TW201725386A - Method for screening circulating tumor cells in blood capable of improving a detection rate of circulating tumor cells in blood, shortening analysis time, and accelerating cancer screening and recovery assessment - Google Patents

Abstract

The present invention relates to a method which uses a blood cell removal technique together with a multi-parameter flow cytometry to detect and quantify circulating tumor cells in blood. The method includes: obtaining blood which contains suspicious rare cell mixes from a testing object; removing red blood cells and white blood cells which may interfere the analysis, and adding at least one fluorescent antibody for identifying rare cells into the blood; using the multi-parameter flow cytometry to detect, qualify, purify, and separate the circulating tumor cells that show fluorescent conjugated specific antibodies; and finally, collecting the circulating tumor cells that include the fluorescent conjugated specific antibodies. The method improves a detection rate of circulating tumor cells in blood, shortens analysis time, and accelerates cancer screening and recovery assessment.

Description

Using hematocrit removal technology combined with multi-parameter flow cytometry to detect and quantify circulating tumor cells in the blood

The invention is used for detecting and quantifying circulating tumor cells, in particular, using a hematocrit removal technique combined with a multi-parameter flow cytometer as a method for detecting and quantifying circulating tumor cells in blood, capable of quickly and accurately identifying, quantifying and separating out Circulating tumor cells in the blood as a judgment of the correlation between disease detection tools and severity.

One of the commonly used techniques is the use of Cell Search technology, which uses immunochemical methods to identify and quantify circulating tumor cells (CTC) in whole blood, which can be used to assess cancer levels and therapeutic response. This technology has been used by US food drugs. The Authority has verified the detection of metastatic breast cancer, prostate cancer and colorectal cancer, and the immunological identification kits used are epithelial cell adhesion protein (EpCAM), cytokeratin 8 (CK8), cytokeratin 18 (CK18), cells. Keratin protein 19 (CK19), CD45, calculates the number of CTCs with EpCAM+, CK8+, CK18+, CK19+ and CD45- from whole blood, and this method has a large amount of hematocrit interference, and not all tumor cells will express EpCAM+. , CK8+, CK18+, CK19+, so the tumor cell capture efficiency will be lower, the specificity is also poor, and the addition of a large number of antibodies increases the experimental cost.

Another related technology is Isoflux System, which mainly removes blood cells in blood, and then uses immunochemical methods to identify circulating tumor cells, and then captures tumor cells with immune identification via magnetic fluid, and the immune antibody set used is EpCAM. Epidermal growth factor Receptors (EGFR), human epidermal growth factor receptor (HER2) and cadherin (N-cadherin) are used to identify tumor cells, which can only capture tumor cells by magnetic fluids and cannot directly provide scientific quantitative methods. Although these detection techniques are to directly identify tumor cells by antigen and antibody, the number of circulating tumor cells is small, and if there are some slight errors in the number, it will easily lead to false positive or false negative results, leading to misjudgment and other problems. .

Circulating tumor cells are very rare in the blood of cancer patients, so the amount 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 determine the metastasis and characteristics of cancer, and it can become one of the current cancer treatment indicators. Because of the scarcity of tumor cells in the blood, many scientists are focusing on techniques for detecting and purifying circulating tumor cells.

At present, in scientific research, it is still developing and effectively detecting and purifying circulating tumor cells in the blood, identifying with cell surface antigen antibodies or tumor cell characteristics including purifying CTCs using specific antigen antibodies or utilizing cell size, metastatic ability and cells. The density is different to separate CTCs. 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 experience of related products for many years. After the detailed design and careful evaluation of the above objectives, the author has finally achieved a practical and practical creation.

The invention relates to a method for detecting and quantifying circulating tumor cells in blood by using a hematocrit removal technology combined with a multi-parameter flow cytometer, the steps comprising: Step 1: obtaining blood containing a mixture of suspected rare cells in the test body; Two: remove red blood from the blood Ball; Step 3: Remove white blood cells in the blood; Step 4: Add at least one fluorescent antibody that recognizes rare cells to the blood; Step 5: Detect, quantify, purify and separate blood in the blood by multi-parameter flow cytometry Circulating tumor cells with photo-engaged specific antibodies; Step 6: Collection of circulating tumor cells with specific antibodies expressing fluorescein.

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

The step of physically separating the red blood cells in the blood is as follows: a) taking a substance which can cause the difference in cell density to be separated into a new tube and then slowly adding the blood, the volume ratio of the substance to the blood is 3:4, a mixture; b) centrifugation at 300g for 30 minutes; the mixture is stratified, the uppermost layer is the serum layer, the middle layer is the transparent solution layer, the bottom layer is the red blood cell layer; c) the serum layer and the transparent solution layer are taken out The milky white bordering peripheral blood mononuclear cell layer to a new centrifuge tube; d) using physiological saline for several times; e) obtaining blood without red blood cells.

Among them, the above substances may be sucrose or other polysaccharides.

Among them, the chemical removal method for removing red blood cells in the blood uses the principle of osmotic pressure to cause the red blood cells without nucleus in the blood to rupture, and the white blood cells with nucleus remain, by: a) Configuring the reaction reagent 1X RBC lysis buffer (red blood cell lysis buffer) 1000 ml, using 8.26 g of NH ₄ Cl, 1.19 g of NaHCO ₃ , 200 μL of 0.5 M pH 8 EDTA (ethylenediaminetetraacetic acid) and adding 1000 ml of sterile distilled water, and then adjusting the final pH to 7.3 after dissolution; b Adding blood and reagents in a volume ratio of 1:5, and allowing to react for 10 minutes; c) then centrifuging at 400 g for five minutes to remove the supernatant; d) adding 10 ml of PBS (phosphate buffered saline) The cells were washed once and centrifuged again for 400 g for 5 minutes; e) the supernatant was finally removed and the remainder was white blood cells.

Among them, step three uses the CD45 cocktail magnetic antibody set to identify and remove all white blood cells expressing CD45 in peripheral blood mononuclear cells.

Wherein, the fluorescent anti-system described in the fourth step is a fluorescent-conjugated specific antibody, and the above antibody is combined with the third step to obtain CD45 white blood cells which exhibit weak or no expression.

Wherein, the specific antibody to the fluorescent junction is epithelial cell adhesion protein (EpCAM), cytokeratin (CK), cancer stem cell surface antigen (CD133, CD44 or CD24), epidermal growth factor receptor (EGFR), human epidermal growth factor Body (HER2), cadherin (N-cadherin) and the like.

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

The above-described objects, structures and features of the present invention will be apparent from the following detailed description of the preferred embodiments of the invention.

Figure 1 is a schematic diagram of the block flow of the present invention.

The above and other objects, features and advantages of the present invention will become more <RTIgt; Blood cell removal technology combined The parameter flow cytometer is used for detecting and quantifying circulating tumor cells in blood, and the steps thereof include: Step 1: obtaining blood containing a mixture of suspected rare cells in the test body; Step 2: removing red blood cells in the blood; Step 3: Removing white blood cells from the blood; Step 4: Adding at least one fluorescent antibody that recognizes rare cells to the blood; Step 5: Detecting, quantifying, purifying, and isolating specific antibodies expressing fluorescent junctions in the blood by multi-parameter flow cytometry Circulating tumor cells; Step 6: Collecting circulating tumor cells with specific antibodies expressing fluorescein.

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

The physical separation method for removing red blood cells in the blood is as follows: a) taking a substance which can cause the difference in cell density to be separated into a new tube and then slowly adding blood, and the volume ratio of the substance to the blood is 3:4, and a Mixing solution; b) Centrifugation at 300g for 30 minutes; stratification of the mixture, the upper layer is the serum layer, the middle layer is the transparent solution layer, the bottom layer is the red blood cell layer; c) the serum layer and the clear solution layer are milky white The peripheral blood mononuclear cells of the junction are connected to a new centrifuge tube; d) washed several times with physiological saline; e) blood without red blood cells is obtained.

Among them, the above substances may be sucrose or other polysaccharides.

Among them, the chemical removal method for removing red blood cells in the blood uses the principle of osmotic pressure to cause the red blood cells without nucleus in the blood to rupture, and the white blood cells with nucleus remain, by: a) Configuring the reaction reagent 1X RBC lysis buffer (red blood cell lysis buffer) 1000 ml, using 8.26 g of NH ₄ Cl, 1.19 g of NaHCO ₃ , 200 μL of 0.5 M pH 8 EDTA (ethylenediaminetetraacetic acid) and adding 1000 ml of sterile distilled water, and then adjusting the final pH to 7.3 after dissolution; b Adding blood and reagents in a volume ratio of 1:5, and allowing the reaction to stand for 10 minutes; c) then centrifuging at 400 g for five minutes to remove the supernatant; d) adding 10 ml of PBS (phosphate buffered saline) The cells were washed once and centrifuged again for 400 g for 5 minutes; e) the supernatant was finally removed and the remainder was white blood cells.

Among them, step three uses the CD45 cocktail magnetic antibody set to identify and remove all white blood cells expressing CD45 in peripheral blood mononuclear cells.

Wherein, the fluorescent anti-system described in the fourth step is a special antibody capable of binding and recognizing at least one of circulating tumor cell surface antigens, and the fluorescent antibodies are combined with the white blood cells of the CD45 which are weakly expressed or not expressed in the third step. .

Wherein, the specific antibody to the fluorescent junction is epithelial cell adhesion protein (EpCAM), cytokeratin (CK), cancer stem cell surface antigen (CD133, CD44 or CD24), epidermal growth factor receptor (EGFR), human epidermal growth factor Body (HER2) and cadherin (N-cadherin).

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.

The invention relates to a method for measuring and purifying circulating tumor cells in blood, in particular, after removing most blood cells in the blood, detecting and quantifying circulating tumor cells in blood by flow cytometry, thereby improving The rate of circulating tumor cells in the blood. In order to detect and quantify the number of circulating tumor cells in the blood, and apply it to cancer diagnosis and post-treatment evaluation. The method described herein can not only detect rare tumor cells in blood, but also purify and isolate tumor cells from blood, and is not limited to a single cancer, as long as it exhibits a special surface antigen of circulating tumor cells, such as epithelial cell adhesion protein (EpCAM), Cytokeratin (CK), cancer stem cell surface antigen (CD133, CD44 or CD24), epidermal growth factor receptor (EGFR), human epidermal growth Both long-term receptor (HER2) and cadherin (N-cadherin) tumor cells can be identified and isolated.

It is to be noted that the above-described embodiments are merely illustrative of the principles of the invention and its advantages, and are not intended to limit the scope of the invention. Modifications and variations of the embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be as described in the appended claims.

Claims (9)

A method for detecting and quantifying circulating tumor cells in blood by using a hematocrit removal technique combined with a multi-parameter flow cytometer comprises the following steps: Step 1: obtaining blood containing a mixture of suspected rare cells in the test body; Step 2: removing Red blood cells in the blood; Step 3: Remove white blood cells in the blood; Step 4: Add at least one fluorescent antibody that recognizes rare cells to the blood; Step 5: Detect, quantify, purify and separate blood through multi-parameter flow cytometry Circulating tumor cells showing fluorescein-specific antibodies; Step 6: Collecting circulating tumor cells with specific antibodies expressing fluorescein. As described in claim 1, the method for removing blood cells in a blood cell is combined with a multi-parameter flow cytometer for detecting and quantifying circulating tumor cells in blood, wherein the method for removing red blood cells in blood is physical separation or Chemical removal method. The method for removing the tumor cells in the blood by using a hemocytocyte removal technique combined with a multi-parameter flow cytometer as described in claim 2, wherein the physical separation method for removing red blood cells in the blood is: a Take a substance that can cause the difference in cell density to be separated into a new tube and then slowly add blood. The volume ratio of the above substance to blood is 3:4 to obtain a mixed solution; b) Centrifuge at 300g for 30 minutes; The liquid is stratified, 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; c) the peripheral blood mononuclear cell layer which is milky-white junction between the serum layer and the transparent solution layer is taken out to a new centrifuge tube; d) rinsing several times with physiological saline; e) obtaining blood without red blood cells. The multi-parameter flow cytometer is used for detecting and quantifying circulating tumor cells in blood as described in claim 3 of the patent application, wherein the substance is sucrose or other polysaccharides. As described in claim 2, the use of hematocrit removal technology combined with multi-parameter flow cytometry as a method for detecting and quantifying circulating tumor cells in blood, wherein the chemical removal method for removing red blood cells in blood utilizes the principle of osmotic pressure The red blood cells without blood nucleus in the blood burst and the white blood cells with the nucleus remain. The method is: a) configure the reaction reagent 1X RBC lysis buffer (red blood cell lysis buffer) 1000ml, using 8.26g NH ₄ Cl (ammonium chloride) , 1.19g of NaHCO ₃ (sodium bicarbonate), 200μL of 0.5M pH 8 EDTA (ethylenediaminetetraacetic acid) and add 1000ml of sterile distilled water, and then adjust the final pH to 7.3 after dissolution; b) Add blood and reaction reagent , the volume ratio is 1:5, and the reaction is allowed to stand for 10 minutes; c) then centrifuged at 400 g for five minutes to remove the supernatant; d) the cells are washed once with 10 ml of PBS (phosphate buffered saline), and Centrifuge again for 400 g for 5 minutes; e) Finally remove the supernatant and the remainder are white blood cells. As described in claim 1, the use of hematocrit removal technology combined with multi-parameter flow cytometry as a detection and quantification of circulating tumor cells in the blood, wherein the third step is to identify and remove the CD45 cocktail magnetic antibody set. All white blood cells expressing CD45 in peripheral blood mononuclear cells. As described in claim 1, the blood cell removal technique is combined with a multi-parameter flow cytometer for detecting and quantifying circulating tumor cells in the blood, wherein the fluorescent reaction system described in the fourth step is a fluorescent junction specific one. The antibody is collected and the above antibody is collected in step three to obtain a white blood cell which exhibits weak or no expression of CD45. The method for detecting and quantifying circulating tumor cells in blood by using a hematocrit removal technique combined with a multi-parameter flow cytometer according to the sixth aspect of the patent application, wherein the fluorescent-conjugated specific antibody is epithelial cell adhesion protein, cytokeratin Protein, cancer stem cell surface antigen, epidermal growth factor receptor, human epidermal growth factor receptor, cadherin. As described in claim 1, the use of hematocrit removal technology combined with multi-parameter flow cytometry is used to detect and quantify circulating tumor cells in the blood, wherein the number of circulating tumors can be detected and determined for cancer detection. The treatment and judgment of postoperative healing can be extended to genetic analysis, drug resistance testing and drug testing.