Immune cell full-dimensional analysis method
阅读说明：本技术 一种免疫细胞全维分析方法 (Immune cell full-dimensional analysis method ) 是由 张娈 常菁 张瀚心 虞强 于 2018-07-13 设计创作，主要内容包括：本发明公开了一种免疫细胞全维分析方法,包括：取不同的荧光标记的抗体,与待检测样品混合,孵育后,经流式细胞术检测,得检测数据,分析所得检测数据包括中各类免疫细胞的百分比及计数,本发明的方法实现了对免疫细胞进行更加全面的精细免疫分型,所需待测样品少,所需时间短、准确性高、分析全面,可广泛应用于免疫细胞全维分析。(The invention discloses a full-dimensional analysis method of immune cells, which comprises the following steps: the method provided by the invention realizes more comprehensive fine immunization typing on the immune cells, has the advantages of less required samples to be detected, short required time, high accuracy and comprehensive analysis, and can be widely applied to immune cell full-dimensional analysis.)
1. An immune cell full-dimensional analysis method is characterized by comprising T lymphocyte immunophenotyping, B lymphocyte immunophenotyping, activated and suppressed cell immunophenotyping, NK cell immunophenotyping and DC cell immunophenotyping;
the method for full-dimensional analysis of the immune cells comprises the following steps:
mixing different fluorescence labeled antibodies with a sample to be detected, incubating, detecting by flow cytometry to obtain detection data, and analyzing the detection data;
when immunophenotyping said subpopulation of T lymphocytes, the antibodies used include:
anti-CD 45, anti-CD 3, anti-CD 8, anti-CD 4, anti-CD 25, anti-CD 127, anti- γ δ;
in the immunotyping of the B lymphocytes, the antibodies used include:
anti-CD 45, anti-CD 3, anti-CD 19, anti-CD 38, anti-CD 27, anti-CD 24, anti-IgD +;
in the immunophenotyping of the activated and suppressed cells, the antibodies used include:
antibodies against CD3, CTLA-4, PD-1, NKG2D, CD16, 56, NKP 46;
when the NK cells are immunophenotyped, the antibodies used comprise:
antibodies against CD45, CD3, CD16, 56;
in the DC cell immunophenotyping, the antibodies used include:
antibodies against LIN, HLA-DR, CD123, CD11 c;
in the immunotyping of the T lymphocyte, the method of analyzing comprises:
cell surface markers CD45+ CD3+ SSC-represent total T lymphocytes;
the cell surface marker CD45+ CD3+ CD8+ represents killer T cells;
the cell surface marker CD45+ CD3+ CD4+ represents helper T cells;
the cell surface marker CD3+ γ δ + represents γ δ T cells;
the cell surface marker CD4+ CD25+ CD127+ represents regulatory CD4+ T cells;
the cell surface marker CD3+ CD8+ CD28+ represents cytotoxic T cells;
cell surface marker CD3+ CD8+ CD 28-represents suppressor T cells;
in the immunotyping of the B lymphocyte, the method for analyzing comprises:
cell surface markers CD3-CD19+ represent total B lymphocytes;
the cell surface marker CD3-CD19+ CD27+ represents memory B lymphocytes;
the cell surface marker CD3-CD19+ CD27-IgD + represents naive B lymphocytes;
the cell surface markers CD3-CD19+ CD24-CD38+ represent plasmablasts;
the activated and suppressed cell immunophenotyping, the method of analysis comprising:
the cell surface marker CD3+ CTLA-4+ represents CTLA-4@ T;
the cell surface marker CD3+ PD-1+ represents PD-1@ T;
the cell surface marker CD3+ NKG2D + represents NKG2D @ T;
the cell surface markers CD3-CD16, 56+ NKG2D + represent NKG2D @ NK;
the cell surface markers CD3-CD16, 56+ NKP46+ represent NKP46@ NK;
the NK cell immunophenotyping, the method of analyzing comprising:
cell surface markers CD3-CD16, 56+ represent NK cells;
cell surface markers CD3+ CD16, 56+ represent NK-T cells;
when the DC cell is used for immune typing, the analysis method comprises the following steps:
the cell surface marker LIN-HLA-DR + CD11c + represents myeloid DC;
the cell surface marker LIN-HLA-DR + CD123+ represents lymphoid lineage DC.
2. The method for full-dimensional analysis of immune cells according to claim 1, wherein: the full-dimensional analysis method also comprises the step of counting the number of various immune cells in the sample to be detected.
3. The method for full-dimensional analysis of immune cells according to claim 2, wherein: the step of counting the number of the various immune cells in the sample to be detected comprises the following steps:
and detecting the percentage of various immune cells in the sample to be detected, and obtaining the number of various immune cells in the sample to be detected after calculation through counting microspheres pre-coated in a flow tube.
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to a full-dimensional analysis method for immune cells.
Background
The immune system is a defense network covering the whole body, can resist the invasion of pathogenic bacteria and is a defense system of the body. Its main functions mainly include immune defense, self-stabilization and immune surveillance. The immune dysfunction of the body can be manifested as over-strong or over-weak immune response, resulting in autoimmune damage or decreased resistance to disease. The immune cells are the main undertaker of immunity, and can give early warning for diseases through qualitative and quantitative analysis of various immune cells (subclasses).
With the continuous progress of scientific research, scientists find more cell surface markers, and can more accurately evaluate the immune function of an organism and better research the occurrence, development and treatment effect evaluation of certain diseases by carrying out immune typing and quantitative analysis on various immune cells in a blood sample. At present, the methods commonly used for the typing and quantitative analysis of immune cells in blood samples are mainly: the method comprises the steps of immunoenzyme labeling, common lymphocyte classification and fine lymphocyte subgroup detection. The immunoassay method only performs specific reaction aiming at a certain or similar specific antigen or antibody, has narrow detection surface, complex operation, large demand on a sample to be detected and time and labor waste. The general lymphocyte classification is generally applied to lymphocyte detection, but the lymphocyte can be only classified into T cells, Tc cells, Th cells, B cells and NK cells, and the lymphocyte cannot be further classified. The detection of the finer lymphocyte subpopulation depends on flow cytometry, and a multicolor flow method is adopted to perform more quantity of lymphocyte. However, if further immunophenotyping and quantitative analysis of cell subsets are desired, other methods for immunophenotyping must be used, which is complicated to operate, requires a large amount of sample to be tested, and is not suitable for practical use. Therefore, there is a need for studies on a method for immunophenotyping and quantitative analysis of lymphocytes, in order to more finely immunophenotyping and quantitative analysis of lymphocytes by a simple method as much as possible.
In the application number: CN201410485865.1, having an application date of 20140922, with the name: the invention discloses a method and a kit for B lymphocyte immunophenotyping, and provides the method for B lymphocyte immunophenotyping, which comprises the following steps: mixing different fluorescence-labeled antibodies with a sample to be detected, incubating, detecting by flow cytometry to obtain detection data, and analyzing the obtained detection data; the antibody comprises: antibodies against CD19, IgD, CD38, CD24 did not fully classify and quantify immune cells in blood samples.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a full-dimensional analysis method for immune cells.
The purpose of the invention can be realized by the following technical scheme:
an immune cell full-dimensional analysis method is characterized by comprising T lymphocyte immunophenotyping, B lymphocyte immunophenotyping, activated cell immunophenotyping, suppressed cell immunophenotyping, NK cell immunophenotyping and DC cell immunophenotyping;
the method for full-dimensional analysis of the immune cells comprises the following steps:
mixing different fluorescence labeled antibodies with a sample to be detected, incubating, detecting by flow cytometry to obtain detection data, and analyzing the detection data;
when immunophenotyping said subpopulation of T lymphocytes, the antibodies used include:
anti-CD 45, anti-CD 3, anti-CD 8, anti-CD 4, anti-CD 25, anti-CD 127, anti- γ δ;
in the immunotyping of the B lymphocytes, the antibodies used include:
anti-CD 45,
in the immunophenotyping of the activated and suppressed cells, the antibodies used include:
antibodies against CD3, CTLA-4, PD-1, NKG2D, 16, 56, NKP 46;
when the NK cells are immunophenotyped, the antibodies used comprise:
antibodies against CD45, CD3, CD16, 56;
in the DC cell immunophenotyping, the antibodies used include:
anti-LIN, anti-HLA-DR, anti-CD 123, anti-CD 11c antibodies;
in the immunotyping of the T lymphocyte, the method of analyzing comprises:
cell surface markers CD45+ CD3+ SSC-represent total T lymphocytes;
the cell surface marker CD45+ CD3+ CD8+ represents killer T cells;
the cell surface marker CD45+ CD3+ CD4+ represents helper T cells;
the cell surface marker CD3+ γ δ + represents γ δ T cells;
the cell surface marker CD4+ CD25+ CD127+ represents regulatory CD4+ T cells;
the cell surface marker CD3+ CD8+ CD28+ represents cytotoxic T cells;
cell surface marker CD3+ CD8+ CD 28-represents suppressor T cells;
in the immunotyping of the B lymphocyte, the method for analyzing comprises:
cell surface markers CD3-CD19+ represent total B lymphocytes;
the cell surface marker CD3-CD19+ CD27+ represents memory B lymphocytes;
the cell surface marker CD3-CD19+ CD27-IgD + represents naive B lymphocytes;
the cell surface markers CD3-CD19+ CD24-CD38+ represent plasmablasts;
the activated and suppressed cell immunophenotyping, the method of analysis comprising:
the cell surface marker CD3+ CTLA-4+ represents CTLA-4@ T;
the cell surface marker CD3+ PD-1+ represents PD-1@ T;
the cell surface marker CD3+ NKG2D + represents NKG2D @ T;
the cell surface markers CD3-CD16, 56+ NKG2D + represent NKG2D @ NK;
the cell surface markers CD3-CD16, 56+ NKP46+ represent NKP46@ NK;
the NK cell immunophenotyping, the method of analyzing comprising:
cell surface markers CD3-CD16, 56+ represent NK cells;
cell surface markers CD3+ CD16, 56+ represent NK-T cells;
when the DC cell is used for immune typing, the analysis method comprises the following steps:
the cell surface marker LIN-HLA-DR + CD11c + represents myeloid DC;
the cell surface marker LIN-HLA-DR + CD123+ represents lymphoid lineage DC.
Preferably, the full-dimensional analysis method further comprises the step of counting the number of each type of immune cells in the sample to be tested.
More preferably, the step of counting the number of each type of immune cells in the test sample comprises:
and detecting the percentage of various immune cells in the sample to be detected, and obtaining the number of various immune cells in the sample to be detected after calculation through counting microspheres pre-coated in a flow tube.
The invention has the beneficial effects that:
the method realizes more comprehensive fine immunization typing of immune cells, requires few samples to be detected, is simple to operate, short in required time, high in accuracy and comprehensive in analysis, and can be widely applied to full-dimensional analysis of immune cells.
Drawings
FIG. 1 shows the results of typing of T lymphocytes under the CD3/CD8/CD45/CD4 antibody labeling; FIG. 1-A, FIG. 1-B, and FIG. 1-C are the results of typing of T lymphocyte subpopulations.
In the figure: r1 is total lymphocytes; r2 is a killer T lymphocyte; r3 is a T lymphocyte; r4 is a helper T lymphocyte.
FIG. 2 shows the results of typing of T lymphocytes under the CD3/γ δ antibody label; FIG. 2-A and FIG. 2-B show the results of typing of γ δ cells.
In the figure: r1 is a lymphocyte; r2 is γ δ T cell.
FIG. 3 shows the results of typing of T lymphocytes under the CD4/CD25/CD127 antibody labeling; FIG. 3-A, FIG. 3-B, FIG. 3-C are the results of typing regulatory CD4+ T cells.
In the figure: r1 is a lymphocyte; r3 is derived from R1; r4 is a regulatory CD4+ T cell.
FIG. 4 shows the results of typing of T lymphocytes under the CD3/CD8/CD28 antibody labeling; FIG. 4-A, FIG. 4-B, and FIG. 4-C are the results of typing cytotoxic/inhibitory T lymphocytes.
In the figure: r1 is a lymphocyte; r2 is a T lymphocyte; r3 is a cytotoxic T cell; r4 is an suppressor T cell.
FIG. 5B lymphocyte immunophenotyping results under the labeling of CD3/CD45/CD19/IgD/CD27/CD38/CD24 antibody; FIG. 5-A, FIG. 5-B, FIG. 5-C, FIG. 5-D, and FIG. 5-E are the results of typing of B lymphocytes.
In the figure: r1 is a lymphocyte; r2 is B and NK cells; r3 is total B lymphocytes; r6 is memory B lymphocyte; r7 is naive B lymphocytes; r8 is a plasmablast.
FIG. 6 typing results of activated/inhibited cells under CD3/CTLA-4/PD-1 antibody labeling; FIG. 6-A, FIG. 6-B, and FIG. 6-C are the results of typing inhibitory cells.
In the figure: r1 is a lymphocyte; r2 is CTLA-4@ T; r3 is PD-1@ T.
FIG. 7 typing results of activated/inhibited cells under CD3/CD16+56/NKG2D/NKP46 antibody labeling; FIGS. 7-A, 7-B, 7-C, 7-D and 7-E show the results of typing activated cells.
In the figure: r1 lymphocytes; r3 is an NK cell; r4 is NKG2D @ T cells; r5 is NKG2D @ NK; r6 is NKP46@ NK cell.
FIG. 8 shows the result of NK cell immunophenotyping classification under CD3/CD16+56 antibody labeling; FIG. 8-A, FIG. 8-B, and FIG. 8-C are the results of typing NK cells under different fluorescent labels. In the figure: r1 lymphocytes; r2 is B and NK cells; r4 is an NK cell; r5 is NK-T cells.
FIG. 9 shows the results of DC cell immunotyping classification under LIN/CD123/HLR-DR/CD11c antibody marker; FIGS. 9-A, 9-B, 9-C and 9-D are the results of typing DC cells.
In the figure: r1 is a nucleated cell; r2 was derived from R1, LIN expression negative; r3 is myeloid DC; r4 is lymphoid DC.
Detailed Description
The technical scheme of the invention is further described by combining the attached drawings:
the reagent and the raw material used in the method for the full-dimensional analysis of the immune cells provided by the invention can be purchased from the market.
The fluorescence labels FITC, PE, APC, Percp and BV510 used in the invention are all common fluorescence labels which can be purchased from the market, and antibodies of each fluorescence label can also be purchased from the market.
In order to make the technical solutions of the present invention better understood, the present invention is further described below with reference to the following embodiments:
experimental materials:
a sample to be tested: the anticoagulant external blood sample is derived from healthy volunteers and is external blood of normal people. [ sample requirements ]
1. And (4) collecting the whole blood to a scale marking position by using an EDTA-K2 anticoagulation tube vein.
2. The samples were stored at room temperature and were kept from shaking vigorously and were tested within 24 hours after blood collection.
3. The sample should be free of microbial contamination, lipemia, coagulation, etc. If the above situation exists, please note in the result report.
Other materials to be prepared:
1. a flow cytometer;
2. vacuum blood collection tubes (EDTA-K2 anticoagulation);
3. the range is suitable for 10ul, 100ul and 1000ul pipettors and disposable suckers;
4. a vortex oscillator;
5. hemolysin.
The experimental method comprises the following steps:
[ MEASUREMENT METHOD ]
1. Preheating for 20min after starting up the flow cytometer, and cleaning;
2. respectively adding 50ul of uniformly mixed anticoagulated blood into 10 counting micro-sphere tubes, adding a certain amount of cell immunophenotyping staining solution according to the requirement in the table 1, and standing for 20min in a dark place;
3. adding 500 μ l hemolysin, mixing with vortex oscillator, standing in dark for 20min, and allowing hemolysis to succeed when blood is transparent;
4. detecting on a computer to obtain various immune cell typing results;
the items to be examined and the amount of staining solution are shown in Table 1 below:
TABLE 1 test items and staining solution addition
The results of the tests are shown in FIGS. 1-9 for lymphocyte classification.
Finally, it should be noted that:
the method may be carried out and its application may be carried out by those skilled in the art with reference to the disclosure herein, and it is expressly intended that all such alternatives and modifications as would be apparent to those skilled in the art are deemed to be included within the invention. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application of the invention as described herein may be made and equivalents employed without departing from the spirit and scope of the invention.