Method and kit for detecting natural killer cells on flow cytometer

Abstract

The present invention provides a method for detecting natural killer cells on a flow cytometer and a kit thereof. The method is a staining method for detecting natural killer cells. The method promotes CD3 ‑ (CD56+CD16) + Cell populations and CD3 ‑ (CD56+CD16) ‑ The cell groups are separated; the method uses a buffer solution to wash the whole blood to obtain a cell suspension, then mixes the CD45 staining antibody, CD3 staining antibody, CD16 staining antibody and CD56 staining antibody with the cell suspension, incubates and stains, adds a lysate after staining, lyses the red blood cells, and finally detects on a flow cytometer. The method of the present invention significantly increases the separation degree of NK cells and non-NK cells, and the cell grouping of unclear NK grouping samples is significantly improved after using the washing first and then staining mode.

Description

Technical Field

[0001] The present invention relates to the field of cell detection, and in particular to a method for detecting natural killer cells on a flow cytometer and a kit thereof. Background Art

[0002] Flow cytometry is currently recognized as the "gold standard" for lymphocyte subsets. Flow cytometer detection of lymphocyte subsets has become a routine laboratory test item. Among them, the detection of natural killer cells (NK cells) is of great reference significance for the evaluation of the body's immune function, disease diagnosis and monitoring of tumor patients, management of infectious diseases, immunodeficiency and autoimmune diseases, hematopoietic stem cell transplantation and CAR-T therapy. The detection of NK cells can help doctors better understand the patient's immune status.

[0003] NK cells are cytotoxic lymphocytes with direct killing function in the body's innate immune system. They are not restricted by MHC and do not require antigen sensitization. They can directly kill tumor cells and virus-infected cells, and have the function of regulating other immune cells and promoting tissue production. NK cells in the human body are CD3 - CD56 + cells, which can be divided into two major cell subtypes during development CD56 dim and CD56 bright , the former is more mature in function than the latter. dim NK cells show stronger cytotoxicity, killing target cells by expressing a large amount of CD16 and secreting cytotoxic molecules such as perforin and granzyme. bright The main function of NK cells is to release cytokines and kill tumor cells through immune regulation. NK cells can also affect the results of adaptive immune responses. In the actual detection of NK cells, four antibodies, CD45, CD3, CD16 and CD56, need to be used simultaneously. CD45 has different expression intensities on the surface of granulocytes, monocytes and lymphocytes. When using flow cytometry to detect peripheral blood cells, it is often used for lymphocyte gate analysis. CD3 is mainly used to obtain CD3 - Cell population, further analysis (CD16+CD56) + Compared with CD45 and CD3 lymphocyte subset surface antigens, CD56 and CD16 antigens are less abundant, and are usually used with high-brightness fluorescent dyes to increase the fluorescence intensity of the stained cells. Currently, the staining method of directly mixing single-labeled antibodies with whole blood is used. However, for some clinical samples with low CD56 expression, the current staining method still cannot achieve clear grouping of NK cells, that is, it is impossible to separate CD3 - (CD56+CD16) +Cell populations and CD3 - (CD56+CD16) - Cell population separation, CD3 - (CD56+CD16) + The cell population is natural killer cells, CD3 - (CD56+CD16) - The cell population is non-natural killer cells, so it is necessary to find a new staining method to improve the NK cell clustering effect, thereby providing clinicians with more accurate NK cell detection results. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for detecting natural killer cells on a flow cytometer, wherein the method is a staining method for detecting natural killer cells, and the method promotes CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The cell groups are separated; the method comprises washing whole blood with a buffer to obtain a cell suspension, then mixing CD45 staining antibody, CD3 staining antibody, CD16 staining antibody and CD56 staining antibody with the cell suspension, incubating and staining, adding a lysis solution after staining, lysing red blood cells, and finally detecting on a flow cytometer.

[0005] In one embodiment, the method comprises:

[0006] Step 1: Add CD45 staining antibody, CD3 staining antibody, CD16 staining antibody and CD56 staining antibody into the flow tube;

[0007] Step 2: Wash the whole blood to obtain a cell suspension, add it to the above flow tube, and incubate at room temperature for staining;

[0008] Step 3: Take out the flow tube incubated and stained in step 2, add lysis buffer, and lyse the red blood cells;

[0009] Step 4: After lysis, detect on flow cytometer.

[0010] In one embodiment, the whole blood is washed with at least one of PBS buffer, MOPS buffer, acetic acid-sodium acetate buffer, Tris-HCl buffer, disodium hydrogen phosphate-citric acid, and potassium dihydrogen phosphate-sodium hydroxide buffer.

[0011] In one embodiment, whole blood is washed with MOPS buffer.

[0012] In one embodiment, whole blood is washed with a 0.04M-0.4M MOPS solution.

[0013] In one embodiment, whole blood is washed with a 0.1 M to 0.4 M MOPS solution.

[0014] In one embodiment, whole blood is washed with a MOPS solution having a pH in the range of 5.0-7.5.

[0015] In one embodiment, whole blood is washed with a MOPS solution having a pH in the range of 5.5-6.5.

[0016] In one embodiment, a kit for use in the above method is provided.

[0017] In the present invention, compared with the direct staining test results, the CD3 cell clustering of all specimens in the test results of washing first and then staining did not change significantly, while the NK cell clustering was significantly improved, and the separation degree of NK cells and non-NK cells was significantly increased. The cell clustering of unclear NK clustering samples was significantly improved after using the washing first and then staining mode. Compared with the direct staining method, the effect of washing first and then staining is better. It is inferred that the washing process exposes the antigens on the surface of NK cells more or the physical and chemical properties of the washing solution are more conducive to the binding of cell surface antigens and detection antibodies, resulting in increased reactivity of antigens and antibodies, showing CD3 - (CD56+CD16) + The fluorescence intensity of the cell population increases, so CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The cell populations are clearly separated. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The results of the test in two modes in blood sample No. 1 are shown in Figure 1 , Figure 1 A is the direct mode result graph and Figure 1 B is the result of the washing-then-dyeing mode;

[0020] Figure 2 The results of the test in two modes in blood sample No. 2 are shown in Figure 2 , Figure 2 A is the direct mode result graph and Figure 2 B is the result of the washing-then-dyeing mode;

[0021] Figure 3 The washing effect of different concentrations of MOPS solution at pH 7.2 Figure 3 ,in Figure 3 A is 0.04M, Figure 3 B is 0.08M, Figure 3 C is 0.1M, Figure 3 D is 0.2M, Figure 3 E is 0.4M and Figure 3 F is the 0.8M washing effect diagram;

[0022] Figure 4 The washing effect of 0.2M MOPS solution with different pH values Figure 4 ,in Figure 4 A is pH 5.0, Figure 4 B is pH 5.5, Figure 4 C is pH 6.2, Figure 4 D is pH 6.5, Figure 4 E is pH 7.2, Figure 4 F is pH 7.5 and Figure 4 G is the washing effect diagram when the pH value is 7.9. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the following embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should belong to the scope of protection of the present application. The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0024] In the present invention, flow cytometer (model: CytoFocus), PerCP-Cy5.5 Mouse Anti-Human CD45 (lot number: 3180272), FITC Mouse Anti-Human CD3 (lot number: 3222318), PE Mouse Anti-HumanCD16 (lot number: 3255645), PE Mouse Anti-Human CD56 (lot number: 3352849); auxiliary reagent: BD FACSTM Lysing solution (lot number: 3248587), all reagents were used within the validity period.

[0025] Example 1 Data comparison between washing and not washing

[0026] Wash-free process: Add 5μL PerCP-Cy5.5 Mouse Anti-Human CD45, 20μL FITC Mouse Anti-Human CD3, 5μL PE Mouse Anti-Human CD16 and 20μL PE Mouse Anti-HumanCD56 to the flow tube, add 100μL of the mixed peripheral whole blood sample, vortex to mix, and incubate at room temperature in the dark for 15 minutes. Be careful not to let the blood hang on the inner wall of the flow tube; take out the flow tube and add 450μL BD FACS TM Lysing solution, vortex mix, and place at room temperature away from light for 15 min for red blood cell lysis; detect by flow cytometry.

[0027] Washing process: Add 5μL of PerCP-Cy5.5 Mouse Anti-Human CD45, 20μL of FITCMouse Anti-Human CD3, 5μL of PE Mouse Anti-Human CD16 and 20μL of PE Mouse Anti-HumanCD56 to the flow tube, add about 100μL of cell suspension (100μL of mixed peripheral whole blood specimen, add 500μL of 0.01M PBS (pH7.2) for washing, centrifuge at 500g and remove the supernatant to retain a total volume of about 100μL). The other processes are the same as the no-wash process.

[0028] Table 1: Comparison of two staining modes

[0029]

[0030] Note: Except for step 2, the other steps are exactly the same, and the amount of reagents added, the amount of samples added and the total staining system are all the same.

[0031] The NK cell separation degree of the reagent in the two modes of direct staining and washing before staining was calculated according to the following formula, that is, NK cells (CD3 - (CD56+CD16) + ) and non-NK cells (CD3 - (CD56+CD16) - ) In the flow cytometry scatter plot, if the reagent test results show that the washing-before-staining mode is better, it is considered that the washing-before-staining mode has better NK cell separation than the direct staining mode.

[0032]

[0033] Table 2: Experimental results of blood sample No. 1

[0034]

[0035] Table 3: Experimental results of blood sample No. 2

[0036]

[0037] From the above results, it can be seen that when natural killer cells are detected on a flow cytometer, in the case of comparison between the two staining modes of the present invention, the washing-before-staining mode can detect CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - Specifically, in direct mode, CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The cell populations could not be separated at all, but in the wash-before-staining mode, the cell separation degree reached 158; the fluorescence intensity of lymphocyte PerCP and CD3 + Lymphocyte FITC fluorescence intensity and CD3 - The difference in the fluorescence intensity of lymphocyte FITC in the two modes is not big, which shows that different staining methods have different effects on different cells. In the mode of washing first and then staining, the CD3 - (CD56+CD16) + Cell population and CD3-(CD56+CD16) - The separation of cell populations provides more accurate NK cell detection results. The results of the test in two modes for blood sample No. 1 are shown in Figure 1 , Figure 1 A is the direct mode result graph and Figure 1 B is the result of the washing-then-staining mode.

[0038] In blood sample No. 2, CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The cell population separation degree was 72, while in the wash-before-staining mode, the cell separation degree reached 316; similarly, the lymphocyte PerCP fluorescence intensity, CD3 + Lymphocyte FITC fluorescence intensity and CD3 -The fluorescence intensity of lymphocyte FITC is not much different in the two modes, and the results in the two modes are not much different, which further illustrates that different staining methods have different effects on different cells. In the mode of washing first and then staining, the CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The separation of cell populations provides more accurate NK cell detection results. The results of the test in two modes for blood sample No. 2 are shown in Figure 2 , Figure 2 A is the direct mode result graph and Figure 2 B is the result of the washing-then-staining mode.

[0039] Example 2. Washing results with different buffers

[0040] The specific experimental method is as in the “washing process” in Example 1. Blood sample No. 3 was washed using commonly used buffer solutions as shown in the following table. The washing effects of different buffer solutions are shown in the following table.

[0041] Table 4 Washing effect of different buffers

[0042]

[0043] From the washing effects of the commonly used buffers above, PBS buffer, MOPS buffer, acetic acid-sodium acetate buffer, Tris-HCl buffer, disodium hydrogen phosphate-citric acid and potassium dihydrogen phosphate-sodium hydroxide buffer can be used in the method of the present invention, among which MOPS (pH7.2, 0.08M) buffer has the best washing effect, with a NK cell separation degree of 248.

[0044] Example 3 Washing experiments with different molar concentrations and different pH values ​​of MOPS

[0045] 1. Based on Example 2, the specific experimental method is the same as the "washing process" in Example 1. Blood sample No. 4 is washed with MOPS solutions of pH 7.2 with different molar concentrations as shown in the following table. The washing effects of MOPS solutions of pH 7.2 with different molar concentrations are shown in Table 5 below.

[0046] Table 5 Washing effect of MOPS solution with different molar concentrations and pH 7.2

[0047]

[0048] When washed with MOPS solution at pH 7.2, the separation degree was more than 100 in the range of 0.04M-0.4M. - (CD56+CD16)+ Cell populations and CD3 - (CD56+CD16) - The separation effect of cell population is relatively good; in the concentration range of 0.1M-0.4M, CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The separation of cell populations is better. The washing effect of different concentrations of MOPS solution at pH 7.2 can be found in Figure 3 , where 3A is 0.04M, 3B is 0.08M, 3C is 0.1M, 3D is 0.2M, 3E is 0.4M and 3F is 0.8M washing effect diagram.

[0049] 2. Specific Experimental Methods As in the “Washing Process” in Example 1, blood sample No. 5 was washed with 0.2 M MOPS solutions with different pH values ​​as shown in the following table. The washing effects of different buffer solutions are shown in Table 6 below.

[0050] Table 6 Washing effect of 0.2M MOPS solution with different pH values

[0051]

[0052] When washed with 0.2M MOPS solution, the separation degree was more than 100 in the pH range of 5.0-7.5. - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The separation effect of cell population is relatively good; in the pH range of 5.5-6.5, CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The separation effect of cell groups is better, with a separation degree of more than 300. The washing effect of 0.2M MOPS solution with different pH values ​​can be found in Figure 4 , where 4A is pH 5, 4B is pH 5.5, 4C is pH 6.2, 4D is pH 6.5, 4E is pH 7.2, 4F is pH 7.5 and 4G is pH 7.9 washing effect diagram.

[0053] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0054] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for detecting natural killer cells on a flow cytometer, characterized in that: The method is to detect natural killer cells by staining, and the method promotes CD3 - (CD56+CD16) + Cell populations and CD3 - (CD56+CD16) - The cell groups are separated; the method comprises washing whole blood with a buffer, wherein the buffer is at least one of PBS buffer, MOPS buffer, acetic acid-sodium acetate buffer, Tris-HCl buffer, disodium hydrogen phosphate-citric acid and potassium dihydrogen phosphate-sodium hydroxide buffer; when washing whole blood with the buffer, the buffer is added to the whole blood sample, a part of the supernatant is removed after centrifugation, and then the cell suspension is obtained by resuspending, and then CD45 staining antibody, CD3 staining antibody, CD16 staining antibody and CD56 staining antibody are mixed with the cell suspension, incubated and stained, and a lysis solution is added after staining to lyse the red blood cells, and finally the whole blood is detected by flow cytometry.

2. The method according to claim 1, characterized in that The method comprises: Step 1, adding CD45 staining antibody, CD3 staining antibody, CD16 staining antibody and CD56 staining antibody into the flow tube; Step 2, washing the whole blood to obtain a cell suspension, adding the cell suspension into the above-mentioned flow tube, and incubating and staining at room temperature; Step 3, taking out the flow tube incubated and stained in step 2, adding lysis solution, and lysing red blood cells; Step 4: After lysis, the cells were detected by flow cytometry.

3. The method according to claim 1, characterized in that The buffer is MOPS buffer.

4. The method according to claim 2, characterized in that: The buffer is 0.04M-0.4M MOPS buffer.

5. The method according to claim 3, characterized in that: The buffer is 0.1M-0.4M MOPS buffer.

6. The method according to claim 4, characterized in that The buffer is a MOPS buffer with a pH value in the range of 5.0-7.

5.

7. The method according to claim 5, characterized in that The buffer is a MOPS buffer with a pH value in the range of 5.5-6.5.

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