the red blood cell layer at the bottom of the lymphatic separation tube. Centrifuge at 300g for 10min, discard the supernatant;

[0074] 2. According to 1 volume o...

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