A quality control product for circulating tumor cell separation instrument under liquid phase and preparation method thereof

Abstract

The invention discloses a quality control product for a circulating tumor cell separator under liquid phase and a preparation method thereof. The quality control product comprises a quality control reagent 1 and a quality control reagent 2. The quality control reagent 1 is contained in a 2ml microcentrifuge tube with a capacity of 2ml, and the original liquid contained is called tumor cells, which comes from a cultured tumor cell line; the quality control reagent 2 is contained in a 2ml microcentrifuge tube with a capacity of 2ml, and the original liquid contained is called WBC white blood cells, which comes from a blood sample of a healthy person; the quality control reagent 1 and the quality control reagent 2 are operated and analyzed simultaneously with the sample to judge whether the equipment and the detection method are in normal operation and within the controllable quality range; the preparation method comprises the preparation of the quality control reagent 1 and the quality control reagent 2, is easy to operate, and is used to ensure the quality assurance in the detection of circulating tumor cells, thereby ensuring the detection quality.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to a method for preparing a quality control product of a circulating tumor cell separator under liquid phase, and also relates to a quality control product of a circulating tumor cell separator under liquid phase prepared by the preparation method. Background Art

[0002] Clinical tests usually require the use of a control method to be tested simultaneously with the method to be evaluated, and then the results are analyzed to ensure the accuracy of the test, thereby ensuring the quality of the test. The basic elements that quality control materials need to have are stability, uniformity, and no matrix effect. Matrix effect refers to components other than the analyte in the sample. Since the matrix often significantly interferes with the analysis process of the analyte and affects the accuracy of the analysis results, these effects and interferences are called matrix effects. At present, there are many different methods for the identification and analysis of circulating tumor cells, but there is a lack of corresponding quality control products to ensure the accuracy of the test.

[0003] Chinese patent CN202111139287.2 discloses a kit for preparing circulating tumor cell quality control products and a method for preparing circulating tumor cell quality control products, in which DMSO is used, and the quality control products prepared are stored at -20°C for a long time. In this method, DMSO has certain toxic effects. After long-term storage at -20°C, cells will be dormant and cell activity will be reduced. After freezing and thawing, they need to be processed before use to remove DMSO, which is cumbersome.

[0004] Chinese patent CN201811307835.6 discloses a cell freezing solution for preparing dry tumor cell quality control products and its application. In this method, 5-25% protein, 3-15% cell stabilizer A, and 1-10% cell stabilizer B are added. The protein is whey protein or serum albumin, stabilizer A is serum, and stabilizer B is glucose, etc. Protein and serum are protein substances and the serum composition is complex, which will interfere with the subsequent tumor cell immunoassay. In addition, this method is a freeze-dried preparation, and the freeze-drying and thawing steps are cumbersome and the cost is increased. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a quality control product for a circulating tumor cell separator under liquid phase, which is easy to implement and simple to operate, and is used to ensure quality assurance in circulating tumor cell detection, thereby ensuring the detection quality.

[0006] Another object of the present invention is to provide a quality control product for a circulating tumor cell separation instrument under liquid phase, which includes quality control reagent 1 (i.e., positive quality control product) and quality control reagent 2 (i.e., negative quality control product), which are run and analyzed simultaneously with the sample to determine whether the equipment and the detection method are in normal operation and within the controllable quality range.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: a method for preparing a quality control product of a circulating tumor cell separator under liquid phase, the steps of which are as follows:

[0008] Quality control reagent 1:

[0009] (1) Open the clean bench, put in culture medium, trypsin digestion solution, 1x PBS solution, 15 ml centrifuge tube, and sterile pipette tip, and turn on UV irradiation for 30 min.

[0010] (2) Turn off the UV lamp and turn on the lighting and exhaust. Take a bottle of cultured tumor cells and place it on the clean workbench. Aspirate the culture medium, add 3 ml of 1xPBS solution and gently shake to wash. Aspirate and discard. Add 0.5 ml of trypsin digestion solution, cover the lid tightly, and place it in a carbon dioxide incubator. Take it out after 30 seconds and tap the side of the culture bottle to suspend the cells. At this time, the solution in the bottle is white and turbid. Quickly add 2 ml of culture medium to terminate the enzyme reaction, and resuspend the cells in the bottle by blowing. Transfer to a 15 ml centrifuge tube, centrifuge at 1500 rpm for 3 min to collect the cells, discard the supernatant, and resuspend with 2 ml of 1xPBS to obtain a single cell suspension.

[0011] Note: All the operations in the above clean workbench should be completed around the flame of the alcohol lamp.

[0012] (3) Allow the above tumor single cell suspension to stand for 5 minutes and discard 1 ml of the upper layer of liquid (containing floating dead cells and debris) by aspiration.

[0013] (4) Vortex to mix, pipette 2 μl of liquid onto a clean glass slide, count and calculate the concentration of single-cell suspension under a microscope.

[0014] (5) Take a 1.5 ml EP tube, add 1 ml of 1x PBS solution, and pipette an appropriate amount of cell suspension (containing about 5,000 cells) into it.

[0015] (6) Take out the EpCAM magnetic beads and place them on an ice box. Vortex them for 30 seconds to 1 minute using a vortex oscillator. Pipette 5 ul into a 1.5 ml EP tube and cover it tightly. Mix and react on a 3D shaker at 7 rpm for 30 minutes.

[0016] (7) Remove the 1.5ml EP tube, place it in a palm-type centrifuge for instant centrifugation, aspirate and discard 500ul of supernatant (containing naked cells that are not bound to magnetic beads), centrifuge for another 10s, aspirate and discard the supernatant to a remaining volume of 50ul, add 500ul of fixative, tap the bottom of the EP tube to resuspend the cells, let it stand for 15min, place it in a palm-type centrifuge for centrifugation for 10s, carefully aspirate and discard the supernatant to a remaining volume of 100ul, add 1ml of 1xPBS solution, invert 4 to 5 times to mix, and set aside.

[0017] (8) Transfer the prepared magnetic bead-cell solution to the quality control reagent tube 1, then wash the original EP tube with 1 ml of 1xPBS and transfer the washed solution to the quality control reagent tube 1, and tighten the bottle cap.

[0018] Quality control reagent 2:

[0019] (1) Take 4 ml of fresh healthy human peripheral blood, transfer it to a 15 ml centrifuge tube, add 3 times the volume of red blood cell lysis buffer, invert 4 to 5 times to mix, and place on a 3D shaker to mix for 8 to 10 minutes until the solution turns into a transparent cherry red;

[0020] (2) Collect the cells by centrifugation at 1900 rpm for 10 min, discard most of the supernatant, centrifuge again briefly, discard all the supernatant, and add 2 ml of 1x PBS to resuspend the cells;

[0021] (3) Transfer the prepared WBC cell suspension into the quality control reagent tube 2 and tighten the cap.

[0022] A liquid phase circulating tumor cell separation instrument quality control product prepared by the aforementioned preparation method comprises:

[0023]

[0024] From the above, the quality control reagent 1 can display the positive quality control cell specific marker fluorescence (DAPI and CK positive) when being processed with the sample, but not the negative cell specific marker fluorescence (CD45 negative); the quality control reagent 2 can display the negative quality control cell specific marker fluorescence (DAPI and CD45 positive) when being processed and identified with the sample, but not the positive quality control cell specific marker fluorescence (CK negative). The present invention mainly solves the following problems innovatively: (1) The quality control product prepared by this method is easy to identify. The magnetic beads in the quality control reagent 1 are used as the specific markers of positive quality control cells, which can be clearly observed under a microscope, which is conducive to separating the required cells from a large number of cells, such as separating extremely rare target abnormal cells from billions of red blood cells and white blood cells in the blood; in addition, the method can also be expanded to mark other specific markers to separate cells of different purposes. (2) Different from the conventional immunofluorescence method of observation after coverslipping, the positive quality control cells in this method can be displayed as DAPI and CK positive in the liquid phase, but not CD45; the negative quality control cells in this method can be displayed as DAPI and CD45 in the liquid phase, but not CK, and identification is carried out through the specific biomarkers of positive and negative quality control cells; (3) The quality control product is in a liquid phase buffer environment throughout the process, without a freeze-thaw process and a single buffer composition, and can be used for a variety of downstream applications such as drug sensitivity testing, gene analysis and gene sequencing, and will not interfere with downstream analysis. The quality control product obtained by this method does not damage the cell morphology, cell integrity, protein markers and nucleic acids, and guarantees the integrity of the cells to the greatest extent, so that the next step of cell, protein and gene analysis can be continued.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects: the method of the present invention mainly prepares and preserves the quality control product in liquid phase, which ensures that the cell morphology is kept original to the greatest extent and is not damaged, and the cell target can be protected from interference. In addition, the quality control product prepared by the method is easy to store at 2-8°C and can be stored for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a positive quality control cell image observed under a microscope of the quality control reagent 1 in Example 1;

[0027] Figure 2 This is the immunofluorescence identification image of the positive quality control cells in liquid phase of the quality control reagent 1 in Example 1;

[0028] Figure 3 This is the immunofluorescence identification image of negative quality control cells in liquid phase for quality control reagent 2 in Example 1;

[0029] Figure 4 This is a positive quality control cell image observed under a microscope of the quality control reagent 1 in Example 2;

[0030] Figure 5 This is the immunofluorescence identification image of the positive quality control cells in liquid phase of the quality control reagent 1 in Example 2;

[0031] Figure 6 This is the immunofluorescence identification image of negative quality control cells in liquid phase of quality control reagent 2 in Example 2. DETAILED DESCRIPTION

[0032] The content of the present invention is further described below through specific implementation cases, but the present invention is not limited thereto.

[0033] Embodiment 1:

[0034] A method for preparing a quality control product of breast cancer tumor cells MCF7 in a liquid phase, the steps of which are:

[0035] Quality control reagent 1:

[0036] (1) Open the clean bench, put in culture medium, trypsin digestion solution, 1x PBS solution, 15 ml centrifuge tube, and sterile pipette tip, and turn on UV irradiation for 30 min.

[0037] (2) Turn off the UV lamp and turn on the lighting and exhaust. Take a bottle of cultured MCF-7 cells and place it on the clean bench. Aspirate the culture medium, add 3 ml of 1xPBS solution and gently shake to wash. Aspirate and discard. Add 0.5 ml of trypsin digestion solution, cover the lid tightly, and place it in a carbon dioxide incubator. Take it out after 30 seconds and tap the side of the culture bottle to suspend the cells. At this time, the solution in the bottle is white and turbid. Quickly add 2 ml of culture medium to terminate the enzyme reaction, and transfer the cells in the bottle to a 15 ml centrifuge tube after blowing to suspend them. Centrifuge at 1500 rpm for 3 min to collect the cells, discard the supernatant, and resuspend them in 2 ml of 1xPBS to obtain a single cell suspension.

[0038] Note: All the operations in the above clean workbench should be completed around the flame of the alcohol lamp.

[0039] (3) Allow the above tumor single cell suspension to stand for 5 minutes and discard 1 ml of the upper layer of liquid (containing floating dead cells and debris) by aspiration.

[0040] (4) Vortex to mix, pipette 2 μl of liquid onto a clean glass slide, count and calculate the concentration of single-cell suspension under a microscope.

[0041] (5) Take a 1.5 ml EP tube, add 1 ml of 1x PBS solution, and pipette an appropriate amount of cell suspension (containing about 5,000 cells) into it.

[0042] (6) Take out the EpCAM magnetic beads and place them on an ice box. Vortex them for 30 seconds to 1 minute using a vortex oscillator. Pipette 5 ul into a 1.5 ml EP tube and cover it tightly. Mix and react on a 3D shaker at 7 rpm for 30 minutes.

[0043] (7) Remove the 1.5ml EP tube, place it in a palm-type centrifuge for instant centrifugation, aspirate and discard 500ul of supernatant (containing naked cells that are not bound to magnetic beads), centrifuge for another 10s, aspirate and discard the supernatant to a remaining volume of 50ul, add 500ul of fixative, tap the bottom of the EP tube to resuspend the cells, let it stand for 15min, place it in a palm-type centrifuge for centrifugation for 10s, carefully aspirate and discard the supernatant to a remaining volume of 100ul, add 1ml of 1xPBS solution, invert 4 to 5 times to mix, and set aside.

[0044] (8) Transfer the prepared magnetic bead-cell solution to the quality control reagent tube 1, then wash the original EP tube with 1 ml of 1xPBS and transfer the washed solution to the quality control reagent tube 1, and tighten the bottle cap.

[0045] (9) Quality control reagent 1 is stored in liquid phase at 2-8°C and can be used for immunofluorescence identification and observation analysis in liquid phase.

[0046] Quality control reagent 2:

[0047] (1) Take 4 ml of fresh healthy human peripheral blood, transfer it to a 15 ml centrifuge tube, add 3 times the volume of red blood cell lysis buffer, invert 4 to 5 times to mix, and place on a 3D shaker to mix for 8 to 10 minutes until the solution turns into a transparent cherry red;

[0048] (2) Collect the cells by centrifugation at 1900 rpm for 10 min, discard most of the supernatant, centrifuge again briefly, discard all the supernatant, and add 2 ml of 1x PBS to resuspend the cells;

[0049] (3) Transfer the prepared WBC cell suspension into the quality control reagent tube 2 and tighten the cap.

[0050] (4) Perform immunofluorescence identification and observation on the quality control reagent 2 under liquid phase.

[0051] Figure 1 This is a positive quality control cell image observed under a microscope of the quality control reagent 1 in Example 1. A positive quality control cell with two adhered magnetic beads was observed under a bright field microscope. The cell showed magnetic beads adhered to the cell surface under the bright field to form a cell-magnetic bead complex, which was easy to observe.

[0052] Figure 2This is the immunofluorescence identification image of the positive quality control cells in the liquid phase of the quality control reagent 1 in Example 1. Immunofluorescence identification image of the positive quality control cells: these two cells are shown to adhere to the magnetic beads to form a cell-magnetic bead complex under the bright field; the positive quality control cell-specific marker fluorescence (DAPI and CK positive) is displayed under the fluorescent channel, and the negative cell-specific marker fluorescence (CD45 negative) is not displayed.

[0053] Figure 3 This is the immunofluorescence identification image of negative quality control cells in liquid phase for quality control reagent 2 in Example 1. Immunofluorescence identification image of negative quality control cells: under bright field, it can be seen that these cells do not adhere to the magnetic beads to form a cell-magnetic bead complex; under the fluorescent channel, the fluorescence of negative quality control cell-specific markers (DAPI and CD45 positive) is displayed, and the fluorescence of positive quality control cell-specific markers (CK negative) is not displayed.

[0054] Embodiment 2:

[0055] A method for preparing a quality control product of liver cancer tumor cells HepG2, the steps of which are:

[0056] Quality control reagent 1:

[0057] (1) Open the clean bench, put in culture medium, trypsin digestion solution, 1x PBS solution, 15 ml centrifuge tube, and sterile pipette tip, and turn on UV irradiation for 30 min.

[0058] (2) Turn off the UV lamp and turn on the lighting and exhaust. Take a bottle of cultured HepG2 cells and place it on the clean bench. Aspirate the culture medium, add 3 ml of 1xPBS solution and gently shake to wash. Aspirate and discard. Add 0.5 ml of trypsin digestion solution, cover the lid tightly, and place it in a carbon dioxide incubator. Take it out after 30 seconds and tap the side of the culture bottle to suspend the cells. At this time, the solution in the bottle is white and turbid. Quickly add 2 ml of culture medium to terminate the enzyme reaction, and blow the cells in the bottle to suspend them completely. Then transfer them to a 15 ml centrifuge tube, centrifuge at 1500 rpm for 3 min to collect the cells, discard the supernatant, and resuspend them in 2 ml of 1xPBS to obtain a single cell suspension.

[0059] Note: All the operations in the above clean workbench should be completed around the flame of the alcohol lamp.

[0060] (3) Allow the above tumor single cell suspension to stand for 5 minutes and discard 1 ml of the upper layer of liquid (containing floating dead cells and debris) by aspiration.

[0061] (4) Vortex to mix, pipette 2 μl of liquid onto a clean glass slide, count and calculate the concentration of single-cell suspension under a microscope.

[0062] (5) Take a 1.5 ml EP tube, add 1 ml of 1x PBS solution, and pipette an appropriate amount of cell suspension (containing about 5,000 cells) into it.

[0063] (6) Take out the EpCAM magnetic beads and place them on an ice box. Vortex them for 30 seconds to 1 minute using a vortex oscillator. Pipette 5 ul into a 1.5 ml EP tube and cover it tightly. Mix and react on a 3D shaker at 7 rpm for 30 minutes.

[0064] (7) Remove the 1.5ml EP tube, place it in a palm-type centrifuge for instant centrifugation, aspirate and discard 500ul of supernatant (containing naked cells that are not bound to magnetic beads), centrifuge for another 10s, aspirate and discard the supernatant to a remaining volume of 50ul, add 500ul of fixative, tap the bottom of the EP tube to resuspend the cells, let it stand for 15min, place it in a palm-type centrifuge for centrifugation for 10s, carefully aspirate and discard the supernatant to a remaining volume of 100ul, add 1ml of 1xPBS solution, invert 4 to 5 times to mix, and set aside.

[0065] (8) Transfer the prepared magnetic bead-cell solution to the quality control reagent tube 1, then wash the original EP tube with 1 ml of 1xPBS and transfer the washed solution to the quality control reagent tube 1, and tighten the bottle cap.

[0066] (9) Quality control reagent 1 is stored in liquid phase at 2-8°C and can be used for immunofluorescence identification and observation analysis in liquid phase.

[0067] Quality control reagent 2:

[0068] (1) Take 4 ml of fresh healthy human peripheral blood, transfer it to a 15 ml centrifuge tube, add 3 times the volume of red blood cell lysis buffer, invert 4 to 5 times to mix, and place on a 3D shaker to mix for 8 to 10 minutes until the solution turns into a transparent cherry red;

[0069] (2) Collect the cells by centrifugation at 1900 rpm for 10 min, discard most of the supernatant, centrifuge again briefly, discard all the supernatant, and add 2 ml of 1x PBS to resuspend the cells;

[0070] (3) Transfer the prepared WBC cell suspension into the quality control reagent tube 2 and tighten the cap.

[0071] (4) Perform immunofluorescence identification and observation on the quality control reagent 2 under liquid phase.

[0072] Figure 4 This is a positive quality control cell image observed under a microscope of the quality control reagent 1 in Example 2. A positive quality control cell with one magnetic bead attached was observed under the bright field of the microscope. The cell showed magnetic beads attached to the cell surface under the bright field to form a cell-magnetic bead complex, which was easy to observe.

[0073] Figure 5This is the immunofluorescence identification image of the positive quality control cells in the liquid phase of the quality control reagent 1 in Example 2. Immunofluorescence identification image of the positive quality control cells: these two cells are shown to adhere to the magnetic beads to form a cell-magnetic bead complex under the bright field; the positive quality control cell-specific marker fluorescence (DAPI and CK positive) is displayed under the fluorescent channel, and the negative cell-specific marker fluorescence (CD45 negative) is not displayed.

[0074] Figure 6 This is the immunofluorescence identification image of negative quality control cells in liquid phase for quality control reagent 2 in Example 2. Immunofluorescence identification image of negative quality control cells: under bright field, it can be seen that these cells do not adhere to the magnetic beads to form a cell-magnetic bead complex; under the fluorescent channel, the fluorescence of negative quality control cell-specific markers (DAPI and CD45 positive) is displayed, and the fluorescence of positive quality control cell-specific markers (CK negative) is not displayed.

[0075] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be understood by anyone familiar with the technology within the technical scope disclosed by the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a quality control product for a circulating tumor cell separator in a liquid phase, the steps of which are as follows: Quality control reagent 1: (1) Open the clean bench, put in culture medium, trypsin digestion solution, 1xPBS solution, 15ml centrifuge tube, sterile pipette tip, and turn on UV irradiation for 30 minutes; (2) Turn off the UV lamp and turn on the lighting and exhaust. Take a bottle of cultured tumor cells and place it on the clean workbench. Aspirate the culture medium, add 3 ml of 1xPBS solution and gently shake to wash, aspirate and discard, add 0.5 ml of trypsin digestion solution, cover the lid tightly, and place it in a carbon dioxide incubator. Take it out after 30 seconds, tap the side of the culture bottle to suspend the cells. At this time, the solution in the bottle is white and turbid. Quickly add 2 ml of culture medium to terminate the enzyme reaction, and blow the cells in the bottle to suspend them completely, then transfer them to a 15 ml centrifuge tube. Centrifuge at 1500 rpm for 3 min to collect the cells, discard the supernatant, and resuspend them with 2 ml of 1xPBS to obtain a single cell suspension. (3) Let the tumor single cell suspension stand for 5 minutes and discard 1 ml of the upper layer of liquid; (4) Vortex to mix, pipette 2 μl of liquid onto a clean glass slide, count and calculate the concentration of single-cell suspension under a microscope; (5) Take a 1.5 ml EP tube, add 1 ml of 1x PBS solution, and pipette an appropriate amount of cell suspension into it; (6) Take out the EpCAM magnetic beads and place them on an ice box. Vortex them for 30 seconds to 1 minute using a vortex oscillator. Pipette 5ul into a 1.5ml EP tube and cover it tightly. Mix and react on a 3D shaker at 7rpm for 30 minutes. (7) Take out the 1.5ml EP tube, put it into a palm-type centrifuge for instant centrifugation, aspirate and discard 500ul of supernatant, centrifuge again for 10s, aspirate and discard the supernatant until the remaining volume is 50ul, add 500ul of fixative, tap the bottom of the EP tube to resuspend the cells, let it stand for 15min, put it into a palm-type centrifuge for centrifugation for 10s, carefully aspirate and discard the supernatant until the remaining volume is 100ul, add 1ml of 1xPBS solution, invert 4~5 times to mix, and obtain the magnetic bead-cell solution for use; (8) Transfer the prepared magnetic bead-cell solution to the quality control reagent tube 1, then wash the original EP tube with 1 ml of 1xPBS and transfer the washed solution to the quality control reagent tube 1, and tighten the bottle cap; Quality control reagent 2: (1) Take 4 ml of fresh healthy human peripheral blood, transfer it to a 15 ml centrifuge tube, add 3 times the volume of red blood cell lysis buffer, invert 4 to 5 times to mix, and place it on a 3D shaker to mix for 8 to 10 minutes until the solution turns into a transparent cherry red; (2) Centrifuge at 1900 rpm for 10 min to collect the cells, discard most of the supernatant, centrifuge briefly again, discard all the supernatant, and add 2 ml of 1xPBS to resuspend the cells; (3) Transfer the prepared blood leukocyte suspension into the quality control reagent tube 2 and tighten the cap.

Images