HK1183061A - Method for obtaining individual circulating tumor cells - Google Patents

Abstract

The present invention discloses a method for obtaining circulating tumor single cells, comprising the following steps: a) enriching circulating tumor cells in a collective solution sample; b) Identify the circulating tumor cells; c) Separate individual circulating tumor cells. By isolating circulating tumor cells from body fluid samples individually, it facilitates subsequent detection and scientific research.

Description

Method for obtaining circulating tumor single cell

Technical Field

The invention relates to the field of circulating tumor cell detection.

Background

Annual reports published by the american tumor association in 2006 indicate that currently 1 out of every 3 worldwide will have a certain tumor in their lifetime and that the incidence of disease in developing countries will be higher than 1/3 with increasing levels of contamination.

The current worldwide methods for tumor diagnosis can be classified into three major groups: pathology (biopsy); imaging (ultrasound, X-ray, CT or PET, etc.) and serology (serum tumor-associated proteins, such as CA-125, CA-199 or CEA, etc.), but all of these methods have inevitable disadvantages, such as poor sensitivity and specificity and excessive reliance on the subjective judgment of the physician. Therefore, an accurate and rapid tumor detection means is urgently needed for doctors and patients. After a lot of research for a long time, the detection of Circulating Tumor Cells (CTC) in the peripheral blood of human beings certified by FDA in the united states is currently recognized as one of the best and most objective detection methods. At present, more and more doctors at home and abroad begin to apply the technology as an important auxiliary means for tumor examination. Not only here, CTC detection is also recognized as the only effective detection index for blocking tumor cell hematogenous metastasis, which is the current tumor treatment method recently popularized abroad. In addition to the above-mentioned use of CTCs as a means of tumor detection, CTCs have also proven to be successfully used in a range of other applications: (1) the rapid evaluation of the efficacy of the chemotherapy drugs in vivo, wherein CTC can display the efficacy result in 2 weeks, which is particularly important for clinicians and saving the lives of patients, compared with the CT diagnosis of every 12-week examination; (2) judging the condition and survival time of the patient after healing according to the number of CTCs: the company Immunicon/Veridex (Johnson & Johnson) in the United states, after strict validation on several hundred tumor patient specimens, has accurately predicted the survival time of breast, colorectal and prostate cancer patients according to the number change of CTCs before and after treatment, which is approved by the FDA in the United states and is beginning to be widely applied in numerous hospitals in the United states; (3) monitoring and timely diagnosing the recurrence of tumor patients: the vast majority of tumor recurrences are actually the tumor metastasis process fundamentally, so the aim of monitoring the tumor recurrence of the patient can be achieved by continuously detecting CTC of the patient after treatment; (4) screening of in vitro tumor treatment drugs for individualized treatment; and (5) general physical examination of normal population to facilitate early diagnosis of tumor as much as possible. In addition to clinical applications, CTC can also be used in basic research in pharmaceutical factories and scientific research institutions, including the search for new tumor markers and the development of new anti-tumor drugs.

The FDA in the united states has now certified the CTC detection technology of the american marketing company, municon/Veridex, and its clinical application in 2004, which is currently used for circulating tumor cell detection in multiple hospitals in the united states.

However, the detection technique of Immunicon/Veridex has obvious drawbacks. The technology for separating and detecting the tumor cells is mainly based on a specific marker on the surface of the tumor cells. However, it is well known that this tumor cell surface marker is abnormally expressed with pathological growth of tumor cells and invasion of tumor cells into blood. Moreover, the expression level of the specific marker on the cell surface varies greatly according to the tumor, including high expression and non-expression. All this makes the technique in many cases unable to capture tumor cells, limiting the wide spread and application of the technique.

Disclosure of Invention

The invention provides a method for acquiring circulating tumor single cells, which is convenient for circulating tumor cell detection and research.

The invention provides a method for obtaining circulating tumor single cells, which comprises the following steps:

a) enriching circulating tumor cells in a body fluid sample;

b) identifying the circulating tumor cells;

c) a single circulating tumor cell was isolated.

The concentration of the circulating tumor cells is improved by removing non-circulating tumor cell substances in the body fluid sample, and the effect of enriching the circulating tumor cells is achieved. Identifying and screening circulating tumor cells in the enriched body fluid sample; the identified and screened circulating tumor cells are separated one by one, so that the subsequent detection and research are facilitated.

Body fluids generally refer to extracellular fluids.

Further, in the step a), the circulating tumor cells are enriched by removing white blood cells and red blood cells from the body fluid sample.

Further, in the step a), red blood cells in the body fluid sample are removed by a centrifugation technology, and white blood cells in the body fluid sample are removed by an antibody magnetic bead labeling technology.

Further, in the step b), the circulating tumor cells are identified by an epithelial cell-specific marker by using an immunofluorescence staining method.

Further, in the step b), the circulating tumor cells are identified by comparing the particle sizes of the tumor cells and the normal cells by using a microfluidic chip.

Further, in the step b), the circulating tumor cells are identified by using tumor cell surface markers and a fluorescence in situ hybridization method.

Further, in the step c), a single circulating tumor cell is isolated by using a micropipette.

The invention has the beneficial effects that: circulating tumor cells in the body fluid sample are separated individually, so that the subsequent detection and scientific research are facilitated.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments.

A method for obtaining circulating tumor single cell comprises enriching Circulating Tumor Cell (CTC) in body fluid sample by differential method; then, identifying circulating tumor cells; finally, single circulating tumor cells were isolated.

In one embodiment, the method comprises the steps of: a) removing red blood cells in a body fluid sample of a tumor patient by using the existing centrifugation technology, and removing white blood cells by using the existing antibody magnetic bead marking technology, so that circulating tumor cells are enriched in the body fluid sample; b) identifying circulating tumor cells by an immunofluorescence staining method and using epithelial cell specific markers; c) using a micropipette, the procedure was performed under a fluorescent microscope to aspirate single circulating tumor cells. The separated single circulating tumor cell can be used for tumor analysis and detection or other scientific researches.

In this embodiment, the antibody magnetic bead labeling technique is to use fluorescent labeled antibody coated magnetic beads to couple with leukocytes to remove leukocytes from a body fluid sample. Since leukocytes contain monocytes, granulocytes, macrophages, lymphocytes, etc., different combinations of antibodies can be used to remove leukocytes.

In the present embodiment, the diameter of the micropipette is generally in the order of micrometers. During the suction operation, a manual light suction mode with a mouth can be adopted. Due to the adoption of the micro suction pipe, the suction force can be effectively controlled, and the suction force cannot be overlarge.

In another embodiment, the method comprises the steps of: a) removing red blood cells in a body fluid sample of a tumor patient by using a centrifugation technology, and removing white blood cells in the body fluid sample by using an antibody magnetic bead labeling technology, so as to enrich circulating tumor cells in the body fluid sample; b) identifying circulating tumor cells by comparing the particle sizes of the tumor cells and normal cells by adopting a microfluidic chip; c) a single circulating tumor cell was isolated.

In this embodiment, the normal cell has a particle size range, and if the particle size is outside the particle size range, the normal cell is a tumor cell by comparing the particle size of the tumor cell with the particle size range.

In yet another embodiment, the method comprises the steps of: a) enriching circulating tumor cells in a body fluid sample; b) identifying the tumor cells of the urinary system by using the existing tumor cell surface marker and a fluorescence in situ hybridization method; c) using a micropipette, the procedure was performed under a fluorescent microscope to aspirate single circulating tumor cells. The separated single circulating tumor cell can be used for tumor analysis and detection or other scientific researches.

In the present embodiment, when a tumor cell of the urinary system is identified, a tumor cell surface marker of the urinary system, for example, OCT, FGFR3 or Ras gene of bladder cancer, VHL gene of kidney cancer, or the like, is used. In specific identification, the bladder cancer specific surface markers can be used for identifying bladder cancer cells, for example, multiple genes such as OCT4, FGFR3 or Ras are used as multiple targets for identification, namely, two or more specific surface markers can be used for identification and identification.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.

Claims (7)

1. A method for obtaining circulating tumor single cells, comprising the steps of:

a) enriching circulating tumor cells in a body fluid sample;

b) identifying the circulating tumor cells;

c) a single circulating tumor cell was isolated.

2. The method of claim 1, wherein in step a), the circulating tumor cells are enriched by removing leukocytes and erythrocytes in the body fluid sample.

3. The method of claim 2, wherein in step a), the red blood cells in the body fluid sample are removed by centrifugation, and the white blood cells in the body fluid sample are removed by antibody magnetic bead labeling.

4. The method of claim 2, wherein in step b), the circulating tumor cells are identified by epithelial cell-specific markers using immunofluorescence staining.

5. The method of claim 2, wherein in step b), the circulating tumor cells are identified by comparing the sizes of the tumor cells and normal cells using a microfluidic chip.

6. The method of claim 2, wherein in step b), the circulating tumor cells are identified by using tumor cell surface markers and fluorescence in situ hybridization.

7. The method of obtaining single circulating tumor cells according to any one of claims 1 to 6, wherein in step c), single circulating tumor cells are isolated using a micropipette.