WO2023115702A1 - 一种t淋巴细胞发育亚群免疫分型的方法和试剂盒 - Google Patents

一种t淋巴细胞发育亚群免疫分型的方法和试剂盒 Download PDF

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WO2023115702A1
WO2023115702A1 PCT/CN2022/078965 CN2022078965W WO2023115702A1 WO 2023115702 A1 WO2023115702 A1 WO 2023115702A1 CN 2022078965 W CN2022078965 W CN 2022078965W WO 2023115702 A1 WO2023115702 A1 WO 2023115702A1
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cells
antibody
cd45ro
memory
cell
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French (fr)
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赵耀
牛琳琳
谭冰倩
杨宇婷
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重庆医科大学附属儿童医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biotechnology, in particular to a method and a kit for immunotyping T lymphocyte development subgroups.
  • T lymphocytes thymus-dependent lymphocytes, referred to as T cells, are a type of cell population with extremely active functions, which mediate cellular immune responses. T cells are derived from hematopoietic stem cells and mature in the thymus. In peripheral blood, T cells account for about 60% of the total number of lymphocytes.
  • T cells can be divided into several subgroups: helper T cells (Th), cytotoxic T cells (Cytotoxic T cells, Tc), regulatory T cells (Regulatory T cells, Tr).
  • Th helper T cells
  • Tc cytotoxic T cells
  • Tr regulatory T cells
  • T cells express a receptor that has the potential to recognize multiple antigens from pathogens, tumors and the environment, and maintain immune memory and self-tolerance.
  • T cells are also considered a major driver of many inflammatory and autoimmune diseases, and T cells are critical for inducing protective immunity against pathogens or cancer.
  • T lymphocytes account for about 60% of peripheral blood lymphocytes, so CD3+ T cells also account for 60%.
  • CD3+ T cells also account for 60%.
  • the detected T lymphocytes often do not reach the total number of peripheral blood lymphocytes.
  • the positive rate of the monoclonal rosette test adopted by the technique is the lowest, about 50%, while the positive rate of the monoclonal antibody fluorescence microscopy method is increased.
  • flow cytometry is often used to detect T lymphocytes, but there is no further detection of the development of T lymphocytes. Summarizing the above T cell detection methods, the detection area is narrow, the operation is complicated, the sample needs a large amount, and it takes a long time. If more refined immunophenotyping of T cell development is to be performed, methods and quantitative analyzes that are simpler, time-saving, and require less sample volume are required.
  • the present invention provides a method and kit for immunotyping of T lymphocyte developmental subgroups, which can realize more comprehensive and fine immunophenotyping and quantitative analysis of T lymphocytes, with high efficiency and saving
  • the amount of samples to be tested is short, and it is more suitable for the typing and quantitative analysis of T lymphocyte developmental subgroups.
  • the invention provides a method for immunophenotyping of T lymphocyte developmental subgroups, comprising:
  • the antibody combination 1 includes:
  • the determination method of described cell type is:
  • Cell surface markers CD3 + CD4 + CD25 + CD127 - represent Treg cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 - represent helper naive T cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + represent helper stem cell memory T cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 + represent helper center memory T cells
  • Cell surface markers CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 + represent helper effector memory T cells
  • Cell surface markers CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + represent helper transitional memory T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 - represent cytotoxic naive T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 + represent cytotoxic stem cell memory T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO + CD28 + CD95 + represent cytotoxic central memory cells
  • Cell surface markers CD3 + CD8 + CD197 - CD45RO + CD28 - CD95 + represent cytotoxic effector memory T cells
  • Cell surface markers CD3 + CD8 + CD197 - CD45RO + CD28 + CD95 + represent cytotoxic transitional memory T cells.
  • the antibody used in flow cytometry can be an antibody homologous to the sample to be tested or an antibody non-homologous to the sample to be tested, as long as the antibody can bind to the cell surface markers in the sample to be tested
  • the substance can produce antigen-antibody specific binding reaction.
  • the surface markers for identification are expressed on the surface of the cell membrane.
  • fluorescein-labeled monoclonal antibodies as molecular probes, flow cytometry of these cell surface markers can be carried out. Then, the types, subtypes and functional characteristics of cells are analyzed.
  • the present invention provides a method for immunophenotyping of specific T cell developmental subgroups: when the cell surface markers are CD3 + CD4 + CD25 + CD127 - , Treg cells can be immunophenotyped ; When the cell surface markers are CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 - , helper naive T cells can be immunotyped; when the cell surface markers are CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + , can be used for Immunophenotyping of helper stem cell memory T cells; when cell surface markers are CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 + , helper center memory T cells can be immunotyped; cell surface markers are CD3 + CD4 + CD197 - When CD45RO + CD28 - CD95 + , helper effector memory T cells can be immunotyped; when cell surface markers are CD3 + CD4 + CD197 - CD45RO + CD
  • the present invention designs an antibody combination with optimal cell surface markers, and conducts a more comprehensive immune typing of T lymphocyte developmental subgroups through flow cytometry.
  • the method for developing immune typing of T lymphocyte subgroups provided by the present invention requires less samples to be tested, is simple to operate, has high accuracy, takes a short time, and can be used for immune typing of T lymphocyte subgroup development.
  • a fluorescence minus one control (FMO control) is also set.
  • the significance of setting the FMO control is to evaluate the interference of other fluorescent dyes on the target channel, which can be more accurately Determines the threshold for positive staining to help properly set the positive gate.
  • the control group is a fluorescence minus one control (FMO control).
  • FMO control fluorescence minus one control
  • the FMO control tube is to add all fluorescent antibodies except BV421-CD25.
  • the FMO control tube is to add all fluorescent antibodies except BV605-CD28.
  • the method for immunophenotyping T lymphocyte developmental subgroups provided by the present invention is specifically:
  • the sample to be tested with erythrocyte lysate, place the resulting mixture in a water bath at 36.5°C to 37.5°C for 6min to 8min, and centrifuge to obtain the first product, which is fluorescently labeled anti-CD45 antibody, anti-CD3 antibody, anti-CD4 Antibody, anti-CD8 antibody, anti-CD25 antibody, anti-CD127 antibody, anti-CD45RO antibody, anti-CD197 antibody, anti-CD28 antibody and anti-CD95 antibody, mixed with the first product, room temperature (ie 20 °C ⁇ Under the condition of 25°C), incubate for 15min-20min, wash, and perform flow cytometry detection on the machine to obtain detection data, and determine the cell type according to the detection data;
  • the first product which is fluorescently labeled anti-CD45 antibody, anti-CD3 antibody, anti-CD4 Antibody, anti-CD8 antibody, anti-CD25 antibody, anti-CD127 antibody, anti-CD45RO antibody, anti-CD197 antibody, anti-CD28 antibody and anti
  • the determination method of described cell type comprises:
  • Cell surface markers CD3 + CD4 + CD25 + CD127 - represent Treg cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 - represent helper naive T cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + represent helper stem cell memory T cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 + represent helper center memory T cells
  • Cell surface markers CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 + represent helper effector memory T cells
  • Cell surface markers CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + represent helper transitional memory T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 - represent cytotoxic naive T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 + represent cytotoxic stem cell memory T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO + CD28 + CD95 + represent cytotoxic central memory cells
  • Cell surface markers CD3 + CD8 + CD197 - CD45RO + CD28 - CD95 + represent cytotoxic effector memory T cells
  • Cell surface markers CD3 + CD8 + CD197 - CD45RO + CD28 + CD95 + represent cytotoxic transitional memory T cells
  • the fluorescent labels in each fluorescently labeled antibody are not limited by the present invention, and those skilled in the art can select appropriate fluorescent labels and control methods according to actual conditions.
  • the method for immunotyping T lymphocyte developmental subgroups provided by the present invention further includes the step of detecting the number of T lymphocytes in the sample to be tested.
  • the present invention provides a method for immunophenotyping T lymphocyte developmental subgroups, the step of detecting the number of T lymphocyte developmental groups in the sample to be tested includes:
  • the cell number of the T lymphocyte development subgroup in the test sample is obtained.
  • the total number of lymphocytes is multiplied by the percentage of T lymphocytes therein to obtain the number of T lymphocytes.
  • the absolute number of cells in each T cell subgroup can be obtained by multiplying the absolute number of T lymphocytes by the immunotype and relative statistics of each T cell development subgroup in the sample to be tested.
  • the step of detecting the percentage of T lymphocytes in the sample includes:
  • the antibody combination 2 used in the detection of the percentage of T lymphocytes in the sample to be tested includes:
  • Anti-CD45 antibody anti-CD3 antibody, anti-CD4 antibody and anti-CD8 antibody.
  • the anti-CD45 antibody in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, is fluorescently labeled as FITC.
  • the fluorescent label of the anti-CD3 antibody is APC-Cy7.
  • the fluorescent label of the anti-CD4 antibody is Percp-Cy5.5 or BV650.
  • the fluorescent label of the anti-CD8 antibody is BV510.
  • the step of detecting the percentage of T lymphocytes in the sample is a routine lymphocyte immunophenotyping and quantitative analysis method, which is not limited by the present invention, and those skilled in the art can according to The actual situation chooses the method of determining the percentage of T lymphocytes in the sample to be tested.
  • the method of detecting the total number of lymphocytes in the sample to be tested is counting by using a blood cell counter or a cell counter.
  • the method for detecting the total number of lymphocytes in the sample to be tested is a conventional method, which is not limited by the present invention, and those skilled in the art can choose to detect the sample to be tested according to the actual situation. The total number of lymphocytes in the method.
  • the present invention also provides a method for immunophenotyping of T lymphocyte developmental subgroups, which includes the step of immunotyping of T lymphocyte developmental subgroups provided by the present invention;
  • the methods for immunophenotyping of T lymphocyte developmental subsets include:
  • This antibody includes:
  • Methods for this analysis include:
  • Cell surface markers CD3 + CD4 + CD25 + CD127 - represent Treg cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 - represent helper naive T cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + represent helper stem cell memory T cells
  • Cell surface markers CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 + represent helper center memory T cells
  • Cell surface markers CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 + represent helper effector memory T cells
  • Cell surface markers CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + represent helper transitional memory T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 - represent cytotoxic naive T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 + represent cytotoxic stem cell memory T cells
  • Cell surface markers CD3 + CD8 + CD197 + CD45RO + CD28 + CD95 + represent cytotoxic central memory cells
  • Cell surface markers CD3 + CD8 + CD197 - CD45RO + CD28 - CD95 + represent cytotoxic effector memory T cells
  • Cell surface markers CD3 + CD8 + CD197 - CD45RO + CD28 + CD95 + represent cytotoxic transitional memory T cells
  • the present invention also provides a kit for immunophenotyping of T lymphocyte developmental subgroups, including the following different fluorescently labeled antibodies:
  • the fluorescently labeled antibody in the kit for immunophenotyping of T lymphocyte developmental subpopulations provided by the present invention, can be placed separately with the fluorescent marker and the antibody, and the two are coupled to obtain fluorescence when used. Labeled antibody; it can also be directly fluorescently labeled antibody, which can be used directly when used.
  • the kit for immunophenotyping of T lymphocyte developmental subpopulations includes fluorescent markers and antibodies:
  • This antibody includes:
  • the fluorescent labels include FITC, APC-Cy7, Percp-cy5.5, BV510, BV421, Alexa Fluor 700, BV785, PE, BV605, and APC, including but not limited to the above labels, which can be selected according to the actual situation and needs in the field .
  • the fluorescent label of the anti-CD45 antibody is FITC.
  • the fluorescent label of the anti-CD3 antibody is APC-Cy7.
  • the fluorescent label of the anti-CD4 antibody is Percp-Cy5.5 or BV650.
  • the fluorescent label of the anti-CD8 antibody is BV510.
  • the fluorescent label of the anti-CD25 antibody is BV421.
  • the fluorescent label of the anti-CD127 antibody is Alexa Fluor 700.
  • the fluorescent label of the anti-CD45RO antibody is BV785.
  • the fluorescent label of the anti-CD197 antibody is PE.
  • the fluorescent label of the anti-CD28 antibody is BV605.
  • the fluorescent label of the anti-CD95 antibody is APC.
  • the control group is a fluorescence minus one control (FMO control).
  • FMO control fluorescence minus one control
  • the FMO control tube is to add all fluorescent antibodies except BV421-CD25.
  • the FMO control tube is to add all fluorescent antibodies except BV605-CD28.
  • the fluorescent markers in each fluorescently labeled antibody are not limited by the present invention, and those skilled in the art can select appropriate fluorescent markers and corresponding controls according to the actual situation.
  • the accuracy of the target cells can be judged by the target cell grouping and the fluorescence intensity of the target cells; the target cell grouping is clear, and the target cell fluorescence intensity is accurate and high, indicating that the accuracy is high .
  • the invention provides a method and kit for immunotyping T lymphocyte developmental subgroups.
  • the method for immunophenotyping of T lymphocyte developmental subgroups provided by the present invention comprises: taking different fluorescently labeled antibodies, mixing them with the samples to be tested, and after incubation, detecting by flow cytometry to obtain detection data, and analyzing the obtained detection data
  • the antibodies include: anti-CD45 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD25 antibodies, anti-CD127 antibodies, anti-CD45RO antibodies, anti-CD197 antibodies, anti-CD28 antibodies and Antibodies against CD95.
  • Experimental results confirm that the present invention uses the optimal cell surface marker antibody combination to achieve a more comprehensive immune typing and quantitative analysis of T lymphocyte developmental subgroups through flow cytometry.
  • the method provided by the present invention requires a small amount of samples to be tested, simple operation, obvious clustering, and short time-consuming. In some embodiments of the present invention, the method provided by the present invention has good repeatability, obvious grouping and high accuracy, and can be widely used in immunophenotyping and quantitative analysis of T lymphocyte developmental subgroups.
  • Fig. 1 is the result of T lymphocyte classification in embodiment 1;
  • Fig. 2 is the typing result of T lymphocyte development subpopulation in embodiment 1;
  • Fig. 2-A, Fig. 2-B are the typing result of Treg cell;
  • Fig. 2-D, 2-E, 2-F are The typing results of helper T cell developmental subgroups;
  • 2-H, 2-I, 2-J are the typing results of cytotoxic T cell developmental subgroups;
  • Fig. 3 is the result of T lymphocyte classification in embodiment 2;
  • Fig. 4 is the typing result of T lymphocyte development subpopulation in embodiment 2; Wherein, Fig. 4-A, Fig. 4-B are the typing result of Treg cell; Fig. 4-D, 4-E, 4-F are The typing results of helper T cell developmental subgroups; 4-H, 4-I, 4-J are the typing results of cytotoxic T cell developmental subgroups;
  • Fig. 5 is the result of T lymphocyte classification in embodiment 3.
  • Fig. 6 is the typing result of T lymphocyte development subpopulation in embodiment 3; Wherein, Fig. 6-A, Fig. 6-B are the typing result of Treg cell; Fig. 6-D, 6-E, 6-F are The typing results of helper T cell developmental subgroups; 6-H, 6-I, 6-J are the typing results of cytotoxic T cell developmental subgroups;
  • Fig. 7 is the result of T lymphocyte classification in embodiment 4.
  • Fig. 8 is the typing result of T lymphocyte development subpopulation in embodiment 4; Wherein, Fig. 8-A, Fig. 8-B are the typing result of Treg cell; Fig. 8-D, 8-E, 8-F are The typing results of helper T cell developmental subgroups; 8-H, 8-I, and 8-J are the typing results of cytotoxic T cell developmental subgroups;
  • Fig. 9 is the result of T lymphocyte classification in embodiment 5.
  • Fig. 10 is the typing result of T lymphocyte development subpopulation in embodiment 5; Wherein, Fig. 10-A, Fig. 10-B are the typing result of Treg cell; Fig. 10-D, 10-E, 10-F are The typing results of helper T cell developmental subgroups; 10-H, 10-I, and 10-J are the typing results of cytotoxic T cell developmental subgroups.
  • the invention provides a method and kit for immunotyping T lymphocyte developmental subgroups.
  • Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • the reagents and raw materials used in the method and kit for immunotyping T lymphocyte developmental subgroups provided by the present invention can be purchased from the market.
  • the fluorescent labels FITC, APC-Cy7, Percp-cy5.5, BV510, BV421, Alexa Fluor 700, BV785, PE, BV605, APC, and BV650 used in the present invention are common fluorescent labels that can be purchased from the market. Fluorescently labeled antibodies are also commercially available.
  • Sample to be tested an anticoagulated peripheral blood sample, derived from a healthy volunteer (female, 36 years old), which is the peripheral blood of a normal person.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect T lymphocyte developmental subpopulations, and carry out immunophenotyping and quantitative analysis of T lymphocyte developmental subpopulations.
  • T-1 and T-2 are the test groups
  • T-1 and T-2 are the test groups
  • the relative number (percentage) and the absolute number of lymphocytes of each T lymphocyte development subgroup are obtained.
  • T lymphocyte immunophenotyping The results of T lymphocyte immunophenotyping are shown in Figure 1. It can be seen from the figure that the relative numbers (percentage) of T cells, Tc cells, Th cells, and Treg cells; according to the absolute number of lymphocytes and the relative numbers of each T cell subset (percentage), the absolute number of each T cell subset was calculated, and the specific experimental results are shown in Table 3.
  • T lymphocyte developmental subgroups The typing results of T lymphocyte developmental subgroups are shown in Figure 2-A, 2-B, 2-C, 2-D, 2-E, 2-F, 2-G, 2-H, 2-I, 2-J , as can be seen from the figure, the relative number (percentage) of various T cell developmental subgroups, the relative number is the percentage relative to the number of T cells; according to the absolute number of T lymphocytes, the relative number (percentage) of each cell subgroup , calculate the absolute number of each T lymphocyte development subgroup.
  • Treg cells 1. Regulatory T cells (Treg cells)
  • CD3 + CD4 + CD25 + CD127 - is the target group.
  • the target cells accounted for 18.86% of the CD4 + T cells measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of Treg cells was 116/ ⁇ L.
  • helper naive T cells ie CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 -
  • helper stem cell memory T cells ie TSCM, CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 +
  • the relative number was 2.62%, and the absolute number was 16 cells/ ⁇ L
  • the relative number of helper center memory T cells that is, TCM, CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 +
  • the relative number of helper effector memory T cells i.e.
  • CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 + is 3.16%, and the absolute number is 20 / ⁇ L; the relative number of helper transitional memory T cells (ie TTM, CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + ) was 30.93%, and the absolute number was 190/ ⁇ L.
  • cytotoxic naive T cells i.e. CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 -
  • CD8+T relative number 26.39%, absolute number 93/ ⁇ L
  • cytotoxic stem cell memory T cells TSCM, CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 +
  • the relative number is 3.15%, the absolute number is 11/ ⁇ L;
  • the relative number of cytotoxic effector memory T cells (i.e., TEM, CD3 + CD8 + CD197 - CD45RO + CD28 - CD95 + ) was 4.15%, and the absolute number was 15 cells/ ⁇ L; the cytotoxic transitional memory T cells The relative number of cells (ie TTM, CD3 + CD8 + CD197 - CD45RO + CD28 + CD95 + ) was 22.55%, and the absolute number was 80 cells/ ⁇ L.
  • Table 4 The relative number and absolute number of each cell subset in T lymphocytes of normal people
  • the method used in this case can successfully divide T lymphocytes into CD3 + T cells, CD3 + CD4 + cells, and CD3 + CD8 + cells, and perform quantitative analysis to obtain the relative number of each T lymphocyte and absolute numbers. From the results in Table 4, it can be seen that the relative numbers and absolute numbers of each cell subpopulation of the sample to be tested are within the reference range of normal people, indicating that the experimental results of the present invention are stable and accurate.
  • the method used in this case can accurately divide T lymphocytes Divided into Treg cells, helper naive T cells, helper stem cell memory T cells, helper center memory T cells, helper effector memory T cells, helper transitional memory T cells, cytotoxic naive T cells, cytotoxic stem cell memory T cells, cytotoxic center Memory T cells, cytotoxic effector memory T cells, and cytotoxic transitional memory T cells were quantitatively analyzed to obtain the relative and absolute numbers of each T lymphocyte developmental subgroup.
  • the relative number and absolute number of each T cell subset in the test sample are basically within the reference range of normal people, although the relative number of regulatory T cells increases, but its absolute value is within the range of normal people. within the reference range.
  • the relative number and absolute number of cytotoxic effect memory T cells were close to the reference value of normal people. It shows that the experimental results of the present invention are accurate and stable.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of T lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurately and clearly immunotype each T lymphocyte development subgroup.
  • Samples to be tested anticoagulated peripheral blood samples, derived from the venous blood samples of children (male, 14 years old) who came to our hospital (with signed informed consent), and were obtained from children with suspected neutrophil-mediated autoinflammation. peripheral blood.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect T lymphocyte developmental subpopulations, and carry out immunophenotyping and quantitative analysis of T lymphocyte developmental subpopulations.
  • antibodies to the flow tube FITC-labeled anti-CD45 antibody, APC-cy7-labeled anti-CD3 antibody, Percp-cy5.5-labeled anti-CD4 antibody, BV510-labeled anti-CD8 antibody, BV421-labeled anti-CD25 antibody, Alexa Fluor700-labeled anti-CD127 antibody, BV785-labeled anti-CD45RO antibody, PE-labeled anti-CD197 antibody, BV605-labeled anti-CD28 antibody, APC-labeled anti-CD95 antibody, after fully vortexing, incubate at room temperature in the dark for 20 minutes.
  • T lymphocyte immunophenotyping are shown in Figure 3. From the figure, it can be seen that the relative numbers (percentage) of T cells, Tc cells, Th cells, and Treg cells; according to the absolute number of lymphocytes and the relative numbers of each T cell subset (percentage), the absolute number of each T cell subset was calculated, and the specific experimental results are shown in Table 6.
  • Table 6 The cell surface markers used for immunophenotyping of each T cell subset, and the relative and absolute numbers of each T cell subset
  • T lymphocyte developmental subgroups The typing results of T lymphocyte developmental subgroups are shown in Figure 4-A, 4-B, 4-C, 4-D, 4-E, 4-F, 4-G, 4-H, 4-I, 4-J , as can be seen from the figure, the relative number (percentage) of various T cell developmental subgroups, the relative number is the percentage relative to the number of T cells; according to the absolute number of T lymphocytes, the relative number (percentage) of each cell subgroup , calculate the absolute number of each T lymphocyte development subgroup.
  • Treg cells 1. Regulatory T cells (Treg cells)
  • CD3 + CD4 + CD25 + CD127 - is the target group. It can be measured by the flow cytometer that the target cells account for 3.48% of the lymphocytes, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of Treg cells is 102/ ⁇ L.
  • helper naive T cells ie CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 -
  • helper stem cell memory T cells i.e. TSCM, CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 +
  • absolute number 63/ ⁇ L
  • helper center memory T cells i.e. TCM, CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 + ) is 5.75%, and the absolute number is 90 cells/ ⁇ L;
  • helper effector memory T cells ie TEM, CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 +
  • the absolute number is 2/ ⁇ L
  • helper transitional memory T cells That is, TTM, CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + ) relative number 8.59%, absolute number 135/ ⁇ L.
  • cytotoxic naive T cells i.e. CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 -
  • cytotoxic stem cell memory T cells TSCM, CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 +
  • the relative number of cytotoxic central memory T cells i.e. TCM, CD3 + CD8 + CD197 + CD45RO + CD28 + CD95 + ) is 0.10%, and the absolute number is 2 cells/ ⁇ L;
  • the method used in this case can successfully divide T lymphocytes into CD3 + T cells, CD3 + CD4 + cells and CD3 + CD8 + cells, and perform quantitative analysis to obtain the relative number of each T lymphocyte and absolute numbers.
  • the method used in this case can accurately divide T lymphocytes Divided into Treg cells, helper naive T cells, helper stem cell memory T cells, helper center memory T cells, helper effector memory T cells, helper transitional memory T cells, cytotoxic naive T cells, cytotoxic stem cell memory T cells, cytotoxic center Memory T cells, cytotoxic effector memory T cells, and cytotoxic transitional memory T cells were quantitatively analyzed to obtain the relative and absolute numbers of each T lymphocyte developmental subgroup.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of T lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurately and clearly immunotype each T lymphocyte development subgroup.
  • Samples to be tested anticoagulated peripheral blood samples, derived from the venous blood samples of children (male, 10 months old) who came to our hospital for treatment (informed consent has been signed), and peripheral blood of children with suspected liver failure.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect T lymphocyte developmental subpopulations, and carry out immunophenotyping and quantitative analysis of T lymphocyte developmental subpopulations.
  • antibodies to the flow tube FITC-labeled anti-CD45 antibody, APC-cy7-labeled anti-CD3 antibody, Percp-cy5.5-labeled anti-CD4 antibody, BV510-labeled anti-CD8 antibody, BV421-labeled anti-CD25 antibody, Alexa Fluor700-labeled anti-CD127 antibody, BV785-labeled anti-CD45RO antibody, PE-labeled anti-CD197 antibody, BV605-labeled anti-CD28 antibody, APC-labeled anti-CD95 antibody, after fully vortexing, incubate at room temperature in the dark for 20 minutes.
  • T lymphocyte immunophenotyping The results of T lymphocyte immunophenotyping are shown in Figure 5. It can be seen from the figure that the relative numbers (percentages) of T cells, Tc cells, Th cells, and Treg cells; according to the absolute number of lymphocytes and the relative numbers of each T cell subset (percentage), the absolute number of each T cell subset was calculated, and the specific experimental results are shown in Table 7.
  • Table 7 The cell surface markers used for immunophenotyping of each T cell subset, and the relative and absolute numbers of each T cell subset
  • T lymphocyte developmental subgroups The typing results of T lymphocyte developmental subgroups are shown in Figure 6-A, 6-B, 6-C, 6-D, 6-E, 6-F, 6-G, 6-H, 6-I, 6-J , as can be seen from the figure, the relative number (percentage) of various T cell developmental subgroups, the relative number is the percentage relative to the number of T cells; according to the absolute number of T lymphocytes, the relative number (percentage) of each cell subgroup , calculate the absolute number of each T lymphocyte development subgroup.
  • Treg cells 1. Regulatory T cells (Treg cells)
  • CD3 + CD4 + CD25 + CD127 - is the target group.
  • the target cells accounted for 2.04% of the lymphocytes as measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of Treg cells was 105/ ⁇ L.
  • helper naive T cells ie CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 - ) relative number 49.64%, absolute number 1298/ ⁇ L; helper stem cell memory T cells (ie TSCM, CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + ) relative number 0.44 %, absolute number 11/ ⁇ L;
  • helper effector memory T cells ie TEM, CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 +
  • the absolute number is 0.4 cells/ ⁇ L
  • helper transitional memory T cells That is, TTM, CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + ) relative number 2.09%, absolute number 55/ ⁇ L.
  • cytotoxic naive T cells i.e. CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 -
  • cytotoxic stem cell memory T cells TSCM, CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 +
  • the method used in this case can successfully divide T lymphocytes into CD3 + T cells, CD3 + CD4 + cells and CD3 + CD8 + cells, and perform quantitative analysis to obtain the relative number of each T lymphocyte and absolute numbers.
  • the method used in this case can accurately divide T lymphocytes Divided into Treg cells, helper naive T cells, helper stem cell memory T cells, helper center memory T cells, helper effector memory T cells, helper transitional memory T cells, cytotoxic naive T cells, cytotoxic stem cell memory T cells, cytotoxic center Memory T cells, cytotoxic effector memory T cells, and cytotoxic transitional memory T cells were quantitatively analyzed to obtain the relative and absolute numbers of each T lymphocyte developmental subgroup.
  • helper center memory T cells helper effector memory T cells
  • cytotoxic naive T cells cytotoxic center memory T cells
  • cytotoxic effector memory T cells in the samples to be tested were all lower than Normal value, suggesting that the child's T cell development is abnormal.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of T lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurately and clearly immunotype each T lymphocyte development subgroup.
  • Samples to be tested anticoagulated peripheral blood samples, derived from the venous blood samples of children (male, 4 months old) who came to our hospital for treatment (informed consent has been signed), and are peripheral blood of children with biliary atresia to be diagnosed.
  • Red blood cell lysate (cat RT122-02) was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the remaining 100 ⁇ L was used to detect T lymphocyte developmental subpopulations, and carry out immunophenotyping and quantitative analysis of T lymphocyte developmental subpopulations.
  • antibodies to the flow tube FITC-labeled anti-CD45 antibody, APC-cy7-labeled anti-CD3 antibody, Percp-cy5.5-labeled anti-CD4 antibody, BV510-labeled anti-CD8 antibody, BV421-labeled anti-CD25 antibody, Alexa Fluor700-labeled anti-CD127 antibody, BV785-labeled anti-CD45RO antibody, PE-labeled anti-CD197 antibody, BV605-labeled anti-CD28 antibody, APC-labeled anti-CD95 antibody, after fully vortexing, incubate at room temperature in the dark for 20 minutes.
  • T lymphocyte immunophenotyping are shown in Figure 7, as can be seen from the figure, the relative number (percentage) of T cells, Tc cells, Th cells, and Treg cells; according to the absolute number of lymphocytes and the relative numbers of each T cell subset (percentage), the absolute number of each T cell subset was calculated, and the specific experimental results are shown in Table 8.
  • Table 8 The cell surface markers used for immunophenotyping of each T cell subset, and the relative and absolute numbers of each T cell subset
  • T lymphocyte developmental subgroups The typing results of T lymphocyte developmental subgroups are shown in Figure 8-A, 8-B, 8-C, 8-D, 8-E, 8-F, 8-G, 8-H, 8-I, 8-J , as can be seen from the figure, the relative number (percentage) of various T cell developmental subgroups, the relative number is the percentage relative to the number of T cells; according to the absolute number of T lymphocytes, the relative number (percentage) of each cell subgroup , calculate the absolute number of each T lymphocyte development subgroup.
  • Treg cells 1. Regulatory T cells (Treg cells)
  • CD3 + CD4 + CD25 + CD127 - is the target group.
  • the target cells accounted for 0.96% of the lymphocytes can be measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of Treg cells is 70/ ⁇ L.
  • helper naive T cells i.e. CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 -
  • helper stem cell memory T cells ie TSCM, CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + ) relative number 0.71 %, the absolute number is 15/ ⁇ L;
  • the relative number of helper center memory T cells (ie, TCM, CD3 + CD4 + CD197 + CD45RO + CD28 + CD95 + ) is 3.65%, and the absolute number is 75 cells/ ⁇ L;
  • helper effector memory T cells ie TEM, CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 +
  • the absolute number was 30 cells/ ⁇ L
  • helper transitional memory T cells That is, TTM, CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + ) relative number 1.76%, absolute number 36/ ⁇ L.
  • cytotoxic naive T cells i.e. CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 -
  • cytotoxic stem cell memory T cells TSCM, CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 +
  • the method used in this case can successfully divide T lymphocytes into CD3 + T cells, CD3 + CD4 + cells and CD3 + CD8 + cells, and perform quantitative analysis to obtain the relative number of each T lymphocyte and absolute numbers.
  • the method used in this case can accurately divide T lymphocytes Divided into Treg cells, helper naive T cells, helper stem cell memory T cells, helper center memory T cells, helper effector memory T cells, helper transitional memory T cells, cytotoxic naive T cells, cytotoxic stem cell memory T cells, cytotoxic center Memory T cells, cytotoxic effector memory T cells, and cytotoxic transitional memory T cells were quantitatively analyzed to obtain the relative and absolute numbers of each T lymphocyte developmental subgroup.
  • helper center memory T cells helper effector memory T cells
  • cytotoxic naive T cells cytotoxic center memory T cells
  • cytotoxic effector memory T cells in the samples to be tested were all lower than Normal value, suggesting that the child's T cell development is abnormal.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of T lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurately and clearly immunotype each T lymphocyte development subgroup.
  • Sample to be tested an anticoagulated peripheral blood sample, derived from a healthy volunteer (female, 26 years old), which is the peripheral blood of a normal person.
  • Antibodies, anti-CD45RO (BV785), anti-CD197 (PE), anti-CD28 (BV605) and anti-CD95 (APC) were purchased from BioLegend.
  • Lymphocyte separation medium was purchased from Dakowei Biotechnology Co., Ltd.
  • peripheral blood mononuclear cells PBMCs
  • T lymphocyte developmental subgroups T lymphocyte developmental subgroups, and perform immunotype and quantitative analysis of T lymphocyte developmental subgroups.
  • lymphocyte separation medium was added to the diluted peripheral blood, and centrifuged at 800g for 20min. After centrifugation, the buffy coat in the middle was sucked, which was PBMC.
  • FITC-labeled anti-CD45 antibody Take 1 ⁇ 106 flow tube and add antibodies: FITC-labeled anti-CD45 antibody, APC-cy7-labeled anti-CD3 antibody, BV650-labeled anti-CD4 antibody, BV510-labeled anti-CD8 antibody, BV421-labeled anti-CD25 antibody, Alexa Fluor700-labeled anti-CD127 antibody, BV785-labeled anti-CD45RO antibody, PE-labeled anti-CD197 antibody, BV605-labeled anti-CD28 antibody, APC-labeled anti-CD95 antibody, after fully vortexing, incubate at room temperature in the dark for 20 minutes.
  • T lymphocyte immunophenotyping results are shown in Figure 9, as can be seen from the figure, the relative number (percentage) of T cells, Tc cells, Th cells, and Treg cells; according to the absolute number of lymphocytes and the relative number of each T cell subset (percentage), the absolute number of each T cell subset was calculated, and the specific experimental results are shown in Table 9.
  • Table 9 Cell surface markers used for immunophenotyping of each T cell subset, relative and absolute numbers of each T cell subset
  • T lymphocyte development subgroups The typing results of T lymphocyte development subgroups are shown in Figure 10-A, 10-B, 10-C, 10-D, 10-E, 10-F, 10-G, 10-H, 10-I, 10-J , as can be seen from the figure, the relative number (percentage) of various T cell developmental subgroups, the relative number is the percentage relative to the number of T cells; according to the absolute number of T lymphocytes, the relative number (percentage) of each cell subgroup , calculate the absolute number of each T lymphocyte development subgroup.
  • Treg cells 1. Regulatory T cells (Treg cells)
  • CD3 + CD4 + CD25 + CD127 - is the target group.
  • the target cells accounted for 4.12% of the lymphocytes as measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of Treg cells was 25/ ⁇ L.
  • helper naive T cells ie CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 - ) relative number 14.07%, absolute number 87/ ⁇ L; helper stem cell memory T cells (i.e. TSCM, CD3 + CD4 + CD197 + CD45RO - CD28 + CD95 + ) relative number 2.47 %, the absolute number is 15/ ⁇ L;
  • helper effector memory T cells ie TEM, CD3 + CD4 + CD197 - CD45RO + CD28 - CD95 +
  • the absolute number is 2/ ⁇ L
  • helper transitional memory T cells That is, TTM, CD3 + CD4 + CD197 - CD45RO + CD28 + CD95 + ) relative number 14.87%, absolute number 92/ ⁇ L.
  • cytotoxic naive T cells i.e. CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 -
  • cytotoxic stem cell memory T cells TSCM, CD3 + CD8 + CD197 + CD45RO - CD28 + CD95 +
  • the absolute number is 15/ ⁇ L
  • the method used in this case can successfully divide T lymphocytes into CD3+T cells, CD3+CD4+ cells and CD3+CD8+ cells, and perform quantitative analysis to obtain the relative number and absolute number of each T lymphocyte number.
  • the method used in this case can accurately divide T lymphocytes Divided into Treg cells, helper naive T cells, helper stem cell memory T cells, helper center memory T cells, helper effector memory T cells, helper transitional memory T cells, cytotoxic naive T cells, cytotoxic stem cell memory T cells, cytotoxic center Memory T cells, cytotoxic effector memory T cells, and cytotoxic transitional memory T cells were quantitatively analyzed to obtain the relative and absolute numbers of each T lymphocyte developmental subgroup. It can be seen from Table 9 that the relative numbers and absolute numbers of each T cell subsets in the samples to be tested are basically at or close to the reference values of normal people. It shows that the experimental results of the present invention are accurate and stable.
  • the method provided by the present invention can realize the immunophenotyping of the developmental subgroups of T lymphocytes in the samples to be tested by using a small amount of samples to be tested, and count the content of each cell subgroup, and the method can Accurately and clearly immunotype each T lymphocyte development subgroup.

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Abstract

一种T淋巴细胞发育亚群免疫分型的方法和试剂盒。T淋巴细胞发育亚群免疫分型的方法包括：取不同荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据；抗体包括：抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD28的抗体、抗CD197的抗体和抗CD95的抗体。实现了对T淋巴细胞发育亚群进行更加全面的免疫分型，所需待测样品少，操作简单、所需时间短、准确性高。

Description

一种T淋巴细胞发育亚群免疫分型的方法和试剂盒
本申请要求于2021年12月22日提交中国专利局、申请号为202111584011.5、发明名称为“一种T淋巴细胞发育亚群免疫分型的方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，尤其涉及一种T淋巴细胞发育亚群免疫分型的方法和试剂盒。
背景技术
T淋巴细胞即胸腺依赖性淋巴细胞，简称T细胞，是一类功能极为活跃的细胞群体，其介导细胞免疫应答。T细胞来源于造血干细胞，在胸腺中发育成熟。外周血中，T细胞约占淋巴细胞总数的60％左右。
按照免疫应答中的功能不同，可以将T细胞分成若干亚群：辅助性T细胞(Helper T cells,Th)、细胞毒性T细胞(Cytotoxic T cells,Tc)、调节性T细胞(Regulatory T cells，Tr)。随着科学技术的进步，根据不同的表面标记可以定义更加精细的T细胞亚群的表型：初始T细胞、记忆T细胞和效应T细胞。免疫应答、体内平衡和记忆的建立和维持都依赖于T细胞。T细胞表达一种受体，具有从病原体、肿瘤和环境中识别多种抗原的潜能，并保持免疫记忆和自我耐受。T细胞也被认为是许多炎症和自身免疫性疾病的主要驱动因素，T细胞对于诱导对病原体或癌症的保护性免疫至关重要。
血液样品中的T淋巴细胞的亚群与机体的免疫***的关系至关重要。T淋巴细胞在外周血淋巴细胞中约占60％左右，因此CD3+T细胞也相应为60％。但是，由于技术原因，检测的T淋巴细胞常达不到外周血淋巴细胞的总数。采用的技术单克隆玫瑰花结实验的阳性率最低，为50％左右，而采用单克隆抗体荧光显微镜法阳性率有所升高。目前，常采用流式细胞计 数仪检测T淋巴细胞，但是，没有对T淋巴细胞的发育情况进行进一步的检测。综述上述T细胞检测方法，检测面窄，操作复杂，样品需要量大，耗时较长。如果要对T细胞发育进行更加精细的免疫分型，需要采用更为简单、省时和样品量需求小的方法和定量分析。
发明内容
有鉴于此，本发明提供了一种T淋巴细胞发育亚群免疫分型的方法和试剂盒，该方法可实现T淋巴细胞更全面、精细地免疫分型和定量分析，效率高，且节约了待测样品的用量，用时短，更适合T淋巴细胞发育亚群的分型和定量分析。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种T淋巴细胞发育亚群免疫分型的方法，包括：
取不同荧光标记的抗体组合1，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，根据检测数据判定细胞型别；
所述抗体组合1包括：
抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体；
所述细胞型别的判定方法为：
细胞表面标志CD3 +CD4 +CD25 +CD127 -代表Treg细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -代表辅助初始T细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +代表辅助干细胞记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +代表辅助中心记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +代表辅助效应记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +代表辅助过渡记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -代表细胞毒性初始T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +代表细胞毒性干细胞记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +代表细胞毒性中心记忆细胞；
细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +代表细胞毒性效应记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +代表细胞毒性过渡记忆T细胞。
在本发明中，流式细胞术中所用到的抗体可以为与待测样品同源的抗体或也可以为与待测样品非同源的抗体，只要该抗体能够与待测样品中细胞表面标志物产生抗原-抗体特异性结合反应即可。
在本发明中，“+”代表阳性，即表示该抗原在细胞表面有表达；
“++”代表强阳性，即表示该抗原在细胞表面高表达；
“-”代表阴性，即表示该抗原在细胞表面不表达。
淋巴细胞在各自正常分化成熟的不同阶段及活化过程中，其细胞膜表面均表达可供鉴别的表面标志，利用荧光素标记的单克隆抗体作为分子探针，可对这些细胞表面标志进行流式细胞术检测，进而对细胞的种类、亚类以及功能特性进行分析。本发明经过大量创造性的研究和试验验证，提供了一种针对的T细胞发育亚群免疫分型的方法：细胞表面标志为CD3 +CD4 +CD25 +CD127 -时，可对Treg细胞进行免疫分型；细胞表面标志为CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -时，可对辅助初始T细胞进行免疫分型；细胞表面标志为CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +时，可对辅助干细胞记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +时，可对辅助中心记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +时， 可对辅助效应记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +时，可对辅助过渡记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -时，可对细胞毒性初始T细胞进行免疫分型；细胞表面标志为CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +时，可对细胞毒性干细胞记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +时，可对细胞毒性中心记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +时，可对细胞毒性效应记忆T细胞进行免疫分型；细胞表面标志为CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +时，可对细胞毒性过渡记忆T细胞进行免疫分型。本发明根据对各个细胞所对应的细胞表面标志，设计获得了最优的细胞表面标志的抗体组合，通过流式细胞术，对T淋巴细胞发育亚群进行了更为全面的免疫分型。本发明提供的T淋巴细胞亚群发育免疫分型的方法所需待测样品少、操作简单、准确性高，用时短、能够用于T淋巴细胞发育亚群的免疫分型。
优选地，本发明提供T淋巴细胞发育亚群的免疫分型方法中，还设置荧光减一对照(FMO对照)，设置FMO对照的意义在于评估其他荧光染料对目的通道的干扰，能较为准确地确定阳性染色的阈值，帮助正确设置阳性门。
在本发明的实施例中，本发明提供的T淋巴细胞发育亚群的免疫分型的方法中，对照组采用的是荧光减一对照(FMO对照)。当检测BV421-CD25时，FMO对照管就是加入除去BV421-CD25以外的所有荧光抗体。当检测BV605-CD28时，FMO对照管就是加入除去BV605-CD28以外的所有荧光抗体。
本发明的实施例中，本发明提供的T淋巴细胞发育亚群免疫分型的方法，具体为：
取待测样品与红细胞裂解液混合，并将所得混合液至于36.5℃～37.5℃的水浴6min～8min，离心，得第一产品，取荧光标记的抗CD45 的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体，与第一产品混合，室温(即20℃～25℃)条件下，孵育15min～20min，洗涤，流式上机检测，得到检测数据，根据检测数据判定细胞型别；
所述细胞型别的判定方法包括：
细胞表面标志CD3 +CD4 +CD25 +CD127 -代表Treg细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -代表辅助初始T细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +代表辅助干细胞记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +代表辅助中心记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +代表辅助效应记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +代表辅助过渡记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -代表细胞毒性初始T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +代表细胞毒性干细胞记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +代表细胞毒性中心记忆细胞；
细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +代表细胞毒性效应记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +代表细胞毒性过渡记忆T细胞；
在本发明中，本发明提供的方法中，各个荧光标记抗体中的荧光标记不受本发明的限制，本领域的技术人员可以根据实际情况选择合适的荧光标记和对照方法。
本发明提供的T淋巴细胞发育亚群的免疫分型方法中，还包括检测待测样品中T淋巴细胞数目的步骤。
在本发明的实施例中，本发明提供T淋巴细胞发育亚群免疫分型的方法中，检测待测样品中T淋巴细胞发育群数目的步骤包括：
检测待测样品中淋巴细胞的总数；
检测待测样品中T淋巴细胞发育亚群占所述淋巴细胞的百分比；
通过计算，获得待测样品中T淋巴细胞发育亚群的细胞数目。
本发明提供的方法中，检测样品中T淋巴细胞的数目中，淋巴细胞总数乘以其中T淋巴细胞的百分比，即得T淋巴细胞的数目。在本发明中，经过对待测样品中各T细胞发育亚群的免疫分型和相对数统计，再乘以T淋巴细胞的绝对数即可获得每个T细胞亚群的细胞的绝对数。
在本发明的实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤，包括：
取不同的荧光标记的抗体组合2，与所述待测样品混合，孵育后，经流式细胞术检测，得检测数据，分析检测数据，计算待测样品中T淋巴细胞的百分比；
其中，所述检测待测样品中T淋巴细胞的百分比中所用的抗体组合2包括：
抗CD45的抗体、抗CD3的抗体、抗CD4的抗体和抗CD8的抗体。
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD45的抗体荧光标记为FITC。
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD3的抗体的荧光标记为APC-Cy7。
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD4的抗体的荧光标记为Percp-Cy5.5或BV650。
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD8的抗体的荧光标记为BV510。
在本发明中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤为常规的淋巴细胞免疫分型和定量分析方法，该方法不受本发明的限制，本领域技术人员可以根据实际情况选择测定待测样品中T淋巴细胞的百分比的方法。
在本发明的实施例中，本发明提供的方法中，检测待测样品中淋巴细胞的总数的方法为采用血细胞计数仪或细胞计数仪进行计数。在本发明中，本发明提供的方法中，检测待测样品中淋巴细胞的总数的方法为常规的方法，该方法不受本发明的限制，本领域技术人员可以根据实际情况选择检测待测样品中淋巴细胞的总数的方法。
本发明还提供了一种T淋巴细胞发育亚群免疫分型的方法，其包括本发明提供的T淋巴细胞发育亚群免疫分型的步骤；
该T淋巴细胞发育亚群免疫分型的方法包括：
取不同的荧光标记的抗体，与待测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所述检测数据；
该抗体包括：
抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体；
该分析的方法包括：
细胞表面标志CD3 +CD4 +CD25 +CD127 -代表Treg细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -代表辅助初始T细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +代表辅助干细胞记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +代表辅助中心记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +代表辅助效应记忆T细胞；
细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +代表辅助过渡记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -代表细胞毒性初始T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +代表细胞毒性干细胞记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +代表细胞毒性中心记忆细胞；
细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +代表细胞毒性效应记忆T细胞；
细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +代表细胞毒性过渡记忆T细胞；
本发明还提供了一种用于T淋巴细胞发育亚群的免疫分型的试剂盒，包括如下不同的荧光标记的抗体：
抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体。
在本发明中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，荧光标记的抗体，可以是荧光标记物与抗体单独放置，在使用时将两者偶联获得荧光标记的抗体；也可以是直接荧光标记的抗体，在使用时，直接使用即可。
在本发明的一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒包括荧光标记物和抗体：
该抗体包括：
抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体。
所述荧光标记包括FITC、APC-Cy7、Percp-cy5.5、BV510、BV421、Alexa Fluor 700、BV785、PE、BV605和APC，包括但不仅限于以上标记，本领域可根据实际情况和需要进行选择。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD45的抗体的荧光标记为FITC。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD3的抗体的荧光标记为APC-Cy7。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD4的抗体的荧光标记为Percp-Cy5.5或BV650。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD8的抗体的荧光标记为BV510。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD25的抗体的荧光标记为BV421。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD127的抗体的荧光标记为Alexa Fluor 700。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD45RO的抗体的荧光标记为BV785。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD197的抗体的荧光标记为PE。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD28的抗体的荧光标记为BV605。
在本发明一些实施例中，本发明提供的用于T淋巴细胞发育亚群免疫分型的试剂盒中，抗CD95的抗体的荧光标记为APC。
在本发明的实施例中，本发明提供的T淋巴细胞发育亚群的免疫分型的方法中，对照组采用的是荧光减一对照(FMO对照)。当检测BV421-CD25时，FMO对照管就是加入除去BV421-CD25以外的所有荧光抗体。当检测BV605-CD28时，FMO对照管就是加入除去BV605-CD28以外的所有荧光抗体。
在本发明中，本发明提供的试剂盒中，各个荧光标记的抗体中的荧光 标记物不受本发明的限制，本领域技术人员可以根据实际情况选择合适的荧光标记物，以及对应的对照。
在本发明中，流式细胞术检测过程中，通过目标细胞分群和目标细胞的荧光强度可以对目标细胞进行准确性的判断；目标细胞分群清楚、目标细胞荧光强度准确高，则表示准确性高。
本发明提供了一种T淋巴细胞发育亚群免疫分型的方法和试剂盒。本发明提供的T淋巴细胞发育亚群免疫分型的方法，包括：取不同的荧光标记的抗体，与待测样品混合，孵育后，经流式细胞术检测，得到检测数据，分析所得检测数据；该抗体包括：抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体。实验结果证实，本发明采用最优的细胞表面标志的抗体组合，通过流式细胞术，实现了对T淋巴细胞发育亚群进行了更加全面的免疫分型和定量分析。在本发明的一些实施例中，本发明提供的方法，所需待测样本量少、操作简单、分群明显、用时短。在本发明的一些实施例中，本发明提供的方法重复性好、分群明显、准确性高、可广泛用于T淋巴细胞发育亚群免疫分型和定量分析。
附图说明
图1为实施例1中T淋巴细胞分类的结果；
图2为实施例1中T淋巴细胞发育亚群的分型结果；其中，图2-A、图2-B为Treg细胞的分型结果；图2-D、2-E、2-F为辅助T细胞发育亚群分型结果；2-H、2-I、2-J为细胞毒性T细胞发育亚群的分型结果；
图3为实施例2中T淋巴细胞分类的结果；
图4为实施例2中T淋巴细胞发育亚群的分型结果；其中，图4-A、图4-B为Treg细胞的分型结果；图4-D、4-E、4-F为辅助T细胞发育亚群分型结果；4-H、4-I、4-J为细胞毒性T细胞发育亚群的分型结果；
图5为实施例3中T淋巴细胞分类的结果；
图6为实施例3中T淋巴细胞发育亚群的分型结果；其中，图6-A、图6-B为Treg细胞的分型结果；图6-D、6-E、6-F为辅助T细胞发育亚群分型结果；6-H、6-I、6-J为细胞毒性T细胞发育亚群的分型结果；
图7为实施例4中T淋巴细胞分类的结果；
图8为实施例4中T淋巴细胞发育亚群的分型结果；其中，图8-A、图8-B为Treg细胞的分型结果；图8-D、8-E、8-F为辅助T细胞发育亚群分型结果；8-H、8-I、8-J为细胞毒性T细胞发育亚群的分型结果；
图9为实施例5中T淋巴细胞分类的结果；
图10为实施例5中T淋巴细胞发育亚群的分型结果；其中，图10-A、图10-B为Treg细胞的分型结果；图10-D、10-E、10-F为辅助T细胞发育亚群分型结果；10-H、10-I、10-J为细胞毒性T细胞发育亚群的分型结果。
具体实施方式
本发明提供了一种T淋巴细胞发育亚群免疫分型的方法和试剂盒。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的一种T淋巴细胞发育亚群免疫分型的方法和试剂盒中所用到的试剂和原料均可由市场购得。
本发明中用到的荧光标记FITC、APC-Cy7、Percp-cy5.5、BV510、BV421、Alexa Fluor 700、BV785、PE、BV605、APC、BV650均为常见的荧光标记可以由市场购得，各个荧光标记的抗体也可以由市场购得。
表1 中英文对照表
Figure PCTCN2022078965-appb-000001
Figure PCTCN2022078965-appb-000002
下面结合实施例，进一步阐述本发明：
实施例1 T淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于健康志愿者(女，36岁)，为正常人的外周血。
荧光标记FITC的抗CD45抗体、抗CD3(APC-Cy7)的抗体、抗CD4(Percp-cy5.5)的抗体、抗CD8(BV510)的抗体、抗CD25(BV421)的抗体、抗CD127(Alexa Flour 700)的抗体、抗CD45RO(BV785)的抗体、抗CD197(PE)的抗体、抗CD28(BV605)的抗体和抗CD95(APC)的抗体购买于BioLegend。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为2.58×10 9个/L。
剩下100μL用于检测T淋巴细胞发育亚群，进行T淋巴细胞发育亚群免疫分型和定量分析。
T淋巴细胞发育亚群免疫分型
1、取两根流式管，分别标记为T-1、T-2，其中T-1为对照组，T-2为待检测组，向T-1、T-2两根流式管中各加入50μL待测样品，然后分别向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
2、按照表2向T-1、T-2流式管中加入以下抗体，充分涡旋后，室温避光孵育20min；
表2 各个流式管中加入的抗体的类别和加入的体积
Figure PCTCN2022078965-appb-000003
Figure PCTCN2022078965-appb-000004
3、各加入1mL PBS，500g，离心5min，洗涤一次后，加入200μLPBS垂悬，流式上机并分析结果。
结果分析：
通过以上操作，得到各T淋巴细胞发育亚群相对数(百分数)和淋巴细胞绝对数。各T淋巴细胞发育亚群相对数(百分数)×淋巴细胞绝对数，即得到T淋巴细胞各亚群绝对数。
T淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞免疫分型结果见图1，从图中可知，T细胞、Tc细胞、Th细胞、Treg细胞的相对数(百分数)；根据淋巴细胞的绝对数、和各个T细胞亚群的相对数(百分数)，计算获得各个T细胞亚群的绝对数，具体实验结果见表3。
表3 各个T细胞亚群免疫分型所用的细胞表面标志、
各个T细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000005
T细胞发育亚群免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞发育亚群分型结果见图2-A、2-B、2-C、2-D、2-E、2-F、2-G、2-H、2-I、2-J，从图中可知，各类T细胞发育亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据T淋巴细胞的绝对数、各个细胞亚群的相对数(百分数)，计算获得各个T淋巴细胞发育亚群的绝对数。
1、调节性T细胞(Treg细胞)
由图2-B可知，CD3 +CD4 +CD25 +CD127 -即为目标群。由流式细胞仪可测得目标细胞占CD4 +T细胞的18.86％，可由血细胞计数仪测得的淋巴细胞绝对数算出，Treg细胞的绝对数为116个/μL。
1、辅助T细胞亚群
1)从图2-D可得，辅助初始T细胞(即
Figure PCTCN2022078965-appb-000006
CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -)占CD4 +T细胞为相对数26.67％，绝对数164个/μL；辅助干细胞记忆T细胞(即TSCM，CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +)相对数2.62％，绝对数16个/μL；2)从图2-E可得，辅助中心记忆T细胞(即TCM，CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +)相对数23.67％，绝对数146个/μL；3)从图2-F可得，辅助效应记忆T细胞(即TEM，CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +)相对数3.16％，绝对数20个/μL；辅助过渡记忆T细胞(即TTM，CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +)相对数30.93％，绝对数190个/μL。
2、细胞毒性T细胞亚群
1)从图2-H可得，细胞毒性初始T细胞(即
Figure PCTCN2022078965-appb-000007
CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -)CD8+T相对数26.39％，绝对数93个/μL；细胞毒性干细胞记忆T细胞(TSCM，CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +)相对数3.15％，绝对数11个/μL；
2)从图2-I可得，细胞毒性中心记忆T细胞(即TCM，CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +)相对数4.25％，绝对数15个/μL；
3)从图2-J可得，细胞毒性效应记忆T细胞(即TEM，CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +)相对数4.15％，绝对数15个/μL；细胞毒性过渡记忆T细胞(即TTM，CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +)相对数22.55％，绝对数80个/μL。
表4 正常人的T淋巴细胞中各个细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000008
其中，“*”代表目前研究报道的实验结果。
参考文献：Anders H.Kverneland et al..Age and Gender Leucocytes Variances and References Values Generated Using the Standardized ONE-Study Protocol.2016,Cytometry PartA,89A:543-564。
表5 正常儿童的T淋巴细胞中各个细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000009
Figure PCTCN2022078965-appb-000010
其中，“*”代表目前研究报道的实验结果，参考文献：Yuan Ding et al..Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018,J ALLERGY CLIN IMMUNOL.,1-4；Marina Garcia-Prat et al..Extended Immunophenotyping Reference Values in a Healthy Pediatric Population.2018,Cytometry Part B(Clinical Cytometry),DOI:10.1002/cyto.b.21728。
“-”代表未从文献查到Tregs占T细胞的绝对数。
根据图1可知，本案例所用的方法能够成功的将T淋巴细胞分为CD3 +T细胞、CD3 +CD4 +细胞和CD3 +CD8 +细胞，并且进行定量分析，获得了各个T淋巴细胞的相对数和绝对数。从表4中结果可知，待测样本的各个细胞亚群的相对数和绝对数都在正常人的参考范围内，说明本发明的实验结果稳定、准确。
根据图2-B、图2-D、图2-E、图2-F、图2-H、图2-I、图2-J可得，本案例所用的方法能够准确的将T淋巴细胞分为Treg细胞、辅助初始T细胞、辅助干细胞记忆T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、辅助过渡记忆T细胞、细胞毒性初始T细胞、细胞毒性干细胞记忆T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞、细胞毒性过渡记忆T细胞，进行定量分析，获得了各个T淋巴细胞发育亚群的相对数和绝对数。从表3中可知，待测样品的各个T细胞亚群的相对数和绝对数基本都在正常人的参考范围内，虽然调节性T细胞的的相对数增加，但是其绝对值在正常人的参考范围内。细胞毒性效应记忆T细胞的相对数和绝对数接近正常人的参考值。说明本发明实验结果准确和稳定。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的T淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个T淋巴细胞发育亚群进行免疫分型。
实施例2 T淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于来我院就诊患儿(男，14岁)静脉血样品(已签知情同意书)，为拟诊中性粒细胞介导的自身炎症患儿的外周血。
荧光标记FITC的抗CD45抗体、抗CD3(APC-Cy7)的抗体、抗CD4(Percp-cy5.5)的抗体、抗CD8(BV510)的抗体、抗CD25(BV421)的抗体、抗CD127(Alexa Flour 700)的抗体、抗CD45RO(BV785)的抗体、抗CD197(PE)的抗体、抗CD28(BV605)的抗体和抗CD95(APC)的抗体购买于BioLegend。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为2.92×10 9个/L。
剩下100μL用于检测T淋巴细胞发育亚群，进行T淋巴细胞发育亚群免疫分型和定量分析。
T淋巴细胞发育亚群免疫分型
与实施例1中记载的T淋巴细胞分类方法相同，流式管中加入50μL待测样品，然后向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
然后向流式管中加入抗体：FITC标记的抗CD45抗体、APC-cy7标记的抗CD3抗体、Percp-cy5.5标记的抗CD4抗体、BV510标记的抗CD8抗体、BV421标记的抗CD25抗体、Alexa Fluor700标记的抗CD127抗体、BV785标记的抗CD45RO抗体、PE标记的抗CD197抗体、BV605标记的抗CD28抗体、APC标记的抗CD95抗体，充分涡旋后，室温避光孵育20min。
T淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞免疫分型结果见图3，从图中可知，T细胞、Tc细胞、Th细胞、Treg细胞的相对数(百分数)；根据淋巴细胞的绝对数、和各个T细胞亚群的相对数(百分数)，计算获得各个T细胞亚群的绝对数，具体实验结果见表6。
表6 各个T细胞亚群免疫分型所用的细胞表面标志、各个T细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000011
T细胞发育亚群免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞发育亚群分型结果见图4-A、4-B、4-C、4-D、4-E、4-F、4-G、4-H、4-I、4-J，从图中可知，各类T细胞发育亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据T淋巴细胞的绝对数、各个细胞亚群的相对数(百分数)，计算获得各个T淋巴细胞发育亚群的绝对数。
1、调节性T细胞(Treg细胞)
由图4-B可知，CD3 +CD4 +CD25 +CD127 -即为目标群。由流式细胞仪可测得目标细胞占淋巴细胞的3.48％，可由血细胞计数仪测得的淋巴细胞绝对数算出，Treg细胞的绝对数为102个/μL。
2、辅助T细胞亚群
1)从图4-D可得，辅助初始T细胞(即
Figure PCTCN2022078965-appb-000012
CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -)占T细胞的相对数16.90％，绝对数264个/μL；辅助干细胞记忆T细胞(即TSCM，CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +)相对数3.99％，绝对数63个/μL；
2)从图4-E可得，辅助中心记忆T细胞(即TCM，CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +)相对数5.75％，绝对数90个/μL；
3)从图4-F可得，辅助效应记忆T细胞(即TEM，CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +)相对数0.10％，绝对数2个/μL； 辅助过渡记忆T细胞(即TTM，CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +)相对数8.59％，绝对数135个/μL。
3、细胞毒性T细胞亚群
1)从图4-H可得，细胞毒性初始T细胞(即
Figure PCTCN2022078965-appb-000013
CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -)占T细胞的相对数25.98％，绝对数408个/μL；细胞毒性干细胞记忆T细胞(TSCM，CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +)相对数4.43％，绝对数70个/μL；
2)从图4-I可得，细胞毒性中心记忆T细胞(即TCM，CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +)相对数0.10％，绝对数2个/μL；
3)从图4-J可得，细胞毒性效应记忆T细胞(即TEM，CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +)相对数0.86％，绝对数13个/μL；细胞毒性过渡记忆T细胞(即TTM，CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +)相对数3.42％，绝对数54个/μL。
根据图3可知，本案例所用的方法能够成功的将T淋巴细胞分为CD3 +T细胞、CD3 +CD4 +细胞和CD3 +CD8 +细胞，并且进行定量分析，获得了各个T淋巴细胞的相对数和绝对数。
根据图4-B、图4-D、图4-E、图4-F、图4-H、图4-I、图4-J可得，本案例所用的方法能够准确的将T淋巴细胞分为Treg细胞、辅助初始T细胞、辅助干细胞记忆T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、辅助过渡记忆T细胞、细胞毒性初始T细胞、细胞毒性干细胞记忆T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞、细胞毒性过渡记忆T细胞，进行定量分析，获得了各个T淋巴细胞发育亚群的相对数和绝对数。从表4中可知，待测样品的辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞的相对数和绝对数均低于正常值，提示该患儿T细胞发育异常。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的T淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个T淋巴细胞发育亚群进行免疫分型。
实施例3 T淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于来我院就诊患儿(男，10月龄)静脉血样品(已签知情同意书)，为拟诊肝功能衰竭患儿的外周血。
荧光标记FITC的抗CD45抗体、抗CD3(APC-Cy7)的抗体、抗CD4(Percp-cy5.5)的抗体、抗CD8(BV510)的抗体、抗CD25(BV421)的抗体、抗CD127(Alexa Flour 700)的抗体、抗CD45RO(BV785)的抗体、抗CD197(PE)的抗体、抗CD28(BV605)的抗体和抗CD95(APC)的抗体购买于BioLegend。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为5.14×10 9个/L。
剩下100μL用于检测T淋巴细胞发育亚群，进行T淋巴细胞发育亚群免疫分型和定量分析。
T淋巴细胞发育亚群免疫分型
与实施例1中记载的T淋巴细胞分类方法相同，流式管中加入50μL待测样品，然后向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
然后向流式管中加入抗体：FITC标记的抗CD45抗体、APC-cy7标记的抗CD3抗体、Percp-cy5.5标记的抗CD4抗体、BV510标记的抗CD8抗体、BV421标记的抗CD25抗体、Alexa Fluor700标记的抗CD127抗体、BV785标记的抗CD45RO抗体、PE标记的抗CD197抗体、BV605标记的抗CD28抗体、APC标记的抗CD95抗体，充分涡旋后，室温避光孵育20min。
T淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞免疫分型结果见图5，从图中可知，T细胞、Tc细胞、Th细胞、Treg细胞的相对数(百分数)；根据淋巴细胞的绝对数、和各个T 细胞亚群的相对数(百分数)，计算获得各个T细胞亚群的绝对数，具体实验结果见表7。
表7 各个T细胞亚群免疫分型所用的细胞表面标志、各个T细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000014
T细胞发育亚群免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞发育亚群分型结果见图6-A、6-B、6-C、6-D、6-E、6-F、6-G、6-H、6-I、6-J，从图中可知，各类T细胞发育亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据T淋巴细胞的绝对数、各个细胞亚群的相对数(百分数)，计算获得各个T淋巴细胞发育亚群的绝对数。
1、调节性T细胞(Treg细胞)
由图6-B可知，CD3 +CD4 +CD25 +CD127 -即为目标群。由流式细胞仪可测得目标细胞占淋巴细胞的2.04％，可由血细胞计数仪测得的淋巴细胞绝对数算出，Treg细胞的绝对数为105个/μL。
3、辅助T细胞亚群
1)从图6-D可得，辅助初始T细胞(即
Figure PCTCN2022078965-appb-000015
CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -)相对数49.64％，绝对数1298个/μL；辅助干细胞记忆T细胞(即TSCM，CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +)相对数0.44％，绝对数11个/μL；
2)从图6-E可得，辅助中心记忆T细胞(即TCM，CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +)相对数2.87％，绝对数75个/μL；
3)从图6-F可得，辅助效应记忆T细胞(即TEM，CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +)相对数0.02％，绝对数0.4个/μL；辅助过渡记忆T细胞(即TTM，CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +)相对数2.09％，绝对数55个/μL。
1)细胞毒性T细胞亚群
1)从图6-H可得，细胞毒性初始T细胞(即
Figure PCTCN2022078965-appb-000016
CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -)相对数10.47％，绝对数274个/μL；细胞毒性干细胞记忆T细胞(TSCM，CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +)相对数0.08％，绝对数2个/μL；
2)从图6-I可得，细胞毒性中心记忆T细胞(即TCM，CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +)相对数0.11％，绝对数3个/μL；
3)从图6-J可得，细胞毒性效应记忆T细胞(即TEM，CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +)相对数0.01％，绝对数0.2个/μL；细胞毒性过渡记忆T细胞(即TTM，CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +)相对数0.08％，绝对数2个/μL。
根据图5可知，本案例所用的方法能够成功的将T淋巴细胞分为CD3 +T细胞、CD3 +CD4 +细胞和CD3 +CD8 +细胞，并且进行定量分析，获得了各个T淋巴细胞的相对数和绝对数。
根据图6-B、图6-D、图6-E、图6-F、图6-H、图6-I、图6-J可得，本案例所用的方法能够准确的将T淋巴细胞分为Treg细胞、辅助初始T细胞、辅助干细胞记忆T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、辅助过渡记忆T细胞、细胞毒性初始T细胞、细胞毒性干细胞记忆T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞、细胞毒性过渡记忆T细胞，进行定量分析，获得了各个T淋巴细胞发育亚群的相对数和绝对数。从表4中可知，待测样品的辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞的相对数和绝对数均低于正常值，提示该患儿T细胞发育异常。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的T淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个T淋巴细胞发育亚群进行免疫分型。
实施例4 T淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于来我院就诊患儿(男，4月龄)静脉血样品(已签知情同意书)，为拟诊胆道闭锁患儿的外周血。
荧光标记FITC的抗CD45抗体、抗CD3(APC-Cy7)的抗体、抗CD4(Percp-cy5.5)的抗体、抗CD8(BV510)的抗体、抗CD25(BV421)的抗体、抗CD127(Alexa Flour 700)的抗体、抗CD45RO(BV785)的抗体、抗CD197(PE)的抗体、抗CD28(BV605)的抗体和抗CD95(APC)的抗体购买于BioLegend。
红细胞裂红液(cat RT122-02)购买于天根生化科技有限公司。
实验方法：
取300μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为7.31×10 9个/L。
剩下100μL用于检测T淋巴细胞发育亚群，进行T淋巴细胞发育亚群免疫分型和定量分析。
T淋巴细胞发育亚群免疫分型
与实施例4中记载的T淋巴细胞分类方法相同，流式管中加入50μL待测样品，然后向流式管中加入1mL的红细胞裂红液，充分涡旋后，37℃水浴6min～8min，离心500g，5min，倒去上清液。
然后向流式管中加入抗体：FITC标记的抗CD45抗体、APC-cy7标记的抗CD3抗体、Percp-cy5.5标记的抗CD4抗体、BV510标记的抗CD8抗体、BV421标记的抗CD25抗体、Alexa Fluor700标记的抗CD127抗体、BV785标记的抗CD45RO抗体、PE标记的抗CD197抗体、BV605标记 的抗CD28抗体、APC标记的抗CD95抗体，充分涡旋后，室温避光孵育20min。
T淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞免疫分型结果见图7，从图中可知，T细胞、Tc细胞、Th细胞、Treg细胞的相对数(百分数)；根据淋巴细胞的绝对数、和各个T细胞亚群的相对数(百分数)，计算获得各个T细胞亚群的绝对数，具体实验结果见表8。
表8 各个T细胞亚群免疫分型所用的细胞表面标志、各个T细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000017
T细胞发育亚群免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞发育亚群分型结果见图8-A、8-B、8-C、8-D、8-E、8-F、8-G、8-H、8-I、8-J，从图中可知，各类T细胞发育亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据T淋巴细胞的绝对数、各个细胞亚群的相对数(百分数)，计算获得各个T淋巴细胞发育亚群的绝对数。
1、调节性T细胞(Treg细胞)
由图8-B可知，CD3 +CD4 +CD25 +CD127 -即为目标群。由流式细胞仪可测得目标细胞占淋巴细胞的0.96％，可由血细胞计数仪测得的淋巴细胞绝对数算出，Treg细胞的绝对数为70个/μL。
4、辅助T细胞亚群
1)从图8-D可得，辅助初始T细胞(即
Figure PCTCN2022078965-appb-000018
CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -)相对数55.15％，绝对数1141个/μL；辅助干细胞记忆T细胞(即TSCM，CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +)相对数0.71％，绝对数15个/μL；
2)从图8-E可得，辅助中心记忆T细胞(即TCM，CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +)相对数3.65％，绝对数75个/μL；
3)从图8-F可得，辅助效应记忆T细胞(即TEM，CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +)相对数1.46％，绝对数30个/μL；辅助过渡记忆T细胞(即TTM，CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +)相对数1.76％，绝对数36个/μL。
1)细胞毒性T细胞亚群
1)从图8-H可得，细胞毒性初始T细胞(即
Figure PCTCN2022078965-appb-000019
CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -)相对数7.77％，绝对数160个/μL；细胞毒性干细胞记忆T细胞(TSCM，CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +)相对数0.16％，绝对数3个/μL；
2)从图8-I可得，细胞毒性中心记忆T细胞(即TCM，CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +)相对数0.15％，绝对数3个/μL；
3)从图8-J可得，细胞毒性效应记忆T细胞(即TEM，CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +)相对数1.92％，绝对数40个/μL；细胞毒性过渡记忆T细胞(即TTM，CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +)相对数0.14％，绝对数3个/μL。
根据图7可知，本案例所用的方法能够成功的将T淋巴细胞分为CD3 +T细胞、CD3 +CD4 +细胞和CD3 +CD8 +细胞，并且进行定量分析，获得了各个T淋巴细胞的相对数和绝对数。
根据图8-B、图8-D、图8-E、图8-F、图8-H、图8-I、图8-J可得，本案例所用的方法能够准确的将T淋巴细胞分为Treg细胞、辅助初始T细胞、辅助干细胞记忆T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、辅助过渡记忆T细胞、细胞毒性初始T细胞、细胞毒性干细胞记忆T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞、细胞 毒性过渡记忆T细胞，进行定量分析，获得了各个T淋巴细胞发育亚群的相对数和绝对数。从表4中可知，待测样品的辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞的相对数和绝对数均低于正常值，提示该患儿T细胞发育异常。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的T淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个T淋巴细胞发育亚群进行免疫分型。
实施例5 T淋巴细胞发育亚群免疫分型和定量分析
实验材料：
待测样品：抗凝外周血样品，来源于健康志愿者(女，26岁)，为正常人的外周血。
荧光标记FITC的抗CD45抗体、抗CD3(APC-Cy7)的抗体、抗CD4(BV650)的抗体、抗CD8(BV510)的抗体、抗CD25(BV421)的抗体、抗CD127(Alexa Flour 700)的抗体、抗CD45RO(BV785)的抗体、抗CD197(PE)的抗体、抗CD28(BV605)的抗体和抗CD95(APC)的抗体购买于BioLegend。
淋巴细胞分离液购买于达科为生物技术有限公司。
实验方法：
取3mL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为1.13×10 9个/L。
剩下的外周血用于提取外周血单核细胞(PBMC)，PBMC用于检测T淋巴细胞发育亚群，进行T淋巴细胞发育亚群免疫分型和定量分析。
T淋巴细胞发育亚群免疫分型
外周血用PBS稀释后，在稀释的外周血中加入淋巴细胞分离液，离心800g，20min，离心完后，吸取中间的白膜层，即为PBMC。
取1×10 6流式管中加入抗体：FITC标记的抗CD45抗体、APC-cy7标记的抗CD3抗体、BV650标记的抗CD4抗体、BV510标记的抗CD8抗体、BV421标记的抗CD25抗体、Alexa Fluor700标记的抗CD127抗体、BV785标记的抗CD45RO抗体、PE标记的抗CD197抗体、BV605标记的抗CD28抗体、APC标记的抗CD95抗体，充分涡旋后，室温避光孵育20min。
T淋巴细胞免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞免疫分型结果见图9，从图中可知，T细胞、Tc细胞、Th细胞、Treg细胞的相对数(百分数)；根据淋巴细胞的绝对数、和各个T细胞亚群的相对数(百分数)，计算获得各个T细胞亚群的绝对数，具体实验结果见表9。
表9 各个T细胞亚群免疫分型所用的细胞表面标志、各个T细胞亚群的相对数和绝对数
Figure PCTCN2022078965-appb-000020
T细胞发育亚群免疫分型和定量分析结果(采用FlowJo分析软件分析)
T淋巴细胞发育亚群分型结果见图10-A、10-B、10-C、10-D、10-E、10-F、10-G、10-H、10-I、10-J，从图中可知，各类T细胞发育亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据T淋巴细胞的绝对数、各个细胞亚群的相对数(百分数)，计算获得各个T淋巴细胞发育亚群的绝对数。
1、调节性T细胞(Treg细胞)
由图9-B可知，CD3 +CD4 +CD25 +CD127 -即为目标群。由流式细胞仪可测得目标细胞占淋巴细胞的4.12％％，可由血细胞计数仪测得的淋巴细胞绝对数算出，Treg细胞的绝对数为25个/μL。
5、辅助T细胞亚群
1)从图10-D可得，辅助初始T细胞(即
Figure PCTCN2022078965-appb-000021
CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -)相对数14.07％，绝对数87个/μL；辅助干细胞记忆T细胞(即TSCM，CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +)相对数2.47％，绝对数15个/μL；
2)从图10-E可得，辅助中心记忆T细胞(即TCM，CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +)相对数7.71％，绝对数48个/μL；
3)从图10-F可得，辅助效应记忆T细胞(即TEM，CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +)相对数0.26％，绝对数2个/μL；辅助过渡记忆T细胞(即TTM，CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +)相对数14.87％，绝对数92个/μL。
2)细胞毒性T细胞亚群
1)从图10-H可得，细胞毒性初始T细胞(即
Figure PCTCN2022078965-appb-000022
CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -)相对数14.23％，绝对数88个/μL；细胞毒性干细胞记忆T细胞(TSCM，CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +)相对数2.47％，绝对数15个/μL；
2)从图10-I可得，细胞毒性中心记忆T细胞(即TCM，CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +)相对数0.35％，绝对数2个/μL；
3)从图10-J可得，细胞毒性效应记忆T细胞(即TEM，CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +)相对数1.52％，绝对数9个/μL；细胞毒性过渡记忆T细胞(即TTM，CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +)相对数8.90％，绝对数55个/μL。
根据图9可知，本案例所用的方法能够成功的将T淋巴细胞分为CD3+T细胞、CD3+CD4+细胞和CD3+CD8+细胞，并且进行定量分析，获得了各个T淋巴细胞的相对数和绝对数。
根据图10-B、图10-D、图10-E、图10-F、图10-H、图10-I、图10-J可得，本案例所用的方法能够准确的将T淋巴细胞分为Treg细胞、辅助初始T细胞、辅助干细胞记忆T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、辅助过渡记忆T细胞、细胞毒性初始T细胞、细胞毒性干细胞记忆T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞、细胞毒性过渡记忆T细胞，进行定量分析，获得了各个T淋巴细胞发育亚群的相对数和绝对数。从表9中可知，待测样品的各个T细胞亚群的相对数和绝对数基本都在或接近正常人的参考值。说明本发明实验结果准确和稳定。
综上所述，本发明提供的方法采用少量的待测样品就可以实现对待检测样品的T淋巴细胞的发育亚群的免疫分型，并统计了每个细胞亚群的含量，且本方法可以准确、清晰的将各个T淋巴细胞发育亚群进行免疫分型。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (5)

  1. 一种T淋巴细胞发育亚群免疫分型的方法，其特征在于，包括：
    取不同荧光标记的抗体组合1，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，根据检测数据判定细胞型别；
    所述抗体组合1包括：抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD28的抗体、抗CD197的抗体和抗CD95的抗体；
    所述细胞型别的判定方法为：
    细胞表面标志CD3 +CD4 +CD25 +CD127 -代表调节性T细胞；
    细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 -代表辅助初始T细胞；
    细胞表面标志CD3 +CD4 +CD197 +CD45RO -CD28 +CD95 +代表辅助干细胞记忆T细胞；
    细胞表面标志CD3 +CD4 +CD197 +CD45RO +CD28 +CD95 +代表辅助中心记忆T细胞；
    细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 -CD95 +代表辅助效应记忆T细胞；
    细胞表面标志CD3 +CD4 +CD197 -CD45RO +CD28 +CD95 +代表辅助过渡记忆T细胞；
    细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 -代表细胞毒性初始T细胞；
    细胞表面标志CD3 +CD8 +CD197 +CD45RO -CD28 +CD95 +代表细胞毒性干细胞记忆T细胞；
    细胞表面标志CD3 +CD8 +CD197 +CD45RO +CD28 +CD95 +代表细胞毒性中心记忆细胞；
    细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 -CD95 +代表细胞毒性效应记忆T细胞；
    细胞表面标志CD3 +CD8 +CD197 -CD45RO +CD28 +CD95 +代表细胞毒性 过渡记忆T细胞。
  2. 根据权利要求1所述的方法，其特征在于，还包括统计所述待测样品中T淋巴细胞数目的步骤，包括：
    1)、检测所述待测样品中淋巴细胞的总数；
    2)、检测所述待测样品中T淋巴细胞占所述淋巴细胞的百分比；
    3)、通过计算，获得所述待测样品中T淋巴细胞的数目。
  3. 根据权利要求2所述的方法，其特征在于，所述步骤2)包括：
    取不同的荧光标记的抗体组合2，与所述待测样品混合，孵育后，经流式细胞术检测，得检测数据，分析检测数据；
    所述抗体组合2包括：
    抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体。
  4. 一种用于T淋巴细胞发育亚群免疫分型的试剂盒，其特征在于，包括荧光标记的如下抗体：
    抗CD45的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD25的抗体、抗CD127的抗体、抗CD45RO的抗体、抗CD197的抗体、抗CD28的抗体和抗CD95的抗体。
  5. 根据权利要求4所述的试剂盒，其特征在于，所述荧光标记包括FITC、APC-Cy7、Percp-cy5.5、BV510、BV421、Alexa Fluor 700、BV785、PE、BV605和APC。
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