Thymus recent emigration cell marker and application thereof

Abstract

The invention belongs to the technical field of thymus immigration cell detection, and particularly relates to a thymus immigration cell marker and application thereof, the marker comprises CD38 used for representing CD4RTE and CD8RTE, the CD38 can be used as a marker for defining thymus RTE, and a system and a kit for detecting the output function of thymus by CD3 + CD4 + CD45RA + CCR7 + CD31 + CD38 + and CD3 + CD8 + CD45RA + CCR7 + CD38 + cells are provided. Compared with the prior art, the thymus output function is detected more quickly and effortlessly, human peripheral CD4RTE can be more accurately defined by detecting the expression of the T cells CD31 and CD38, human peripheral CD8RTE cells can be more accurately defined by detecting the expression of the T cells CD38, and the CD38 can be used as a new surface molecular marker of the CD8RTE. The discovery of the CD8RTE marker supplements the existing theory, and is beneficial to the evaluation of the human thymus senescence function.

Description

Technical Field

[0001] The present invention belongs to the technical field of thymus recent emigration cell detection, and in particular relates to a thymus recent emigration cell marker and application thereof. Background Art

[0002] It was not until the 1960s that the thymus was widely recognized as an important site for T cell development. Since then, immunologists have gained a deeper understanding of thymic function and T cell development. Generally speaking, the development of T cells in the thymus is that a small number of thymus-seeding progenitors (TSPs) enter the thymus and undergo multiple cell proliferation steps, undergoing processes such as T cell lineage determination, T cell receptor gene rearrangement (TCR rearrangement), and positive selection and negative selection to generate CD4 T cells that can recognize antigens presented by their own MHC molecules and are tolerant to their own peripheral tissue antigens. + 、CD8 + T cells are exported from the thymus to the periphery, forming a T cell pool with huge TCR diversity, which is distributed to tissues and organs throughout the body to respond to complex antigens and exercise immune functions.

[0003] The thymus, a vital immune organ, plays a key role in the production and maturation of T cells, and its function determines the state of a person's immune function. However, the human thymus degenerates and ages very early: significant atrophy occurs after age 4, and thymic output decreases by half every 16 years thereafter. With aging or illness, thymic function gradually declines, leading to a decrease in the output of immature T cells, which in turn impairs the body's immune response. This manifests as age-dependent poor vaccine responsiveness, increased susceptibility to threatening pathogens and emerging pathogens, and decreased immunity, a phenomenon particularly pronounced during major epidemics.

[0004] Currently, the assessment of thymic function in clinical and scientific research mainly relies on indirect indicators, lacking effective detection methods that directly reflect thymic output function. The commonly used thymic function detection indicators are mainly T-cell receptor excision circles (TREC). TRECs are DNA fragments produced by T cells during thymic development. They cannot replicate during subsequent T cell divisions and their number remains unchanged. Therefore, their number reflects the number of cells that have recently migrated out of the thymus. Detecting TREC levels in peripheral blood through quantitative PCR can indirectly assess thymic output function. However, TREC cannot directly assess T cell function, but can only reflect the total amount of thymic output, and may be affected by other factors in the body. In addition, the detection method is time-consuming and labor-intensive, which is not conducive to clinical promotion.

[0005] Recent thymic emigrants (RTEs) are newly developed T cells that leave the thymus and enter the peripheral circulation, replenishing and maintaining the diversity of the peripheral T cell repertoire. The number and proportion of RTEs in peripheral blood are the most direct indicators of thymic output and function. Because their number and function are directly related to thymic activity, they are particularly important in the study of immune reconstitution and aging. However, the precise surface markers that define RTEs remain unclear. Summary of the Invention

[0006] In order to solve the problems in the prior art, the present invention provides a thymus newly emigrated cell marker and its application, CD38 can be used as a marker to define thymus RTE, and proposes CD3 + CD4 + CD45RA + CCR7 + CD31 + CD38 + and CD3 + CD8 + CD45RA + CCR7 + CD38 + Cells can be used in systems and kits for detecting thymic output function. The detection is accurate, easy to operate, and widely applicable, providing a new and effective solution for thymic output function testing.

[0007] The present invention solves the technical problem by adopting the following technical solutions:

[0008] The present invention aims to provide a thymus newly emigrated cell marker, which includes CD38 for characterizing CD4 RTE and CD8 RTE.

[0009] Furthermore, markers characterizing CD4 RTE are CD38 and CD31.

[0010] Preferably, the marker is expression of CD3 + CD4 + CD45RA + CCR7 + CD31 + CD38 + CD4 + RTE cells and CD3 + CD8 + CD45RA + CCR7 + CD38 + CD8 +Any one or more of RTE cells. Biomarkers used to accurately assess human thymic output function. Flow cytometry analysis of CD3 + CD4 + CD45RA + CCR7 + CD31 + CD38 + RTE and CD3 + CD8 + CD45RA + CCR7 + CD38 + The ratio of RTE to peripheral blood, the amount of one and / or more of the biomarkers are significantly reduced in individuals with decreased thymic output function.

[0011] Analyzing the ratio of immune cell subsets by flow cytometry is an important method for clinically evaluating immune function, but there is no accurate detection index system and dedicated flow cytometry kit for thymic output function. Therefore, there is an urgent need to develop immunological indicators and precise, convenient detection systems and tools to accurately evaluate thymic output function. This is especially important in the evaluation of immune function in immune reconstitution, aging, and certain diseases (such as HIV infection and immune recovery after cancer treatment).

[0012] The present invention also aims to provide an application of a thymic recently emigrated cell marker in a thymic output function detection system, comprising a detection module and an analysis module; the detection module is used to detect the content of each biomarker in a sample; and the analysis module is used to analyze the data obtained by the detection module.

[0013] Furthermore, RTE cells in the sample were labeled with fluorescent monoclonal antibodies labeled with CD3, CD4, CD8, CD45RA, CCR7, CD31, and CD38.

[0014] Furthermore, the method for detecting the thymic output function system includes:

[0015] (1) Collect peripheral blood samples;

[0016] (2) Using fluorescent monoclonal antibodies labeled with CD3, CD4, CD8, CD45RA, CCR7, CD31, and CD38 to label RTE cells in the sample; obtaining immune cell marker detection data by flow cytometry;

[0017] (3) Use fluorescent monoclonal antibodies labeled with CD45 to label lymphocytes in the sample; and use BD Trucount Tubes to detect the absolute number of immune cells;

[0018] (4) The detection data obtained in step (2) is brought into the analysis module for analysis, and combined with step (3), the frequency and number of CD4, CD8, naive (TN), effector memory (TEM), central memory (TCM), effector (TEF) and thymic recent emigration (RTE) cell subsets in T lymphocytes are analyzed to analyze the composition of CD4 T cells and CD8 T cells and the output capacity of thymic recent emigration cells. That is, the CD4 + RTE and CD8 + The ratio and absolute number of RTE cells in peripheral blood samples were used to assess thymic output function.

[0019] The present invention also aims to provide an application of a thymic recently emigrated cell marker in the preparation of a kit for detecting thymic output function, comprising CD3 monoclonal antibodies, CD8 monoclonal antibodies, CD4 monoclonal antibodies, CD45RA monoclonal antibodies, CCR7 monoclonal antibodies, CD31 monoclonal antibodies, and CD38 monoclonal antibodies labeled with different fluorescent markers.

[0020] Further, one of fluorescein FITC, APC, RB780, PerCP / Cy5.5, Brilliant Violet 510, Brilliant Violet 421, PE and RB705.

[0021] Furthermore, the fluorescently labeled monoclonal antibodies were CD3-FITC, CD4-Brilliant Violet 510, CD8-RB780, CD45RA-APC, CCR7-PE, CD38-Brilliant Violet 421, and CD31-RB705.

[0022] Furthermore, the detection method of the kit includes the following steps:

[0023] 1) Collect peripheral blood samples for testing; test immediately after collection or store at room temperature;

[0024] 2) Transfer the sample to a flow cytometer, prepare an antibody mixture for each antibody, add it to the flow cytometer, mix thoroughly, and incubate at 4°C in the dark for 30 minutes;

[0025] 3) Transfer the sample to BD Trucount Tubes, add CD45 antibody to the Trucount Tubes, mix thoroughly, and incubate at 4°C in the dark.

[0026] 4) After incubation, add hemolysin (1×) for flow cytometry analysis to the flow tubes and BD Trucount Tubes, mix well, and incubate at 4°C in the dark for 30 minutes;

[0027] 5) After the incubation is completed, the sample mixture prepared in 2) and 3) is centrifuged, the supernatant is discarded, and the cell pellet is added to the buffer solution and mixed until ready for use;

[0028] 6) Detecting the sample prepared in 5) on a flow cytometer;

[0029] 7) Analyze the data after detection and analyze CD4 + RTE and CD8 + The ratio and absolute number of RTE in peripheral blood.

[0030] Compared with the prior art, the beneficial technical effects of the present invention are:

[0031] 1. This invention can more accurately define human peripheral CD4 RTE by detecting CD31 and CD38 on T cells, and simultaneously accurately define human peripheral CD8 RTE by detecting CD38 expression. This CD8 RTE marker complements existing theories and facilitates the evaluation of human thymic aging function.

[0032] 2. The present invention is a detection system and a kit for evaluating thymic output function, which detects CD4 + RTE cells and CD8 + The system and kit utilize antibodies specifically labeled with CD3, CD4, CD8, CD45RA, CCR7, CD31, and CD38, offering simple operation and accurate analysis, making it suitable for both clinical testing and scientific research applications.

[0033] 3. The present invention can directly evaluate thymic output function: using more accurate definition of CD4 + RTE and CD8 + The combination of surface molecular markers of RTE was detected by flow cytometry and analyzed using a specific gating strategy for CD4 + and CD8 + The RTE ratio can directly and accurately reflect the thymus's newly formed T cell output function, thereby better evaluating the individual's immune function status: using a combination of absolute counting microspheres and lymphocyte surface molecular markers, the absolute number of lymphocytes is detected by flow cytometry and analyzed using a specific gating strategy.

[0034] 4. The present invention offers a simple, rapid, and time-sensitive procedure: Peripheral blood is collected intravenously and, using the eight-color antibody panel and flow cytometry-based detection provided by the system and kit, can be rapidly processed in a single test tube and, through specific gating analysis, accurately quantified for the ratio of recently exported thymic cells, effectively assessing the subject's thymic output function. The simple, time-efficient procedure allows for rapid completion and reporting of results. This significantly reduces both testing time and cost, facilitating widespread application in clinical and scientific research.

[0035] 5. The system and kit of the present invention have a wide range of applications: they are suitable for people of different age groups, health conditions and pathological conditions, and are particularly suitable for the elderly, patients with impaired immune function and individuals undergoing immunotherapy, and have broad clinical application value.

[0036] 6. The present invention can promote personalized medicine: the test results can provide clinicians with important immune function assessment information, which is helpful for personalized diagnosis and treatment and health management, especially the prognosis assessment and treatment effect monitoring of patients with immune-related diseases (such as immunodeficiency diseases and cancer).

[0037] 7. This invention fills the technical gap in existing thymic function assessment methods. Through precise cell marker detection, it opens up a new path for thymic output function assessment, which has innovative significance in the field of immunology.

[0038] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a technical scheme diagram for the development and identification of newly emigrated thymic cells of the present invention.

[0040] Figure 2 This is a bioinformatics analysis diagram of the characterization of RTE of the present invention and CD38 as a new RTE marker.

[0041] Figure 3 The peripheral blood CD4 + RTE and CD8 + Flow cytometry analysis of RTE cell detection.

[0042] Figure 4 This is an analysis chart of the absolute number of peripheral blood lymphocytes in the present invention. DETAILED DESCRIPTION

[0043] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0044] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0045] Example 1

[0046] A detection system for thymic output function biomarkers includes a detection module and an analysis module; the detection module uses a flow cytometer to detect the content of each biomarker in a sample; the analysis module is used to analyze the data obtained by the detection module, including changes in the content of each biomarker.

[0047] The specific steps of the test are as follows: (1) Collect human peripheral blood samples; (2) Use fluorescent monoclonal antibodies labeled with CD3, CD4, CD8, CD45RA, CCR7, CD31 and CD38 to label RTE cells in the sample; obtain immune cell marker detection data through flow cytometry; (3) Use fluorescent monoclonal antibodies labeled with CD45 to label lymphocytes in the sample; obtain the absolute number of immune cells through BD Trucount Tubes detection; (4) Bring the detection data obtained in step (2) into the analysis module for analysis. Use a specific gating strategy to analyze CD4 + RTE and CD8 + The ratio of RTE cells in the peripheral blood sample is analyzed by bringing the detection data obtained in step (3) into the analysis module for analysis. The absolute number of lymphocytes in the peripheral blood sample is analyzed using a specific gating strategy to evaluate the thymic output function.

[0048] Preferably, the fluorescently labeled monoclonal antibodies are CD3-FITC, CD4-Brilliant Violet 510, CD8-RB780, CD45RA-APC, CCR7-PE, CD38-Brilliant Violet 421, and CD31-RB705.

[0049] Example 2

[0050] A detection kit for thymic output function biomarkers includes detection reagents for detecting each biomarker, including different fluorescently labeled CD3 monoclonal antibodies, CD8 monoclonal antibodies, CD4 monoclonal antibodies, CD45RA monoclonal antibodies, CCR7 monoclonal antibodies, CD31 monoclonal antibodies, and CD38 monoclonal antibodies.

[0051] The fluorescein is selected from any one of FITC, Brilliant Violet 510, APC, PE, Brilliant Violet 421, RB780, PE-Cy7, and RB705.

[0052] Preferably, the fluorescently labeled monoclonal antibodies are CD3-FITC, CD4-Brilliant Violet 510, CD8-RB780, CD45RA-APC, CCR7-PE, CD38-Brilliant Violet 421, and CD31-RB705.

[0053] A kit for detecting human thymic output function, the implementation scheme of which is as follows:

[0054] 1) Collect no less than 200 μL of EDTA-anticoagulated human peripheral blood as the test sample; the test sample should be tested immediately after collection or stored at room temperature (20-25°C) and tested within 24 hours;

[0055] 2) Preparation of hemolysin for cell analysis (1×): Add 100 μL of hemolysin for flow cytometry analysis (10×) to 900 μL of sterile water, dilute and mix.

[0056] 3) Prepare FACS working solution: Add 1 mL of FBS to 49 mL of 1× PBS, mix well, and store at 4°C.

[0057] 4) Antibody preparation: Add the flow cytometry antibodies required for detection into a 1.5 mL microcentrifuge tube according to Table 1 and mix well to prepare a flow cytometry antibody mixture (Mix).

[0058] Table 1: Flow cytometry antibody preparation

[0059] markers CD45RA CD45 CD31 CD38 CD4 CCR7 CD3 CD8a Fluorescein APC APC-Cy7 RB705 BV421 BV510 PE FITC PE Cy7 Dosage 0.25 μL 0.25 μL 0.25 μL 0.25 μL 0.25 μL 0.25 μL 0.25 μL 0.25 μL .

[0060] 5) Staining: Transfer 50 mL of fresh whole blood to a flow cytometry tube, add flow cytometry antibody mix, mix thoroughly with a pipette, and incubate at 4°C in the dark for 30 minutes.

[0061] 6) Lysis: After staining, add 450 μL of diluted 1× hemolysin for flow cytometry analysis into the flow tube, mix with a pipette, and incubate at 4°C for 15 minutes to lyse the red cells.

[0062] 7) Detection on the machine: After the red lysis is completed, use a pipette to mix the sample tube to ensure that the cells are evenly suspended and then detect on the machine.

[0063] 8) Sample data collection: Adjust SSC-A / CD45 to determine lymphocyte population (see Figure 3 ). Cells were acquired at a low speed (≤6000 events / s), and 20,000 events of lymphocytes were collected for analysis.

[0064] 9) After the test, the data collected by the detection module is brought into the analysis module, and the CD4 + RTE and CD8 + The ratio of RTE to peripheral blood.

[0065] Example 3

[0066] Human peripheral blood CD4 + RTE and CD8 + RTE cell assay

[0067] 1) Human blood sample collection: Follow the clinical venous blood sample collection guidelines to collect peripheral venous blood: use blood routine tubes (purple cap, EDTA-K2 anticoagulant, BD After sampling, the sample should be stored at room temperature (20-25 degrees) and tested within 24 hours.

[0068] 2) The antibodies and fluorescent channels can be paired as follows: CD3 antibody (SK7, FITC channel), CD4 antibody (SK3, Brilliant Violet 510 channel), CD8 antibody (RPA-T8, RB780 channel), CD45RA antibody (HI100, AP C channel), CCR7 antibody (3D12, PE channel), CD31 antibody (WM59, RB705 channel), CD38 antibody (HI T2, Brilliant Violet 421 channel).

[0069] 3) Pipette 50 μL of the mixed peripheral blood sample into a flow cytometer tube. Prepare an antibody cocktail with 0.25 μL of each fluorescently labeled antibody for CD3, CD4, CD8, CD45RA, CCR7, CD31, and CD38. Add the antibody cocktail to the blood sample, mix thoroughly, and incubate at 4°C in the dark for 30 minutes. After incubation, add 400 μL of 1× flow cytometry hemolysin to the tube, mix thoroughly, and incubate at 4°C in the dark for 30 minutes. Perform multiparameter flow cytometry using a BD LSRFortess a X 20. Analyze the results using FlowJo V10 software (Tree Star).

[0070] 4) The flow cytometry results were analyzed in FlowJo software according to a specific gating strategy. Representative results are shown in Figure 2. Figure 3 As shown, Figure 3 middle:

[0071] ① In the FSC-SSC Plot, set the gate to take lymphocytes with low FSC and low SSC as the first gate (P1), i.e. Lym.

[0072] ② Under the P1 gate, in the CD3-SSC Plot, set the gate to take CD3-positive cells as the second-level gate (P2), that is, Total T cells.

[0073] ③ Under the P2 gate, in the CD4-CD8 Plot, set the gates to take CD4-positive cells as the 3rd gate (P3) and CD8-positive cells as the 4th gate (P4).

[0074] ④ Under the P3 gate, in the CCR7-CD45RAPlot, set the gate to take CD45RA and CCR7 double positive cells (CD4 T cells) were set as the 5th gate (P5), and a gate was further set on P5 to take CD31 and CD38 double-positive cells as the 6th gate (P6), which were CD4+ Recent Thymic Emigrant (RTE) cells.

[0075] ⑤ Under the P4 gate, in the CCR7-CD45RAPlot, set the gate to take CCR7 and CD45RA double positive cells as the 7th gate (P7), i.e. CD8 T cells, and further set a gate on P7 to obtain CD31 and CD38 positive cells (P8), namely CD8+Recent Thymic Emigrant (RTE) cells.

[0076] Figure 4 middle:

[0077] ① In the FSC-CD45 Plot, set the gate to take lymphocytes with low FSC and high CD45 as the first gate (P1), i.e. Ly m.

[0078] ② Under the P1 gate, in the APC-FITC Plot, set the gate to take high APC and high FITC double-positive cells as the second-level gate (P2), that is, Count Beads.

[0079] according to Figure 3 and Figure 4 The experimental results obtained by joint calculation are analyzed as follows:

[0080] Absolute number of T lymphocytes = 2936 × 64.7% = 1900 cells / μL

[0081] CD4 + Absolute number of T cells = 1900 × 50.7% = 963 cells / μL

[0082] CD4 + Absolute number of TEF cells = 963 × 6.71% = 65 cells / μL

[0083] CD4 + Absolute number of TEM cells = 963 × 35.6% = 343 cells / μL

[0084] CD4 + Absolute number of TCM cells = 963 × 16.3% = 157 cells / μL

[0085] CD4 + Absolute number of TN cells = 963 × 40.3% = 388 cells / μL

[0086] CD4 + Absolute number of RTE cells = 346 × 44.8% = 174 cells / μL

[0087] CD8 + Absolute number of T cells = 1900 × 27.3% = 519 cells / μL

[0088] CD8 + Absolute number of TEF cells = 519 × 13.2% = 69 cells / μL

[0089] CD8 + Absolute number of TEM cells = 518 × 47.6% = 247 cells / μL

[0090] CD8 + Absolute number of TCM cells = 518 × 3.29% = 17 cells / μL

[0091] CD8 + Absolute number of TN cells = 518 × 34.7% = 180 cells / μL

[0092] CD8 + The absolute number of RTE cells = 180 × 36.9% = 66 cells / μL.

[0093] The fluorescently labeled CD3, CD4, CD8, CD45RA, CCR7, CD31, and CD38 monoclonal antibodies used in the above examples of the present invention are commercially available American BD brands, but the present invention is not limited to the raw materials of the brands involved in this example.

[0094] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0095] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A thymus recent emigration cell marker, characterized in that: Markers include CD38 for characterization of CD4 RTE and CD8 RTE.

2. A thymic recently emigrated cell marker according to claim 1, characterized in that: The markers that characterize CD4 RTE are CD38 and CD31.

3. Use of a thymic recently emigrated cell marker in a system for detecting thymic output function according to claim 1 or 2, characterized in that: It includes a detection module and an analysis module; the detection module is used to detect the content of each biomarker in the sample; the analysis module is used to analyze the data obtained by the detection module.

4. The use of a thymic recently emigrated cell marker in a system for detecting thymic output function according to claim 3, wherein: RTE cells in the samples were labeled with fluorescent monoclonal antibodies labeled with CD3, CD4, CD8, CD45RA, CCR7, CD31 and CD38.

5. The use of a thymic recently emigrated cell marker in a system for detecting thymic output function according to claim 4, characterized in that: Tests for thymic output function include: (1) Collect peripheral blood samples; (2) Using fluorescent monoclonal antibodies labeled with CD3, CD4, CD8, CD45RA, CCR7, CD31, and CD38 to label RTE cells in the sample; obtaining immune cell marker detection data by flow cytometry; (3) Using fluorescent monoclonal antibodies labeled with CD45 to label lymphocytes in the sample, the absolute number of immune cells was determined using BD TrucountTubes. (4) The detection data obtained in step (2) is brought into the analysis module for analysis, and combined with step (3), the frequency and cell number of CD4, CD8, initial, effector memory, central memory, effector and thymus recently migrated cell subsets in T lymphocytes are analyzed, and the composition of CD4 T cells and CD8 T cells and the output capacity of thymus recently migrated cells are analyzed.

6. Use of a thymic recently emigrated cell marker according to claim 1 or 2 in preparing a kit for detecting thymic output function, characterized in that: Including CD3 monoclonal antibodies, CD8 monoclonal antibodies, CD4 monoclonal antibodies, CD45RA monoclonal antibodies, CCR7 monoclonal antibodies, CD31 monoclonal antibodies, and CD38 monoclonal antibodies labeled with different fluorescent markers.

7. Use of a thymic recently emigrated cell marker in the preparation of a kit for detecting thymic output function according to claim 6, characterized in that: The fluorescein used is one of FITC, APC, RB780, PerCP / Cy5.5, Brilliant Violet510, Brilliant Violet 421, PE and RB705.

8. Use of a thymic recently emigrated cell marker as claimed in claim 7 in preparing a kit for detecting thymic output function, characterized in that: The fluorescently labeled monoclonal antibodies were CD3-FITC, CD4-Brilliant Violet510, CD8-RB780, CD45RA-APC, CCR7-PE, CD38-Brilliant Violet 421, and CD31-RB705.

9. Use of a thymic recently emigrated cell marker according to claim 6 or 8 in preparing a kit for detecting thymic output function, characterized in that: The detection method of the kit includes the following steps: 1) Collect peripheral blood samples for testing; test immediately after collection or store at room temperature; 2) Transfer the sample to a flow cytometer, prepare an antibody mixture for each antibody, add it to the flow cytometer, mix thoroughly, and incubate at 4°C in the dark. 3) Transfer the sample to BD Trucount Tubes, add CD45 antibody to the Trucount Tubes, mix thoroughly, and incubate at 4°C in the dark. 4) After incubation, add hemolysin for flow cytometry analysis to the flow tubes and Trucount Tubes, mix well, and incubate at 4°C in the dark to lyse the red blood cells. 5) Detecting the samples prepared in 2) and 3) on a flow cytometer; 6) Analyze the data after detection and analyze CD4 + RTE and CD8 + The ratio and absolute number of RTE in peripheral blood.

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