TWI656344B - Method for screening circulating tumor cells in blood - Google Patents

Abstract

The present invention is a method for screening circulating tumor cells in blood. First, blood containing a mixture of suspected rare cells is obtained in a specimen, and then red blood cells and white blood cells that interfere with the analysis are removed, and at least one fluorescent antibody that recognizes rare cells is added to In the blood, combined with multi-parameter flow cytometry to detect, quantify, purify, and isolate circulating tumor cells expressing fluorescently-conjugated specific antibodies in the blood, and finally collecting circulating tumor cells displaying fluorescently-specific antibodies; this method improves Detection rate of circulating tumor cells in the blood, and shorten analysis time, speed up cancer screening and post-healing assessment.

Description

Method for screening circulating tumor cells in blood

The invention is used for detecting and quantifying circulating tumor cells, especially a method for screening circulating tumor cells in blood, which can quickly and accurately identify, quantify, and isolate circulating tumor cells in blood. As a disease detection tool, it is related to severity Sexual judgment.

One of the commonly used technologies is the use of Cell Search technology, which mainly uses immunochemical methods to identify and quantify circulating tumor cells (CTC) in whole blood, which can be used to assess the degree of cancer and treatment response. This technology has been used by the US Food and Drug Administration. The Authority has verified the ability to detect metastatic breast, prostate and colorectal cancer, and the immunorecognition kits it uses are epithelial cell adhesion protein (EpCAM), cytokeratin 8 (CK8), cytokeratin 18 (CK18), cells Keratin 19 (CK19), CD45. Calculate the number of CTCs with EpCAM +, CK8 +, CK18 +, CK19 +, and CD45- expression from whole blood. This method has a large number of blood cell interference, and not all tumor cells will express EpCAM +. CK8 +, CK18 +, CK19 +, so the capture efficiency of tumor cells will be lower, the specificity is also poor, and the addition of a large amount of antibodies increases the experimental cost.

Another related technology is the Isoflux System, which mainly removes blood cells from the blood, then uses immunochemical methods to identify circulating tumor cells, and then captures tumor cells with immunorecognition through magnetic fluid. The immune antibody set used is EpCAM. Epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2) and cadherin (N-cadherin) to identify and capture tumor cells, which can only be captured by the magnetic fluid method and cannot be directly provided Scientific quantitative method. Although these detection technologies all need to directly identify tumor cells with antigens and antibodies, the number of circulating tumor cells detected is not large. If there is a slight error in the number, false positive or false negative results will easily occur. Causes problems such as wrong judgment.

Circulating tumor cells are very scarce in the blood of cancer patients, so the quantity and characteristics of circulating tumor cells in the blood of cancer patients are considered to be closely related to cancer metastasis and cancer recurrence. In addition, the detection of circulating tumor cells can be used to judge the metastasis and characteristics of cancer, and it can become one of the current cancer treatment indicators. Because tumor cells are scarce in the blood, many scientists focus on the technology of detecting and purifying circulating tumor cells.

At present, in scientific research, the effective detection and purification of circulating tumor cells in the blood is still being developed. Cell surface antigens and antibodies are identified or the characteristics of tumor cells include the use of specific antigen antibodies to purify CTCs or the use of cell size, metastatic capacity, and cells. CTCs are separated by different density numbers. These methods cannot effectively and accurately detect CTCs and purify surviving CTCs.

In view of this, the inventor has been engaged in the manufacturing, development and design of related products for many years. In view of the above goals, after careful design and careful evaluation, he finally got a practical original creation.

The invention is a method for screening circulating tumor cells in blood. The steps include: Step 1: Obtain blood containing a mixture of suspected rare cells in a specimen; Step 2: Remove red blood cells from the blood; Step 3: Remove blood from the blood White blood cells; Step 4: Add at least one fluorescent antibody that recognizes rare cells to the blood; Step 5: Detect, quantify, purify, and isolate circulating tumor cells expressing fluorescent-conjugated specific antibodies in the blood by multi-parameter flow cytometry ;step Step 6: Collect circulating tumor cells with fluorescent-specific antibodies.

The method for removing red blood cells in the blood in step two is a physical separation method or a chemical removal method.

The steps of the physical separation method for removing red blood cells in blood are: a) take a substance that can cause a difference in cell density into a new tube and then slowly add blood, and the volume ratio of the above substance to blood is 3: 4 to obtain A mixed solution; b) centrifugation at a speed of 300 g for 30 minutes; the mixed solution is delaminated, the uppermost layer is the serum layer, the middle layer is the transparent solution layer, and the bottom layer is the red blood cell layer; The layer of peripheral blood mononuclear cells from the milky white junction to a new centrifuge tube; d) flush several times with physiological saline; e) obtain blood without red blood cells.

The aforementioned substances may be sucrose or other polysaccharides.

Among them, the chemical removal method for removing red blood cells in blood uses the principle of osmotic pressure to burst red blood cells without nucleus in blood and retain white blood cells with nucleus in blood. The method is as follows: a) Configure reaction reagent 1X RBC lysis buffer 1000ml, using 8.26g of NH ₄ Cl, 1.19g of NaHCO ₃ , 200 μL of 0.5M EDTA (ethylenediaminetetraacetic acid) at pH 8 and adding 1000ml of sterile distilled water, adjust the final pH value after dissolution; b ) Add blood and reaction reagents in a volume ratio of 1: 5, and let stand for 10 minutes; c) then centrifuge at 400g for five minutes to remove the supernatant; d) add 10ml PBS (phosphate buffered saline) ) Rinse the cells once and centrifuge again at 400g for 5 minutes; e) Finally remove the supernatant, and the rest are white blood cells.

Among them, the third step is to use the CD45 cocktail magnetic antibody kit to identify and remove all leukocytes expressing CD45 from the peripheral blood mononuclear cells.

Wherein, the fluorescent anti-system described in step 4 is a specific antibody for fluorescent conjugation, and the antibody is combined with the CD45 white blood cell cells showing weak or no expression collected in step 3.

Among them, the specific fluorescent junction antibodies are epithelial cell adhesion protein (EpCAM), cytokeratin (CK), cancer stem cell surface antigen (CD133, CD44 or CD24), epidermal growth factor receptor (EGFR), and human epidermal growth factor. (HER2), cadherin (N-cadherin), etc.

Among them, by detecting and determining the number of circulating tumors, it can be used for cancer detection and postoperative healing judgment, and can be extended to genetic analysis, drug resistance testing and drug testing.

Regarding the technology, means and effects used in the present invention, the preferred embodiments will be described in detail with reference to the drawings. It is believed that the above-mentioned purpose, structure and characteristics of this creation can be understood in depth and concrete.

Figure 1 is a block flow diagram of the present invention

In order to make the above and other objects, features, and advantages of the present invention more comprehensible, the following describes the preferred embodiments and the accompanying drawings in detail, as follows: Referring to FIG. 1, the present invention is a sieve The method for detecting circulating tumor cells in blood includes the following steps: Step 1: Obtain blood containing a mixture of suspicious rare cells in the specimen; Step 2: Remove red blood cells from the blood; Step 3: Remove white blood cells from the blood; Step 4 : Add at least one fluorescent antibody that recognizes rare cells to the blood; Step 5: Through multiple parameters Digital flow cytometry detects, quantifies, purifies, and isolates circulating tumor cells expressing fluorescently-conjugated specific antibodies in blood. Step 6: Collects circulating tumor cells that exhibit fluorescently-specific antibodies.

The method for removing red blood cells in the blood in step two is a physical separation method or a chemical removal method.

The steps of the physical separation method for removing red blood cells from blood are: a) take a substance that can cause differential cell density separation into a new tube and then slowly add blood, and the volume ratio of the above substance to blood is 3: 4 to obtain a The mixed solution; b) centrifugation at a speed of 300g for 30 minutes; the mixed solution was delaminated, the upper layer was a serum layer, the middle layer was a transparent solution layer, and the bottom layer was a red blood cell layer; c) the milky white between the serum layer and the transparent solution layer was taken out Layer of peripheral blood mononuclear cells at the junction to a new centrifuge tube; d) flush several times with physiological saline; e) obtain blood without red blood cells.

The aforementioned substances may be sucrose or other polysaccharides.

Among them, the chemical removal method for removing red blood cells in blood uses the principle of osmotic pressure to burst red blood cells without nucleus in blood and retain white blood cells with nucleus in blood. The method is as follows: a) Configure reaction reagent 1X RBC lysis buffer 1000ml, using 8.26g of NH ₄ Cl, 1.19g of NaHCO ₃ , 200 μL of 0.5M EDTA (ethylenediaminetetraacetic acid) at pH 8 and adding 1000ml of sterile distilled water, adjust the final pH value after dissolution; b ) Add blood and reaction reagents in a volume ratio of 1: 5 in order and let stand for 10 minutes; c) then centrifuge at 400g for five minutes to remove the supernatant; d) add 10ml PBS (phosphate buffered saline) ) Rinse the cells once and centrifuge again at 400g for 5 minutes; e) Finally remove the supernatant, and the rest are white blood cells.

Among them, the third step is using the CD45 cocktail magnetic antibody kit to identify and go to Except for peripheral blood mononuclear cells, all white blood cells expressing CD45.

Wherein, the fluorescent anti-system described in step 4 is a special antibody capable of binding and recognizing at least one of the surface antigens of circulating tumor cells, and combining these fluorescent antibodies with the white blood cells that showed weak or non-expressing CD45 collected in step 3 .

Among them, the specific fluorescent junction antibodies are epithelial cell adhesion protein (EpCAM), cytokeratin (CK), cancer stem cell surface antigen (CD133, CD44 or CD24), epidermal growth factor receptor (EGFR), and human epidermal growth factor. (HER2) and cadherin (N-cadherin).

By detecting and judging the number of circulating tumors, it can be used for cancer detection and postoperative healing judgment, and it can be extended to genetic analysis, drug resistance testing and drug testing.

The invention relates to a method for measuring and purifying circulating tumor cells in blood, and in particular, to detect and quantify circulating tumor cells in blood through flow cytometry after most of the blood cell cells in the blood are removed. Identification rate of circulating tumor cells in blood. This is used to detect and quantify the number of circulating tumor cells in the blood, and use it for cancer diagnosis and healing evaluation. The method described in this article can not only detect rare tumor cells in the blood, but also purify and isolate tumor cells from the blood. It is not limited to a single type of cancer, as long as it displays specific surface antigens of circulating tumor cells, such as epithelial cell adhesion protein (EpCAM), Tumor cells of cytokeratin (CK), cancer stem cell surface antigen (CD133, CD44 or CD24), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2) and cadherin (N-cadherin) are all Can be identified and separated.

It should be noted that the above-mentioned embodiments are merely for illustrative purposes to explain the principles and effects of the present invention, and are not intended to limit the scope of the present invention. Anyone familiar with this technology can make modifications and changes to the embodiments without departing from the technical principles and spirit of the present invention. Therefore the right of the invention The scope of protection shall be as described in the scope of patent application mentioned later.

Claims (6)

A method for screening circulating tumor cells in blood, comprising the steps of: step 1: obtaining blood containing a mixture of suspicious rare cells in a specimen; step 2: removing red blood cells from blood; step 3: removing white blood cells from blood CD45 cocktail magnetic antibody kit identification is used to remove all leukocytes expressing CD45 from peripheral blood mononuclear cells. Step 4: Add at least one fluorescent antibody that recognizes rare cells to the blood. The fluorescent anti-system is Fluorescent junction specific antibody, and combine the above antibodies with white blood cells showing weak or no CD45 collected in step 3. The fluorescent junction specific antibody is epithelial cell adhesion protein, cytokeratin, cancer stem cell surface antigen, epidermal growth. Factor Receptor, Human Epidermal Growth Factor Receptor, Cadherin; Step 5: Detect, quantify, purify, and isolate circulating tumor cells expressing fluorescence-binding specific antibodies in blood by multi-parameter flow cytometry; Step 6: Collect Circulating tumor cells with fluorescent-specific antibodies. The method for screening circulating tumor cells in blood according to item 1 of the scope of the patent application, wherein the method for removing red blood cells in blood in step two is a physical separation method or a chemical removal method. The method for screening circulating tumor cells in blood according to item 2 of the scope of the patent application, wherein the steps of physical separation to remove red blood cells in blood are: a) taking a substance that can cause differential cell density separation to a new tube After the middle period, the blood was slowly added, and the volume ratio of the above substance to blood was 3: 4 to obtain a mixed solution; b) centrifugation at 300 g for 30 minutes; the mixed solution was delaminated, and the upper layer was the serum layer and the middle layer was The transparent solution layer, the bottom layer is the red blood cell layer; c) take the peripheral blood mononuclear cell layer between the serum layer and the transparent solution layer to a new centrifuge tube; d) rinse several times with physiological saline; e) obtain Blood containing red blood cells. The method for screening circulating tumor cells in blood according to item 3 of the scope of patent application, wherein the above-mentioned substance is sucrose or other polysaccharides. The method for screening circulating tumor cells in blood according to item 2 of the scope of the patent application, wherein the chemical removal method for removing red blood cells in blood uses the principle of osmotic pressure to cause the red blood cells without blood cells in the blood to swell. White blood cells are retained by the following methods: a) 1000ml reaction reagent 1X RBC lysis buffer (red blood cell lysis buffer), 8.26g of NH ₄ Cl (ammonium chloride), 1.19g of NaHCO ₃ (sodium bicarbonate), 200μL 0.5 M pH 8 EDTA (ethylenediaminetetraacetic acid) and add 1000ml of sterile distilled water, adjust the final pH value after dissolving; b) add blood and reaction reagents, the volume ratio is 1: 5 in order, and let stand Reaction for 10 minutes; c) followed by centrifugation at 400 g for five minutes to remove the supernatant; d) washed cells by adding 10 ml of PBS (phosphate buffered saline) and centrifuged again at 400 g for 5 minutes; e) finally removed the supernatant The rest are white blood cells. The method for screening circulating tumor cells in blood according to item 1 of the scope of the patent application, wherein the number of circulating tumors can be detected and judged for the detection of cancer and the treatment of postoperative healing, and can be extended to Genetic analysis, drug resistance testing and drug testing.