CN117384289B - Antibody combination and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit - Google Patents

Abstract

The invention discloses an antibody combination for detecting T cell large granule lymphocytes, a kit and application thereof, wherein the antibody combination comprises a first group of antibodies and a second group of antibodies, and the first group of antibodies comprises CD8, CD4, CD16, CD56, CD5, CD3, CD2, CD7 and CD45 antibodies; the second set of antibodies comprises: TRBC1, CD5, tcrγδ, CD45RO, CD3, CD45RA, CD57 and CD45 antibodies. After a large number of experiments, the inventor of the invention screens out two groups of antibody combinations which take CD3+CD5dim area as main target cell groups and cover CD45RA, CD45RO, TRBC1 and other various T cell large granule lymphocytes, and the T cell large granule lymphocytes can be detected rapidly, simply, conveniently, with high accuracy, high sensitivity and high specificity by adopting the antibody combinations, thereby making up the limitations of the prior detection technology.

Description

Antibody combination, kit and application thereof for detecting T cell large granular lymphocytes

Technical Field

The present invention belongs to the technical field of immunological detection, and more specifically, to an antibody combination, a kit and an application thereof for rapid detection of T cell large granular lymphocytes.

Background Art

T-cell large granular lymphocytic leukemia (T-LGLL) is a clonal T lymphocyte proliferative disease, which refers to the continuous increase of T large granular lymphocytes in peripheral blood, usually >2*10 ⁹ /L. It can accumulate in peripheral blood, bone marrow, liver, and spleen, and can lead to hemocyte reduction, especially neutropenia, which can be serious and lead to recurrent infection and even death. In some elderly people, especially those with granulocytopenia, T-cell large granular lymphocytic leukemia (T-LGLL) screening can identify clonal T large granular lymphocytes earlier, which is convenient for better clinical monitoring and diagnosis and treatment.

The current technologies used to detect T-cell large granular lymphocytic leukemia (T-LGLL) include flow cytometry combined with genetic testing. The 2022 NCCN Guidelines for T Lymphoma recommends that flow cytometry should be used to detect the following antibodies for screening T-LGLL: CD3, CD4, CD5, CD7, CD8, CD56, CD57, TCRαβ, TCRγδ, and points out that the typical immunophenotype of T-LGLL is CD3+, CD8+, CD16+, CD57+, CD56+/-, CD5 dim and/or CD7 dim. After using the above antibody combination to identify abnormal T lymphocytes with the typical immunophenotype of T-LGLL, T cell clonality testing (TCR-vβ subset testing and TCR gene rearrangement testing) is performed for further confirmation. Two problems were found when using this method for disease screening. First, it takes a long time to issue a report: TCR-vβ subgroup test and TCR gene rearrangement test are performed only after the flow cytometry test results are reported to determine whether it is clonality, which usually takes about 7 days. Second, the specificity of flow cytometry screening using this antibody combination is not very good. Some patients with viral infections or autoimmune diseases may also have T lymphocytes with the above immune phenotype, but they are not clonally related (TCR rearrangement negative). Reactive CD8+T lymphocytes are detected rather than neoplastic T-LGLL cells. The two cannot be distinguished when using the above antibody combination.

Therefore, a simple, fast, accurate, sensitive and specific detection technology is needed clinically for the initial screening of T-LGLL.

Summary of the invention

Based on this, the purpose of the present invention is to provide an antibody combination, a kit and its application for rapid detection of T cell large granular lymphocytes. The antibody combination of the present invention is used to detect T cell large granular lymphocytes with high accuracy, high sensitivity, strong specificity, and simplicity and convenience.

The technical solutions for achieving the above-mentioned invention objectives include the following.

The first aspect of the present invention provides an antibody combination for detecting T cell large granular lymphocytes, comprising a first group of antibodies and a second group of antibodies, wherein the first group of antibodies comprises: CD8, CD4, CD16, CD56, CD5, CD3, CD2, CD7 and CD45 antibodies; the second group of antibodies comprises: TRBC1, CD5, TCRγδ, CD45RO, CD3, CD45RA, CD57 and CD45 antibodies.

The second aspect of the present invention provides a kit for detecting T cell large granular lymphocytes, the kit comprising the above-mentioned antibody combination for detecting T cell large granular lymphocytes.

The third aspect of the present invention provides the use of the above-mentioned antibody combination or kit for detecting T cell large granular lymphocytes in assisting the detection of T cell large granular lymphocytes.

In a fourth aspect, the present invention provides a system for detecting T cell large granular lymphocytes, comprising: a detection module, which performs flow cytometry detection on the cells to be detected; a data acquisition module, which acquires data of the flow cytometry detection results; and a data analysis module, which analyzes the acquired data and determines whether the acquired T lymphocytes are tumorous T cell large granular lymphocytes based on predetermined analysis logic and judgment criteria.

After a large number of experiments, the inventors of the present invention screened out two groups of antibody combinations with CD3+CD5dim area as the main target cell population, covering CD45RA, CD45RO, TRBC1 and other antibodies that identify T cell large granular lymphocytes. The use of this antibody combination can detect T cell large granular lymphocytes quickly, easily, with high accuracy, high sensitivity and high specificity, making up for the limitations of existing detection technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scatter plot of the CD45-SSC expression of the immunophenotype of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 2 is a scatter plot of the immunophenotype CD3-CD56 expression of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 3 shows the expression of the immunophenotype CD3-CD2 of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 4 shows the expression of the immunophenotype CD3-CD5 of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 5 shows the expression of the immunophenotype CD3-CD7 of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 6 shows the expression of the immunophenotype CD3-CD4 of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 7 shows the expression of the immunophenotype CD3-CD8 of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG8 shows the expression of the immunophenotype CD4-CD8 of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 9 is a scatter plot of the immunophenotype CD3-CD16 expression of normal T lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

FIG. 10 is a scatter plot of the immunophenotype CD3-CD45 expression of normal T lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 11 is a scatter plot of the immunophenotype CD3-TCRγδ expression of normal T lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 12 shows the expression of the immunophenotype CD3-CD5 of normal T lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 13 shows the expression of the immunophenotype CD3-TRBC1 of normal CD3+CD5dimT lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 14 shows the expression of the immunophenotype CD3-CD45RA of normal T lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 15 shows the expression of the immunophenotype CD3-CD45RO of normal T lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 16 shows the expression of the immunophenotype CD3-CD57 of normal CD3+TCRγδT-T lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

FIG. 17 is a scatter plot of the CD45-SSC expression of the immunophenotype of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 18 is a scatter plot of the immunophenotype CD3-CD56 expression of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 19 shows the expression of the immunophenotype CD3-CD2 of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 20 shows the expression of the immunophenotype CD3-CD5 of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 21 shows the expression of the immunophenotype CD3-CD7 of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 22 shows the expression of the immunophenotype CD3-CD4 of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 23 shows the expression of the immunophenotype CD3-CD8 of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 24 shows the expression of the immunophenotype CD4-CD8 of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 25 is a scatter plot of the immunophenotype CD3-CD16 expression of abnormal T lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

FIG. 26 is a scatter plot of the immunophenotype CD3-CD45 expression of abnormal T lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 27 is a scatter plot of the immunophenotype CD3-TCRγδ expression of abnormal T lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 28 shows the expression of the immunophenotype CD3-CD5 of abnormal T lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 29 shows the expression of the immunophenotype CD3-TRBC1 of abnormal CD3+CD5dimT lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 30 shows the expression of the immunophenotype CD3-CD45RA of abnormal T lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 31 shows the expression of the immunophenotype CD3-CD45RO of abnormal T lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 32 shows the expression of the immunophenotype CD3-CD57 of abnormal CD3+TCRγδT-T lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

FIG. 33 is a scatter plot of CD45-SSC expression of the immunophenotype of T lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

FIG. 34 is a scatter plot of the immunophenotype CD3-CD56 expression of the T lymphocytes to be tested using the antibody combination recommended in NCCN in Example 4 of the present invention.

Figure 35 is a scatter plot of the expression of CD3-CD5 immunophenotype of T lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

FIG. 36 is a scatter plot of the expression of the immunophenotype CD3-CD2 of the T lymphocytes to be tested using the antibody combination recommended in NCCN in Example 4 of the present invention.

Figure 37 is a scatter plot of the CD3-CD4 expression of the immunophenotype of the T lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

FIG. 38 is a scatter plot of the expression of the immunophenotype CD3-CD8 of the T lymphocytes to be tested using the antibody combination recommended in NCCN in Example 4 of the present invention.

Figure 39 is a scatter plot of the expression of the immunophenotype CD3-CD7 of the T lymphocytes to be tested using the antibody combination recommended in NCCN in Example 4 of the present invention.

Figure 40 is a scatter plot of the CD4-CD8 expression of the immunophenotype of the T lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

Figure 41 is a scatter plot of the immunophenotype CD3-CD57 expression of the T lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

Figure 42 is a scatter plot of the immunophenotype CD3-TCRγδ expression of the T lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention.

FIG. 43 is a scatter plot of the immunophenotype CD3-TRBC1 expression of T lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention.

FIG. 44 is a scatter plot of the immunophenotype CD3-CD45RA expression of T lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention.

FIG. 45 is a scatter plot of the immunophenotype CD3-CD45RO expression of T lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used in the present invention includes any and all combinations of one or more of the related listed items.

The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

In the present invention, in view of the deficiencies of the prior art, an antibody combination, a kit and a system for rapid detection of T cell large granular lymphocytes are provided. The antibody combination includes two groups of antibodies, with the CD3+CD5dim region as the main target cell population, covering CD45RA, CD45RO, TRBC1 and other antibodies that recognize T cell large granular lymphocytes, and a comprehensive logical analysis strategy, which can better identify tumorous T cell large granular lymphocytes. The present invention reasonably applies various antibody indicators and covers a wide range. The antibody combination can be used through multi-parameter flow detection technology to quickly and easily (only 1 to 2 hours from sample receipt to test results) and highly sensitively detect T cell large granular lymphocytes. The present invention also optimizes the template for result analysis. Before the optimization, only a single gate (CD3+CD5dim) is used to check whether CD57 is expressed. However, because CD3+CD5dim cells are not necessarily all T-large granular lymphocytes, inaccurate gates lead to deviations in the analysis results. After the optimization of the present invention, CD3+CD5dim can be used to circle suspicious cells first, and then CD45RA-CD5dim can be used to further gate, and CD3+CD5dimCD45RA+ cells can be circled to check the expression of TRBC1 and CD57. In addition, the analysis template is fixed, and data can be directly analyzed by dragging in. The operation is simple, the requirements for the professional knowledge level of analysts are reduced, and the limitations of existing detection technologies are compensated.

In some of the embodiments, there is provided an antibody combination for detecting T cell large granular lymphocytes, comprising:

1. The first group of antibodies includes CD8, CD4, CD16, CD56, CD5, CD3, CD2, CD7 and CD45 antibodies; in addition to CD45 as a leukocyte gating antibody, the first group of antibodies can identify suspicious T cell subpopulations (CD3+CD5+dim) through the backbone antibodies CD3 and CD5, circle the T lymphocytes expressing CD3+CD5+dim, and then see whether CD8 or CD4 is expressed singly. If CD8 or CD4 is expressed singly (generally T-LGLL cells often express CD8 singly), it can be initially suspected that there are T-large granular lymphocytes, and then combined with the second group of antibodies to further confirm the clonality of this group of T lymphocytes. The first group of antibodies also adds normal T lymphocyte markers CD2, CD7, and CD5. These markers can be used to see whether the clustering expression pattern of normal T lymphocytes is normal, such as loss of expression or enhanced expression, which is suspected to be abnormal cells. In addition, the first group of antibodies can be combined with CD3, CD4, and CD5 to identify abnormal T lymphocytes in the CD3-CD4+CD5+ region (usually the site where angioimmunoblastic T-cell lymphoma appears). In addition, CD56 and CD16 are added to the first group of antibodies. In addition to checking whether suspicious tumor cells express CD56, abnormal NK cells (CD3-CD56+CD16+) can also be identified. If the expression of NK cell CD56 is enhanced, or the expression of CD56 and CD16 is lost, it is suspected to be abnormal NK, and other NK cell markers (such as: CD158 series markers) need to be further added to identify whether it is tumor NK.

2. The second group of antibodies includes: TRBC1, CD5, TCRγδ, CD45RO, CD3, CD45RA, CD57 and CD45 antibodies. The second group of antibodies must be used in combination with the first group of antibodies. Use CD3+TCRγδ- to circle T lymphocytes expressing TCRab. Use backbone antibodies CD3 and CD5 to identify suspicious T lymphocyte populations (CD3+CD5+dim). Then see if CD57 is expressed to determine whether it is a cytotoxic cell. Combined with TRBC1 (normal T cells are partially expressed, with an expression rate of 15-85%. If the expression rate of TRBC1 is higher than 85% or lower than 15%, it can basically be defined as clonal T lymphocytes) In addition, combined with the uniform expression of CD45RA and the lack of CD45RO (normal T lymphocytes partially express CD45RO but not CD45RA), it can be determined as T-cell large granular lymphocytic leukemia; if the TRBC1 expression rate is between 75% and 84% or between 10% and 14%, CD5 and CD45RA can be used to circle the CD5+dimCD45RA+ cell population, and then see whether the TRBC1 expression rate is higher than 85% or lower than 15% to define whether it is a clonal T-cell large granular cell.

In some preferred embodiments, the antibodies of the antibody combination are monoclonal antibodies.

In some preferred embodiments, the monoclonal antibody is a fluorescein-labeled antibody, so that the above indicators can be simultaneously detected in a ten-color or more flow cytometer, which has a certain convenience; the fluorescein is selected from: FITC, PE, ECD, PE-Cy ^TM 5.5, PE-Cy7, APC, APC-750, PB, KO.

In some preferred embodiments, the CD8 and TRBC1 antibodies are labeled with fluorescein FITC; the CD4 and CD5 antibodies are labeled with fluorescein PE; the CD16 antibody is labeled with fluorescein ECD; the CD56 and TCRγδ antibodies are labeled with fluorescein PE-Cy ^TM 5.5; the CD5 and CD45RO antibodies are labeled with fluorescein PE-Cy7; the CD3 antibody is labeled with fluorescein APC; the CD2 and CD45RA antibodies are labeled with fluorescein APC-750; the CD7 and CD57 antibodies are labeled with fluorescein PB; and the CD45 antibody is labeled with fluorescein KO.

In other embodiments, it relates to a kit for detecting T cell large granular lymphocytes, wherein the kit comprises the above-mentioned antibody combination for detecting T cell large granular lymphocytes.

In other embodiments, it relates to the use of the above-mentioned antibody combination or kit for detecting T cell large granular lymphocytes in assisting the detection of T cell large granular lymphocytes.

In some other embodiments, a system for detecting T cell large granular lymphocytes is provided, comprising:

A detection module, wherein the detection module performs flow cytometry detection on the cells to be detected, and comprises: a single cell suspension module, for preparing a single cell suspension; an incubation module, for incubating the single cell suspension and the reagent composition in a dark environment for antigen-antibody reaction; a resuspension module, for adding hemolysin to the incubated cells, centrifuging, washing, and then resuspending the cells to prepare a suspension; and a determination module, for performing flow cytometry determination on the resuspended suspension;

A data acquisition module, which acquires data of flow cytometry detection results of cells to be tested stained with the antibody combination;

The data analysis module analyzes the acquired data and determines whether the acquired T lymphocytes are tumorous T cell large granular lymphocytes according to a predetermined analysis logic and judgment criteria; it includes: a normal control population antibody expression pattern template and a test cell antibody expression pattern, and the predetermined judgment criteria are: if the test cell antibody expression pattern falls into the normal control population antibody expression pattern template, the test cell is judged as a T lymphocyte population with a normal immune phenotype; if the test cell antibody expression pattern does not fall into the normal control population antibody expression pattern template, the test cell is judged as a suspected tumor cell population.

The technical process of the system for detecting T cell large granular lymphocytes in the present invention can refer to the conventional flow cytometry technology, and the equipment and consumables used are selected from flow cytometry analysis equipment and consumables, such as flow-specific tubes, oscillators, pipettes and other consumables.

In some preferred embodiments, the normal control population antibody expression pattern template is established by the following method: obtaining flow cytometry detection result data of the normal control population cell population, grouping the nucleated cells by setting a gate, circling the target cell population (i.e., T lymphocytes), and then analyzing the expression of each fluorescent antibody in the target cell population to obtain the normal control population antibody expression pattern template.

In some preferred embodiments, the antibody expression pattern of the cells to be tested is established by the following method: obtaining flow cytometry test result data of the cells to be tested, setting gates to group the nucleated cells according to the antibody expression pattern of the normal control population, circling the target cell population (i.e., T lymphocytes), and then analyzing the expression of each fluorescent antibody in the target cell population according to the antibody expression pattern of the normal control population to obtain the antibody expression pattern of the cells to be tested.

In some preferred embodiments, the gating uses CD45-SSC (side scattered light) gating, and divides the cell population into granulocyte population, monocyte population, and lymphocyte population according to the expression of CD45 (CD45 is a common antigen of leukocytes, so CD45 is used as a gating antibody, and the expression intensity of CD45 of normal blood cells is: lymphocyte>monocyte>granulocyte, SSC is side scattered light, which is related to the granularity of the cell, and the normal expression intensity of side scattered light is: granulocyte>monocyte>lymphocyte; combined with the expression characteristics of CD45 and SSC, the lymphocyte, monocyte and granulocyte divisions can be drawn, among which lymphocytes are at the position with the strongest CD45 and the lowest SSC; the CD45 of monocytes is slightly weaker than that of lymphocytes. The SSC of granulocytes is slightly higher than that of lymphocytes; the CD45 of granulocytes is weaker than that of monocytes, but the SSC signal is the highest. According to this expression pattern, the cell population is divided into granulocytes, monocytes, and lymphocytes), which is convenient for further analysis of the target cells in the lymphocyte population; and then the fluorescence expression intensity of the antibody pair in the target cells is analyzed; the antibody pair includes: CD45-SSC, CD3-CD56, CD3-CD2, CD3-CD5, CD3-CD7, CD3-CD4, CD3-CD8, CD4-CD8, CD3-CD16, CD3-CD45, CD3-TCRγδ, CD3-TRBC1, CD3-CD45RA, CD3-CD45RO, CD3-CD57.

The specific sources of some antibodies used in the following examples are: CD8 FITC (Cat. No. A07756), CD16 ECD (Cat. No. B49216), CD56 PECY5.5 (Cat. No. B49189), CD5 PECY7 (Cat. No. A21690), CD3 APC (Cat. No. IM2467), CD2 APC750 (Cat. No. B01681), CD7 PB (Cat. No. B06499), CD45 KO (Cat. No. B36294), CD5 PE (Cat. No. A07753), CD45 RO PECY7 (Cat. No. B13648), CD45 RA APC750 (Cat. No. B49194), CD57 PB (Cat. No. A74779), manufacturer BeckmanCoulter; CD4 PE (Cat. No. 663527), manufacturer BD; TRBC1 FITC (Cat. No. STRBCFI04), manufactured by Kepre; TCRγδPECY5.5 (Cat. No. 331224), manufactured by biolegend.

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1 Antibody combination for detecting T cell large granular lymphocytes

The antibody combination for detecting T cell large granular lymphocytes of this embodiment includes a first group of antibodies and a second group of antibodies, wherein:

The first group of antibodies includes: CD8, CD4, CD16, CD56, CD5, CD3, CD2, CD7 and CD45 antibodies;

The second group of antibodies includes: TRBC1, CD5, TCRγδ, CD45RO, CD3, CD45RA, CD57 and CD45 antibodies.

The fluorescent labels and dosages of the relevant monoclonal antibodies in the above antibodies are shown in Table 1.

Table 1

Note: The commercially available antibodies are tested in a concentration gradient to determine the optimal dosage, and the above-mentioned amounts of monoclonal antibodies are respectively placed in the flow cytometry tubes numbered 1 and 2.

Example 2 System and method for detecting T cell large granular lymphocytes

A system for rapid detection of T cell large granular lymphocytes in this embodiment includes: a detection module, a data acquisition module and a data analysis module. The detection module performs flow cytometry detection on the cells to be detected; the data acquisition module obtains the flow cytometry detection result data of the cells to be detected stained with the detection reagent composition described in Example 1; the data analysis module analyzes the above-obtained data and determines whether the cells to be detected are tumorous T lymphocytes according to a predetermined judgment standard.

The specific workflow for detecting T cell large granular lymphocytes using the above system of this embodiment is as follows:

1. Prepare the antibodies in the antibody combination of Example 1 as shown in Table 1.

2. Sample processing.

The concentration of the sample to be tested (peripheral blood) was adjusted to 1×10 ⁶ cells/ml according to the number of cells to prepare a single cell suspension.

3. Sample testing.

(1) Take a flow tube, mark it as 1 and 2, add the first group of antibodies and the second group of antibodies in Example 1, respectively, in an amount of 23 μl and 28 μl, and then add 100 μl of the suspension in step 2, vortex and mix, and incubate at room temperature in the dark for 15 min.

(2) Add 400 μl of Bc hemolysin to each of the incubated flow tubes 1 and 2, vortex and shake, and let it stand until the hemolysis becomes clear. After the hemolysis becomes clear, centrifuge the flow tubes 1 and 2 at 1500 r/min for 5 min, discard the supernatant, add 2 ml of calf serum, vortex and shake, centrifuge at 1500 r/min for 5 min, and discard the supernatant. Add 400 μl of 1% paraformaldehyde and resuspend.

(3) Use Beckman Coulter Navios ten-color flow cytometer to detect flow tubes 1 and 2 and analyze their immunophenotype.

4. Data analysis.

(1) Establish a template for the antibody expression pattern of healthy people

According to the flow cytometry test results of 40 normal control cell populations, the cells were grouped by setting a gate, preferably using CD45-SSC (side scattered light) to set a gate, circle the target cell population, and then analyze the expression of each fluorescent antibody for this cell population, and select the following antibody pairs: CD45-SSC, CD3-CD56, CD3-CD2, CD3-CD5, CD3-CD7, CD3-CD4, CD3-CD8, CD4-CD8, CD3-CD16, CD3-CD45, CD3-TCRγδ, CD3-TRBC1, CD3-CD45RA, CD3-CD45RO, CD3-CD57.

(2) Establishing the antibody expression pattern of the cells to be tested

The flow cytometry test result data of the cells to be tested are obtained, and the gates are set to group the cells according to the antibody expression pattern of the normal control population, and the target cell population is circled. Then, the expression of each fluorescent antibody in the target cell population is analyzed according to the antibody expression pattern of the normal control population to obtain the antibody expression pattern of the cells to be tested.

(3) Analyze the antibody expression pattern of the cells to be tested

If the antibody expression pattern of the cells to be tested falls into the antibody expression pattern template of the normal control population, the cells to be tested are determined to be a T lymphocyte population with a normal immunophenotype;

If the antibody expression pattern of the cell to be tested does not fall into the antibody expression pattern template of the normal control population, the cell to be tested is determined to be a T-large granular lymphocyte.

Example 3 Verification of the accuracy of the detection method of the present invention

The detection method of Example 2 was used to detect T cell large granular lymphocytes of two cells to be tested (1 normal sample + 1 abnormal sample), and the antibody expression pattern was analyzed to verify the accuracy of the detection method of the present invention.

1. Normal Sample

This sample is a clinical sample with normal physical examination and no obvious large granular lymphocytes in peripheral blood smear morphology analysis. The antibody expression pattern analysis was performed as follows:

Obtain the flow cytometry test result data of the sample to be tested. Use CD45-SSC (side scattered light) to set the gate, and divide the cell population into three regions according to the expression of CD45-SSC, namely granulocytes (the middle upper region in the figure), monocytes (the upper right region in the figure), and lymphocytes (the lower right region in the figure) (as shown in Figure 1). The lymphocyte region is the target cell population. It can be seen that lymphocytes account for 33.50% of the total number of nuclear cells, which is a normal proportion. The following will perform immunophenotypic analysis on this group of cells.

Figure 2 is a scatter plot of the CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3+ T lymphocytes account for 64.33% of lymphocytes, which is a normal proportion. At the same time, this antibody pair can also analyze NK cells (CD3-CD56+), which account for 5.03% of lymphocytes, which is a normal proportion. CD56 expression is weak. If the expression is enhanced (high expression), it may be suspected whether it is an abnormal NK cell. Its nature can be further confirmed by combining the expression of other NK cell immune markers (such as the CD158 series).

Figure 3 is a scatter plot of the CD3-CD2 expression of lymphocyte populations, from which it can be seen that CD3+T lymphocytes are all positive for CD2. If there is a CD3+CD2- cell population, it can be suspected to be an abnormal lymphocyte, and other analysis strategies can be further used to analyze the expression of immune markers such as CD4, CD5, CD7, CD8, TRBC1, Ki67, CD10, CD30, CD25, CD2 to determine its nature.

FIG. 4 is a scatter plot of CD3-CD5 expression of lymphocyte populations, and the presence or absence of CD3+CD5dim cells can be focused on. In this figure, CD3+CD5dim T lymphocyte population accounts for 3.13% of lymphocytes.

Figures 5 to 9 are scatter plots of the expression of CD3-CD7, CD3-CD4, CD3-CD8, CD4-CD8, and CD3-CD16 of the lymphocyte population, respectively. It can be checked whether the CD7 expression of the CD3+CD5dim T cells is weakened, whether CD16 is negative, and whether CD8 is positive. If the positive rate of CD8 is higher than 80% and the expression of CD7 is weakened, it can be preliminarily considered that this group of CD3+CD5dim T lymphocytes is suspected to be T large granular lymphocytes. It is necessary to combine the expression of CD57 in the low impedance body to determine whether it is a cytotoxic cell; combined with TRBC1 (normal T cells are partially expressed, with an expression rate of 15-85%. If the expression rate of TRBC1 is higher than 85% or lower than 15%, it can basically be defined as clonal T lymphocytes), and combined with the uniform expression of CD45RA and the lack of CD45RO (normal T lymphocytes partially express CD45RO and do not express CD45RA), it can be determined to be T cell large granular lymphocytic leukemia.

Figure 10 shows CD3-CD45 in the second group of antibodies, with CD3+ T lymphocytes circled.

FIG. 11 is a scatter plot of CD3-TCRγδ expression of CD3+T lymphocytes, with CD3+TCRγδ- T lymphocytes circled (TCRαβ T lymphocytes by default).

FIG. 12 is a scatter diagram showing the expression of CD3-CD5 in CD3+TCRγδ- T lymphocytes. As shown in the figure, CD3+CD5dim cells account for 3.12%.

FIG. 13 is a scatter diagram of the expression of CD3-TRBC1 in TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, the expression rate of TRBC1 is 37.10%, which is within the normal range (15-85%).

FIG14 is a scatter plot of CD3-CD45RA expression in TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, this group of lymphocytes is almost all negative for CD45RA expression (only 0.51% is expressed);

FIG15 is a scatter plot of CD3-CD45RO expression in TCRγδ-CD3+CD5dimT lymphocytes, as shown in the figure, this group of lymphocytes has a partial expression of CD45RO of 56.25%;

FIG. 16 is a scatter plot of CD3-CD57 expression in TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, CD57 in this group of lymphocytes is partially expressed at 33.45%.

The cell population of the sample to be tested was determined to be a cell population with normal immunophenotype in the antibody expression pattern template of the normal control population, which was consistent with the results of morphological analysis.

2. Abnormal Samples

This sample is a clinical sample that was confirmed to have obvious large granular lymphocytes by peripheral blood smear morphology analysis. The antibody expression pattern analysis was performed according to the following steps:

Obtain the flow cytometry test result data of the sample to be tested. Use CD45-SSC (side scattered light) to set the gate, and divide the cell population into three regions according to the expression of CD45-SSC, as shown in Figure 17, namely granulocytes (the middle upper region in the figure), monocytes (the upper right region in the figure), and lymphocytes (the lower right region in the figure). The lymphocyte region is the target cell population of this implementation case. It can be seen that lymphocytes account for 76.02% of the total number of nuclear cells, and the proportion is significantly increased. The following will perform immunophenotypic analysis on this group of cells.

FIG. 18 is a scatter plot of CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3+ T lymphocytes account for 98.40% of lymphocytes, and the proportion is significantly increased.

FIG. 19 is a scatter plot of CD3-CD2 expression of lymphocyte populations. As shown in the figure, CD2 is expressed on all T lymphocytes without obvious abnormality.

FIG. 20 is a scatter plot of CD3-CD5 expression in lymphocyte populations. As shown in the figure, CD3+CD5dim cells account for 91.11% of lymphocytes and are obviously distributed in groups. They are suspected to be abnormal T lymphocytes and need to be further analyzed in combination with other antibody markers.

Figures 21 to 25 are scatter plots of the expression of CD3-CD7, CD3-CD4, CD3-CD8, CD4-CD8, and CD3-CD16 of the lymphocyte population. It can be seen that the CD7 expression of the CD3+CD5dim T cells is weakened, CD16 is negative, and CD8 is positive. It can be preliminarily considered that this group of CD3+CD5dim T lymphocytes is suspected to be large granular lymphocytes, which needs to be further analyzed and confirmed in combination with the antibody markers in the second group of antibodies.

Figure 26 shows CD3-CD45 in the second panel of antibodies, with CD3+ T lymphocytes circled.

FIG. 27 is a scatter plot of CD3-TCRγδ expression of CD3+ T lymphocytes, with CD3+TCRγδ- T lymphocytes (TCRαβ T lymphocytes by default) circled.

FIG. 28 is a scatter diagram showing the expression of CD3-CD5 in CD3+TCRγδ- T lymphocytes. As shown in the figure, CD3+CD5dim cells account for 91.11%.

FIG. 29 is a scatter plot of CD3-TRBC1 expression of TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, the expression rate of TRBC1 is 0.02% (the expression rate of normal T lymphocytes is 15-85%), which can be judged as clonal T lymphocytes.

FIG30 is a scatter plot of CD3-CD45RA expression of TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, this group of lymphocytes are almost all CD45RA positive, with an expression rate of 99.85%;

FIG31 is a scatter plot of CD3-CD45RO expression of TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, this group of lymphocytes is almost all CD45RO negative, with an expression rate of 0.33%;

FIG32 is a scatter plot of CD3-CD57 expression in TCRγδ-CD3+CD5dimT lymphocytes, as shown in the figure, this group of lymphocytes has a partial expression of CD57 of 59.45%;

The above immune phenotypes all indicate that the cell population of the sample to be tested is not in the antibody expression pattern template of the normal control population, and is a tumor T cell large granular lymphocyte, which is consistent with the results of morphological analysis.

Example 4 Comparison of the detection accuracy of the antibody combination of the present invention and the antibody combination of the existing method

Compare the antibody combination of Example 1 of the present invention and the detection antibody combination recommended in NCCN (CD3, CD4, CD5, CD7, CD8, CD56, CD57, TCRαβ, TCRγδ, the fluorescent labeling of each antibody refers to Table 1, and the dosage also refers to Table 1), and the accuracy when detecting the same cells to be tested.

1. Use the detection antibodies recommended by NCCN (CD3, CD4, CD5, CD7, CD8, CD56, CD57, TCRαβ, TCRγδ) to obtain the flow cytometry test result data of the sample to be tested.

CD45-SSC (side scattered light) was used to set the gate. According to the expression of CD45-SSC, the cell population was divided into three regions, namely, granulocytes (the middle upper region in the figure), monocytes (the upper right region in the figure), and lymphocytes (the lower right region in the figure) (as shown in FIG33 ). The lymphocyte region was the target cell population. It can be seen that lymphocytes accounted for 34.55% of the total number of nuclear cells, which is a normal proportion. The immunophenotype analysis of this group of cells will be performed below.

FIG. 34 is a scatter plot of CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3+ T lymphocytes account for 84.66% of lymphocytes, and the proportion is increased.

Figure 35 is a scatter plot of CD3-CD5 expression of lymphocytes. In this figure, CD3+CD5dim T lymphocytes account for 4.67% of lymphocytes.

Figures 36 to 41 are scatter plots of the expression of CD3-CD2, CD3-CD4, CD3-CD8, CD3-CD7, CD4-CD8, and CD3-CD57 of the lymphocyte population, respectively. It can be seen that the CD3+CD5dim group of T cells has normal expression of CD7 and CD2, is CD8 positive and CD4 negative, and has partial expression of CD57, and can be considered as T-large granular lymphocytes.

2. Use the antibody combination of Example 1 of the present invention to detect the sample to be tested, and obtain flow cytometry test result data of the sample to be tested.

The first group of antibody combination data analysis is the same as above (NCCN recommended combination antibodies), circle the T lymphocytes expressing CD3+CD5dim;

This population of CD3+CD5dim T lymphocytes was further analyzed using a second panel of antibodies as follows:

FIG. 42 shows a scatter plot of CD3-TCRγδ expression of CD3+ T lymphocytes, with CD3+TCRγδ- T lymphocytes circled (TCRαβ T lymphocytes by default).

FIG. 43 is a scatter plot of CD3-TRBC1 expression of TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, the expression rate of TRBC1 is 22.79% (the expression rate of normal T lymphocytes is 15-85%), which can be judged as normal T lymphocytes.

FIG44 is a scatter plot of CD3-CD45RA expression in TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, this group of lymphocytes is partially CD45RA positive, with an expression rate of 40.59%;

FIG45 is a scatter plot of CD3-CD45RO expression in TCRγδ-CD3+CD5dimT lymphocytes. As shown in the figure, this group of lymphocytes is partially CD45RO positive, with an expression rate of 43.62%;

The combined results of TRBC1, CD45RA, and CD45RO antibodies showed that this group of 4.67% CD3+CD5dimT lymphocytes were polyclonal T lymphocytes rather than tumor T lymphocytes.

The results of this embodiment show that the antibody combination designed by the present invention takes the CD3+CD5dim region as the main target cell population, covers a variety of antibodies that recognize T cell large granular lymphocytes such as CD45RA, CD45RO, TRBC1, and a comprehensive logical analysis strategy, which can better identify tumorous T cell large granular lymphocytes, and has better accuracy than the detection antibodies recommended in NCCN.

The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (8)

1. An antibody combination for detecting T cell large particle lymphocytes, comprising a first set of antibodies comprising CD8, CD4, CD16, CD56, CD5, CD3, CD2, CD7 and CD45 antibodies, and a second set of antibodies; the second set of antibodies comprises: TRBC1, CD5, tcrγδ, CD45RO, CD3, CD45RA, CD57 and CD45 antibodies; the antibody of the antibody combination is a monoclonal antibody; the method for detecting the T cell macroparticle lymphocyte comprises the following steps: gating with CD45-SSC, and dividing cell populations into granulocyte populations, monocyte populations and lymphocyte populations according to the expression of CD 45; re-analyzing the fluorescence expression intensity of the antibody pairs in the lymphocyte population; the antibody pair comprises ：CD3-CD56,CD3-CD2,CD3-CD5,CD3-CD7,CD3-CD4,CD3-CD8,CD4-CD8,CD3-CD16,CD3-CD45,CD3-TCRγδ,CD3-TRBC1,CD3-CD45RA,CD3-CD45RO,CD3-CD57.

2. The antibody combination for detecting T cell large particle lymphocytes according to claim 1, wherein the monoclonal antibody is a fluorescein-labeled antibody selected from the group consisting of: FITC, PE, ECD, PE-Cy ^TM 5.5.5, PE-Cy7, APC-750, PB, KO.

3. The antibody combination for detecting T cell large particle lymphocytes according to claim 2, wherein the CD8 and TRBC1 antibodies label fluorescein FITC; the CD5 antibodies in the CD4 antibodies and the second set of antibodies label fluorescein PE; the CD16 antibody labels fluorescein ECD; the CD56 and TCRγδ antibodies label fluorescein PE-Cy ^TM 5.5.5; the CD45RO antibody and the CD5 antibody in the first group of antibodies label fluorescein PE-Cy7; the CD3 antibody marks fluorescein APC; the CD2 and CD45RA antibodies label fluorescein APC-750; the CD7 and CD57 antibodies label fluorescein PB; the CD45 antibody marks fluorescein KO.

4. A kit for detecting T cell large granule lymphocytes, comprising the antibody combination for detecting T cell large granule lymphocytes according to any one of claims 1 to 3.

5. Use of an antibody combination for detecting T cell large granule lymphocytes according to any one of claims 1 to 3 or a kit according to claim 4 for the assisted detection of T cell large granule lymphocytes.

6. A system for detecting T cell large particle lymphocytes, comprising:

the detection module is used for carrying out flow cytometry detection on the cells to be detected;

the data acquisition module acquires data of a flow cytometry detection result;

the data analysis module is used for gating the acquired data by using CD45-SSC, and dividing the cell population into granulocyte population, monocyte population and lymphocyte population according to the expression condition of CD 45; re-analyzing the fluorescence expression intensity of the antibody pairs in the lymphocyte population; the antibody pair comprises ：CD3-CD56,CD3-CD2,CD3-CD5,CD3-CD7,CD3-CD4,CD3-CD8,CD4-CD8,CD3-CD16,CD3-CD45,CD3-TCRγδ,CD3-TRBC1,CD3-CD45RA,CD3-CD45RO,CD3-CD57,, and whether the obtained T lymphocyte is a tumor T cell large granule lymphocyte is judged according to a judging standard.

7. The system for detecting T cell large granule lymphocytes according to claim 6, wherein the judgment criteria comprises:

if the antibody expression pattern of the cells to be detected falls into the expression pattern template of the antibodies of the normal control population, judging the cells to be detected as T lymphocyte groups with normal immunophenotype;

If the antibody expression pattern of the test cell does not fall into the antibody expression pattern template of the normal control population, the test cell is judged to be a suspected tumor cell population.

8. The system for detecting T cell large granule lymphocytes according to claim 7, wherein the antibody expression pattern of the test cell and the expression pattern of the normal control population antibody are established by: dividing nucleated cells into granulocyte groups, monocyte groups and lymphocyte groups according to data of detection results of flow cytometry by setting a gate on CD45-SSC, and analyzing the expression condition of each fluorescent antibody pair in the lymphocyte groups; the fluorescent antibody pair comprises ：CD3-CD56,CD3-CD2,CD3-CD5,CD3-CD7,CD3-CD4,CD3-CD8,CD4-CD8,CD3-CD16,CD3-CD45,CD3-TCRγδ,CD3-TRBC1,CD3-CD45RA,CD3-CD45RO,CD3-CD57.