CN117384289A - Antibody combination and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit - Google Patents

Antibody combination and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit Download PDF

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Publication number
CN117384289A
CN117384289A CN202311488821.XA CN202311488821A CN117384289A CN 117384289 A CN117384289 A CN 117384289A CN 202311488821 A CN202311488821 A CN 202311488821A CN 117384289 A CN117384289 A CN 117384289A
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lymphocytes
antibody
antibodies
cell
expression
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Inventor
韦杰
潘建华
张静文
徐志宏
王青
王佳佳
范昌军
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JINAN KINGMED CENTER FOR CLINICAL LABORATORY CO LTD
Guangzhou Kingmed Diagnostics Central Co Ltd
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JINAN KINGMED CENTER FOR CLINICAL LABORATORY CO LTD
Guangzhou Kingmed Diagnostics Central Co Ltd
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Priority to CN202311488821.XA priority Critical patent/CN117384289A/en
Publication of CN117384289A publication Critical patent/CN117384289A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2806—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD2
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD45
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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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 and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit
Technical Field
The invention belongs to the technical field of immunological detection, and in particular relates to an antibody combination and a kit for rapidly detecting T cell large granule lymphocytes and application thereof.
Background
T-cell large granule lymphocytic leukemia (T-LGLL) is a clonal T-lymphocyte proliferative disorder, which refers to a sustained increase in T-large granule lymphocytes in peripheral blood, usually > 2 x 10 9 and/L. Peripheral blood, bone marrow, liver, spleen can accumulate, which can lead to cytopenia-related, especially neutropenia, which can be severe, leading to recurrent infections and even death. In some elderly, particularly in granulocytopenic patients, T-cell large granule lymphocytic leukemia (T-LGLL) screening can be performed to identify clonal T-large granule lymphocytes early, facilitating better clinical monitoring and diagnosis.
Current techniques for detecting T cell large particle lymphocytic leukemia (T-LGLL) include the use of flow assays in combination with genetic assays. In the NCCN guideline, version 2022 for T-lymphoma, it is recommended that the following antibodies should be detected for screening for T-LGLL using flow cytometry: CD3, CD4, CD5, CD7, CD8, CD56, CD57, tcrαβ, tcrγδ, and indicate that typical T-LGLLs have an immunophenotype of cd3+, cd8+, cd16+, cd57+, cd56+/-, CD5dim, and/or CD7 dim. The identification of abnormal T lymphocytes with the immunophenotype of a typical T-LGLL using the above antibody combination was followed by a T cell clonality assay (TCR-vβ subset assay and TCR gene rearrangement assay) for further confirmation. Two problems are found when the method is used for disease screening, namely, long reporting time: the results of flow cytometry detection are reported and then are used for TCR-vβ subgroup detection and TCR gene rearrangement detection to determine whether the samples are clonality, and generally about 7 days is required; secondly, the specificity was not very good when the antibody combination was used for screening, and patients with partial viral infection or autoimmune diseases could also develop T lymphocytes of the above immunophenotype, but they were not clonogenic (TCR rearrangement negative), and reactive cd8+ T lymphocytes but not neoplastic T-LGLL cells were detected, which could not be identified when the above antibody combination was used.
Therefore, a detection technique which is simple, convenient, rapid, high in accuracy, high in sensitivity and good in specificity is clinically needed to perform primary screening of T-LGLL.
Disclosure of Invention
Based on the above, the invention aims to provide an antibody combination and a kit for rapidly detecting large-particle lymphocytes of T cells and application thereof, and the antibody combination is used for detecting the large-particle lymphocytes of the T cells, so that the accuracy is high, the sensitivity is high, the specificity is strong, and the method is simple and convenient.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the invention, there is provided an antibody combination for detecting T cell large particle lymphocytes, comprising a first set of antibodies and a second set of antibodies, wherein the first set 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.
In a second aspect of the invention, there is provided a kit for detecting T cell large granule lymphocytes, the kit comprising an antibody combination for detecting T cell large granule lymphocytes as described above.
In a third aspect, the invention provides an application of the antibody combination or the kit for detecting the T cell macroparticle lymphocytes in auxiliary detection of the T cell macroparticle lymphocytes.
In a fourth aspect of the invention, there is provided a system for detecting T cell large granule 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; and the data analysis module is used for analyzing the acquired data and judging whether the acquired T lymphocytes are tumor T cell large-particle lymphocytes or not according to preset analysis logic and judgment standards.
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.
Drawings
FIG. 1 is a plot of immunophenotype CD45-SSC expression using a first set of antibodies to detect normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 2 is a plot of immunophenotype CD3-CD56 expression using a first set of antibodies to detect normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 3 shows the use of a first set of antibodies for detecting immunophenotype CD3-CD2 expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 4 shows the use of a first set of antibodies to detect immunophenotype CD3-CD5 expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 5 shows the use of a first set of antibodies to detect immunophenotype CD3-CD7 expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 6 shows the use of a first set of antibodies to detect immunophenotype CD3-CD4 expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 7 shows the use of a first set of antibodies for detecting immunophenotype CD3-CD8 expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 8 shows the immunophenotype CD4-CD8 expression of normal T lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 9 is a plot of immunophenotype CD3-CD16 expression using a first set of antibodies to detect normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 10 is a plot of immunophenotype CD3-CD45 expression using a second set of antibodies to detect normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 11 is a plot of immunophenotype CD3-TCRγδ expression from a second set of antibodies used to detect normal T lymphocytes under test in example 3 of the invention.
FIG. 12 shows the use of a second set of antibodies for detecting immunophenotype CD3-CD5 expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 13 shows the immunophenotype CD3-TRBC1 expression of normal CD3+CD5dimT lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 14 shows the immunophenotype CD3-CD45RA expression of normal T lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 15 shows the use of a second set of antibodies for detecting immunophenotype CD3-CD45RO expression in normal T lymphocytes to be tested in example 3 of the present invention.
FIG. 16 shows the immunophenotype CD3-CD57 expression of normal CD3+ TCRγδT-T lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 17 is a plot of immunophenotype CD45-SSC expression using a first set of antibodies to detect abnormal T lymphocytes under test in example 3 of the present invention.
FIG. 18 is a plot of immunophenotype CD3-CD56 expression using a first set of antibodies to detect abnormal T lymphocytes under test in example 3 of the present invention.
FIG. 19 shows the immunophenotype CD3-CD2 expression of abnormal T lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 20 shows the immunophenotype CD3-CD5 expression of abnormal T lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 21 is a diagram showing the detection of immunophenotype CD3-CD7 expression of abnormal T lymphocytes to be tested using a first set of antibodies in example 3 of the present invention.
FIG. 22 shows the immunophenotype CD3-CD4 expression of abnormal T lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 23 shows the immunophenotype CD3-CD8 expression of abnormal T lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 24 is a diagram showing the detection of immunophenotype CD4-CD8 expression of abnormal T lymphocytes to be tested using a first set of antibodies in example 3 of the present invention.
FIG. 25 is a plot of immunophenotype CD3-CD16 expression using a first set of antibodies to detect abnormal T lymphocytes under test in example 3 of the present invention.
FIG. 26 is a plot of immunophenotype CD3-CD45 expression using a second set of antibodies to detect abnormal T lymphocytes under test in example 3 of the present invention.
FIG. 27 is a plot of immunophenotype CD3-TCRγδ expression from detection of abnormal T lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 28 is a diagram showing the use of a second set of antibodies to detect immunophenotype CD3-CD5 expression of test abnormal T lymphocytes in example 3 of the present invention.
FIG. 29 shows the immunophenotype CD3-TRBC1 expression of abnormal CD3+CD5dimT lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 30 shows the immunophenotype CD3-CD45RA expression of abnormal T lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 31 shows the immunophenotype CD3-CD45RO expression of abnormal T lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 32 is a graph showing the immunophenotype CD3-CD57 expression of abnormal CD3+ TCRγδT-T lymphocytes to be tested using a second set of antibodies in example 3 of the present invention.
FIG. 33 is a plot of immunophenotype CD45-SSC expression using the combination of antibodies recommended in NCCN for detection of T lymphocytes to be tested in example 4 of the present invention.
FIG. 34 is a scatter plot of immunophenotype CD3-CD56 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 35 is a scatter plot of immunophenotype CD3-CD5 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 36 is a scatter plot of immunophenotype CD3-CD2 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 37 is a scatter plot of immunophenotype CD3-CD4 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 38 is a scatter plot of immunophenotype CD3-CD8 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 39 is a scatter plot of immunophenotype CD3-CD7 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 40 is a plot of immunophenotype CD4-CD8 expression using the combination of antibodies recommended in NCCN for detection of T lymphocytes to be tested in example 4 of the present invention.
FIG. 41 is a scatter plot of immunophenotype CD3-CD57 expression of T lymphocytes tested using the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 42 is a plot of immunophenotype CD3-TCRγδ expression from T lymphocytes to be tested using the second set of antibodies of the invention in example 4 of the invention.
FIG. 43 is a plot of immunophenotype CD3-TRBC1 expression using the second set of antibodies of the invention to detect T lymphocytes under test in example 4 of the invention.
FIG. 44 is a plot of immunophenotype CD3-CD45RA expression using the second set of antibodies of the present invention to detect T lymphocytes under test in example 4 of the present invention.
FIG. 45 is a plot of immunophenotype CD3-CD45RO expression of T lymphocytes to be tested using the second set of antibodies of the present invention in example 4 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
In the invention, aiming at the defects of the prior art, an antibody combination, a kit and a system for rapidly detecting T cell large granule lymphocytes are provided. The antibody combination comprises two groups of antibodies, uses CD3+CD5dim zone as main target cell group, covers various antibodies for recognizing T cell large granule lymphocytes such as CD45RA, CD45RO, TRBC1 and the like, and can better recognize neoplastic T cell large granule lymphocytes by comprehensive logic analysis strategy. The invention reasonably applies various antibody indexes, has wide coverage range, and can detect the large-particle lymphocytes of the T cells rapidly, simply and conveniently (only 1-2 hours are needed for receiving the test result from the sample) with high sensitivity by adopting the antibody combination through a multiparameter flow detection technology. The invention optimizes the template of result analysis, only uses a single round gate (CD3+CD5dim) to see whether CD57 is expressed or not before optimizing, but because the cells of CD3+CD5dim are not necessarily T-large granular lymphocytes, the round gate inaccurately leads to deviation of analysis results.
In some embodiments thereof, an antibody combination for detecting T cell large particle lymphocytes is provided, comprising:
1. a first set of antibodies, including 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 a subset of suspicious T cells (CD3+CD5+dim) by the framework antibodies CD3 and CD5, circling T lymphocytes expressed by CD3+CD5+dim, and further checking whether CD8 or CD4 expression is expressed singly, if CD8 or CD4 is expressed singly (CD 8 is expressed singly by general T-LGLL cells), T-large granular lymphocytes can be suspected initially, and then the second group of antibodies can be combined to further confirm the clonality of the group of T lymphocytes. The first set of antibodies also increased the normal T lymphocyte markers CD2, CD7, CD5, which could see if the cluster expression pattern of normal T lymphocytes is normal, such as loss of expression or enhancement of expression is suspected of being abnormal cells. In addition, the first group of antibodies can bind to CD3, CD4, CD5 and recognize abnormal T lymphocytes (often the sites where angioimmunoblastic T cell lymphomas appear) in the CD3-CD4+CD5+ region. In addition, CD56 and CD16 are added in the first group of antibodies, so that whether suspicious tumor cells express CD56 or not can be checked, abnormal NK cells (CD 3-CD56+CD16+), such as the enhancement of the expression of NK cells CD56 or the suspected abnormal NK of the loss of the expression of CD56 and CD16, and other NK cell markers (such as a CD158 series marker) need to be further added for identification to confirm whether the tumor NK cells are tumor NK or not.
2. A second set of antibodies comprising: TRBC1, CD5, tcrγδ, CD45RO, CD3, CD45RA, CD57 and CD45 antibodies. The second group of antibodies needs to be used simultaneously in combination with the first group of antibodies, T lymphocytes expressed by TCRab are circled by using CD3+TCRγδ -, suspicious T lymphocyte groups (CD3+CD5+dim) can be identified by using framework antibodies CD3 and CD5, and then whether CD57 is expressed or not can be checked to determine whether the cells are cytotoxic cells or not; in addition, the T cell large particle lymphoblastic leukemia can be determined by combining TRBC1 (the normal T cell is partially expressed, the expression rate is 15-85%, for example, the expression rate of TRBC1 is higher than 85% or lower than 15%, and basically can be defined as cloned T lymphocyte), and combining CD45RA for homogeneous expression and CD45RO for non-expression (the normal T lymphocyte is partially expressed and CD45RA is not expressed); if TRBC1 expression is between 75-84% or 10-14%, CD5 and CD45RA can be used to combine to circle out CD5+dimCD45RA+ cell populations, and then see if TRBC1 expression is higher than 85% or lower than 15% to define whether it is a clonal T cell large granulosa 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 detected simultaneously in a ten-color or more flow cytometer, with certain convenience; the luciferin 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 label fluorescein FITC; the CD4 and CD5 antibodies label fluorescein PE; the CD16 antibody labels fluorescein ECD; the CD56 and TCRγδ antibodies label fluorescein PE-Cy TM 5.5; the CD5 and CD45RO 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.
In other embodiments, a kit for detecting T cell large granule lymphocytes is provided, comprising the above antibody combination for detecting T cell large granule lymphocytes.
In other embodiments, the use of the above antibody combination or kit for detecting T cell large granule lymphocytes in the auxiliary detection of T cell large granule lymphocytes is contemplated.
In other embodiments, a system for detecting T cell large particle lymphocytes is provided, comprising:
the detection module, the detection module carries out flow cytometry to the cell that awaits measuring and detects, includes: a single cell suspension module for preparing a single cell suspension; the incubation module is used for respectively carrying out light-proof incubation on the single cell suspension and the reagent composition to carry out antigen-antibody reaction; a resuspension module for adding hemolysin into the incubated cells, centrifuging and washing, and then resuspension the cells to prepare suspension; the measuring module is used for carrying out flow cytometry measurement on the resuspended suspension;
the data acquisition module is used for acquiring data of a flow cytometry detection result of the antibody combination-stained cells to be detected;
the data analysis module is used for analyzing the acquired data and judging whether the acquired T lymphocytes are tumor T cell large-particle lymphocytes or not according to preset analysis logic and judgment standards; comprising the following steps: the standard of the predetermined judgment is as follows: if the antibody expression pattern of the cells to be detected falls into the antibody expression pattern template of the normal control population, judging the cells to be detected as T lymphocyte populations with normal immunophenotype; and if the antibody expression pattern of the cell to be detected does not fall into the antibody expression pattern template of the normal control population, judging the cell to be detected as a suspected tumor cell population.
The technical process of the system for detecting the T cell large granule lymphocytes in the invention is just to refer to the conventional technology of flow cytometry, and the equipment and the consumable materials used for flow cytometry analysis are selected from the consumable materials such as a flow special pipe, an oscillator, a liquid-transfering device and the like.
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 normal control crowd cell groups, grouping nucleated cells by setting a gate, circling a target cell group (namely T lymphocyte), and analyzing the expression condition of each fluorescent antibody in the target cell group to obtain a normal control crowd antibody expression pattern template.
In some preferred embodiments, the test cell antibody expression pattern is established by: obtaining flow cytometry detection result data of cells to be detected, setting a gate to divide nucleated cells into groups according to the mode in the antibody expression mode of normal control crowd, circling a target cell group (namely T lymphocyte), and analyzing the expression condition of each fluorescent antibody in the target cell group according to the mode in the antibody expression mode of the normal control crowd to obtain the antibody expression mode of the cells to be detected.
In some preferred embodiments, the gating uses CD45-SSC (side scatter light) gating, and divides the cell population into granulocyte population, monocyte population and lymphocyte population according to the expression condition of CD45 (CD 45 is a leukocyte common antigen, so that CD45 is used as gating antibody, the expression intensity of CD45 of normal blood cells is that lymphocyte > monocyte > granulocyte, SSC is used as side scatter light, the normal expression intensity of the side scatter light is that granulocyte > monocyte > lymphocyte, and the combined expression characteristics of CD45 and SSC can divide the lymphocyte, monocyte and granulocyte into sections, wherein lymphocyte is positioned at the strongest and lowest position of CD45, monocyte CD45 is slightly weaker than lymphocyte and SSC is slightly higher than lymphocyte, and granulocyte CD45 is weaker than monocyte and SSC signal is highest; analyzing the fluorescence expression intensity of the antibody pair in the target cell; the antibody pair comprises: 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.
Specific sources of the partial antibodies used in the following examples are: CD8 FITC (accession number A07756), CD16 ECD (accession number B49216), CD56 PECY5.5 (accession number B49189), CD5 PECY7 (accession number A21690), CD3 APC (accession number IM 2467), CD2 APC750 (accession number B01681), CD7 PB (accession number B06499), CD45 KO (accession number B36294), CD5 PE (accession number A07753), CD45RO PECY7 (accession number B13648), CD45RA APC750 (accession number B49194), CD57 PB (accession number A74779), manufacturer BeckmanCoulter; CD4 PE (cat No. 663527), manufacturer BD; TRBC1 FITC (product number STRBCFI 04), manufacturer Kaplan; tcrγδ PECY5.5 (cat No. 331224), vendor biolegend.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 antibody combinations for detection of T cell large particle lymphocytes
The antibody combination for detecting T cell large granule lymphocytes of the embodiment comprises a first group of antibodies and a second group of antibodies, wherein,
the first set 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.
The fluorescent labeling and the amount of the related monoclonal antibody in the above antibodies are shown in Table 1.
TABLE 1
Note that: the commercially available antibodies were subjected to concentration gradient verification to determine the optimal amounts, and the amounts of monoclonal antibodies were taken and placed in flow-type tubes No. 1 and No. 2, respectively.
Example 2 System and method for detecting T cell large granular lymphocytes
A system for rapid detection of T cell large granule lymphocytes of this embodiment comprises: the system comprises a detection module, a data acquisition module and a data analysis module. The detection module is used for carrying out flow cytometry detection on cells to be detected; the data acquisition module acquires flow cytometry detection result data of a cell to be detected stained with the detection reagent composition described in example 1; the data analysis module analyzes the acquired data and judges whether the cell to be detected is a neoplastic T lymphocyte or not according to a preset judgment standard.
By adopting the system of the embodiment, the specific workflow for detecting the T cell large granule lymphocytes is as follows:
1. each antibody in the antibody combination of example 1 was formulated as shown in table 1.
2. Sample processing.
The concentration of the sample to be tested (peripheral blood) was adjusted to 1X 10 based on the number of cells 6 Single cell suspensions were prepared per ml.
3. And (5) detecting a sample.
(1) Taking flow tubes, marking 1 and 2, respectively adding the first group of antibodies and the second group of antibodies in the example 1, respectively adding 23 mu l and 28 mu l of the first group of antibodies, respectively adding 100 mu l of the suspension in the step 2, uniformly mixing by vortex vibration, and incubating for 15min at room temperature in a dark place.
(2) 400 μl Bc hemolysin is added to each of the incubated flow tubes 1, 2, vortex shaking and standing until hemolysis is clear. After the hemolysis was clear, the flow tubes 1, 2 were centrifuged at 1500r/min for 5min, the supernatant was discarded, 2ml calf serum was added, vortexed and centrifuged at 1500r/min for 5min, and the supernatant was discarded. 400 μl 1% paraformaldehyde was added to resuspend.
(3) Flow tubes 1, 2 were examined using a Beckmann Coulter Navios ten-color flow cytometer and analyzed for immunophenotype.
4. And (5) data analysis.
(1) Building a healthy human antibody expression pattern template
According to the flow cytometry detection result data of 40 normal control population cell groups, the cells are clustered by gating, preferably using CD45-SSC (side scattered light) gating, a target cell group is circled, and then the cell group is analyzed for the expression condition of each fluorescent antibody, and the following antibody pair is selected: 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 antibody expression mode of cell to be tested
Obtaining flow cytometry detection result data of the cells to be detected, setting a gate to divide the cells into groups according to the mode in the normal control crowd antibody expression mode, circling a target cell group, and analyzing the expression condition of each fluorescent antibody in the target cell group according to the mode in the normal control crowd antibody expression mode to obtain the antibody expression mode of the cells to be detected.
(3) Analysis of antibody expression patterns in test cells
If the antibody expression pattern of the cells to be detected falls into the antibody expression pattern template of the normal control population, judging the cells to be detected as T lymphocyte populations with normal immunophenotype;
and if the antibody expression pattern of the cell to be detected does not fall into the antibody expression pattern template of the normal control population, judging the cell to be detected as the T-large granular lymphocyte.
Example 3 verification of the accuracy of the detection method of the present invention
By adopting the detection method of example 2, T cell large granule lymphocytes of two samples of cells to be detected (1 sample of normal cells+1 sample of abnormal cells) are detected, and antibody expression pattern analysis is carried out to verify the accuracy of the detection method of the invention.
1. Normal sample
The sample was normal for physical examination and was analyzed using peripheral blood smear morphology, no clinical samples of significantly large granular lymphocytes were seen. The antibody expression pattern analysis was performed as follows:
and obtaining flow cytometry detection result data of the sample to be detected. The cell population was divided into 3 regions, i.e., granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) 3 regions (as shown in fig. 1), respectively, using a gate for CD45-SSC (side scattered light), and the lymphocyte region was the target cell population, and it was found that the lymphocyte occupied 33.50% of the total nuclear cell count, and the proportion was normal, and the immunophenotype analysis was performed on this cell population.
FIG. 2 shows a CD3-CD56 immunophenotype scatter plot of lymphocytes, wherein it can be seen that CD3+ T lymphocytes account for 64.33% of lymphocytes in normal proportion, and that the antibody pair can analyze NK cells (CD 3-CD56+), account for 5.03% of lymphocytes in normal proportion, and CD56 expression is weaker, if the expression is enhanced (high expression), whether the cells are abnormal NK cells can be suspected, and the properties of the cells can be further confirmed by combining the expression of immune markers (such as CD158 series) of other NK cells.
FIG. 3 is a scatter plot of CD3-CD2 expression of lymphocyte populations, showing that CD3+ T lymphocytes, CD2, are positive. If a cell population such as CD3+CD2-is suspected of being an abnormal lymphocyte, other analysis strategies may be further used to analyze the expression of the immune markers such as CD4, CD5, CD7, CD8, TRBC1, ki67, CD10, CD30, CD25, CD2, etc. to determine its properties.
FIG. 4 is a scatter plot of CD3-CD5 expression of lymphocyte populations, focusing on the presence or absence of CD3+CD5dim cells, where CD3+CD5dim T lymphocyte populations account for 3.13% of lymphocytes.
FIGS. 5-9 are scatter plots of CD3-CD7, CD3-CD4, CD3-CD8, CD4-CD8, CD3-CD16 expression of a lymphocyte population, and can be used to determine whether the cell population is cytotoxic by considering whether CD7 expression in the low steric hindrance antibody is bound to CD57, whether CD3+CD5dim population of T cells has reduced CD7 expression, whether CD16 is negative, whether CD8 is positive, such as CD8 positive rate is higher than 80%, and CD7 expression is reduced, respectively; the T cell large particle lymphocytic leukemia can be determined by combining TRBC1 (the normal T cell is partially expressed, the expression rate is 15-85%, for example, the expression rate of TRBC1 is higher than 85% or lower than 15%, basically can be defined as a cloned T lymphocyte), and combining CD45RA homogeneous expression and CD45RO non-expression (the normal T lymphocyte CD45RO is partially expressed and CD45RA non-expressed).
FIG. 10 is a schematic of CD3-CD45 in the second set of antibodies, circling CD3+ T lymphocytes.
FIG. 11 is a scatter plot of CD3-TCRγδ expression from CD3+ T lymphocytes, circling CD3+ TCRγδ -T lymphocytes (default TCRαβT lymphocytes).
FIG. 12 is a scatter plot of CD3-CD5 expression of CD3+TCRγδ -T lymphocytes, showing 3.12% CD3+CD5dim cells.
FIG. 13 is a scatter plot of CD3-TRBC1 expression in TCRγδ -CD3+CD5dimT lymphocytes, showing that TRBC1 expression rate is 37.10% within the normal range (15-85%).
FIG. 14 is a scatter plot of CD3-CD45RA expression from TCRγδ -CD3+CD5dimT lymphocytes, as shown by nearly all of this group of lymphoid CD45RA being negative expressed (only 0.51% expressed);
FIG. 15 is a scatter plot of CD3-CD45RO expression of TCRγδ -CD3+CD5dimT lymphocytes, as shown by the group of lymphoid CD45RO being partially expressed 56.25%;
FIG. 16 is a scatter plot of CD3-CD57 expression of TCRγδ -CD3+CD5dimT lymphocytes, as shown by the 33.45% partial expression of this group of lymphoid CD57.
In the sample to be tested, the cell population is judged to be the cell population with normal immunophenotype in the normal control crowd antibody expression pattern template, and the cell population is consistent with the morphological analysis result.
2. Abnormal sample
The sample is a clinical sample which is determined to have obviously large granular lymphocytes by adopting peripheral blood smear morphological analysis. The antibody expression pattern analysis was performed as follows:
and obtaining flow cytometry detection result data of the sample to be detected. The cell population was divided into 3 regions based on the expression of CD45-SSC using a gate for CD45-SSC (side scattered light), and as shown in FIG. 17, granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) were each divided into 3 regions, and the lymphocyte region was the target cell population in the present embodiment, and the proportion of the total number of the nuclear cell occupied by lymphocytes was significantly increased, and the immunophenotype analysis was performed on this cell population.
FIG. 18 is a plot of CD3-CD56 immunophenotype scatter of lymphocytes, showing that CD3+ T lymphocytes account for 98.40% of lymphocytes, with a significant increase in proportion.
FIG. 19 is a scatter plot of CD3-CD2 expression of lymphocyte populations, as shown by T lymphocyte CD2 expression, without significant abnormalities.
FIG. 20 is a scatter plot of CD3-CD5 expression of lymphocyte populations, showing that CD3+CD5dim cells account for 91.11% of lymphocytes, with obvious clustered distribution, suspected of abnormal T lymphocytes, and continued further analysis in combination with other antibody markers.
FIGS. 21-25 are scatter plots of the lymphocyte populations for CD3-CD7, CD3-CD4, CD3-CD8, CD4-CD8, and CD3-CD16 expression, and can be examined for a decrease in CD7 expression for the population of CD3+CD5dim, for a negative CD16, and for a positive CD8 expression, and can be analyzed and confirmed by preliminary consideration of the fact that the population of CD3+CD5dim T lymphocytes are suspected to be T large granule lymphocytes, and binding to the antibody markers in the second set of antibodies.
FIG. 26 is a schematic of CD3-CD45 in the second set of antibodies, circling CD3+ T lymphocytes.
FIG. 27 is a scatter plot of CD3-TCRγδ expression of CD3+ T lymphocytes, circling CD3+ TCRγδ -T lymphocytes (default TCRαβT lymphocytes).
FIG. 28 is a scatter plot of CD3-CD5 expression of CD3+TCRγδ -T lymphocytes, as shown by 91.11% CD3+CD5dim cells.
FIG. 29 is a scatter plot of the expression of CD3-TRBC1 in TCRγδ -CD3+CD5dimT lymphocytes, showing that the TRBC1 expression rate was 0.02% (15-85% of the normal T lymphocytes) and it was determined as a clonal T lymphocyte.
FIG. 30 is a scatter plot of CD3-CD45RA expression in TCRγδ -CD3+CD5dimT lymphocytes, showing that the group of lymphoCD45 RA are almost positive with an expression rate of 99.85%;
FIG. 31 is a scatter plot of CD3-CD45RO expression of TCRγδ -CD3+CD5dimT lymphocytes, as shown by the fact that the group of lymphocytes CD45RO is almost negative, the expression rate is 0.33%;
FIG. 32 is a scatter plot of CD3-CD57 expression of TCRγδ -CD3+CD5dimT lymphocytes, as shown by the partial expression of 59.45% of this group of lymphoid CD57;
the immunophenotype indicates that the cell population of the sample to be detected is not in the normal control crowd antibody expression pattern template, is tumor T cell large granular lymphocyte and accords with morphological analysis results.
Example 4 comparison of detection accuracy of antibody combinations of the invention with antibody combinations of prior art methods
Comparing the antibody combination of example 1 of the present invention with the recommended detection antibody combinations (CD 3, CD4, CD5, CD7, CD8, CD56, CD57, tcrαβ, tcrγδ, fluorescein labeling of each antibody is referenced in table 1, and the amounts used are also referenced in table 1) in NCCN, the accuracy in detecting the same test cells.
1. Flow cytometry detection data of the test samples were obtained using the detection antibodies recommended in NCCN (CD 3, CD4, CD5, CD7, CD8, CD56, CD57, tcrαβ, tcrγδ).
The cell population was divided into 3 regions, i.e., granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) 3 regions (as shown in fig. 33), respectively, using a gate for CD45-SSC (side scattered light), and the lymphocyte region was the target cell population, and it was found that the lymphocyte occupied 34.55% of the total nuclear cell count, and the proportion was normal, and the immunophenotype analysis was performed on this cell population.
FIG. 34 is a plot of CD3-CD56 immunophenotype scatter of lymphocytes, showing that CD3+ T lymphocytes account for 84.66% of lymphocytes, with an elevated proportion.
FIG. 35 is a scatter plot of CD3-CD5 expression of lymphocyte populations. In this figure the T lymphocyte population of cd3+cd5dim represents 4.67% of lymphocytes.
FIGS. 36 to 41 are scatter plots of the expression of CD3-CD2, CD3-CD4, CD3-CD8, CD3-CD7, CD4-CD8, and CD3-CD57, respectively, for lymphocyte populations, and it can be seen that the population of T cells CD7 and CD2 of CD3+CD5dim are normally expressed, and that CD8 positive CD4 is negative, and that part of CD57 is expressed, and can be considered as T-large granular lymphocytes.
2. The antibody combination of the embodiment 1 of the invention is used for detecting the sample to be detected, and the flow cytometry detection result data of the sample to be detected is obtained.
The first set of antibody combinatorial data was analyzed as above (NCCN recommended combinatorial antibodies), circling cd3+cd5dim-expressed T lymphocytes;
the population of cd3+cd5dim T lymphocytes was further analyzed using a second panel of antibody combinations, as follows:
FIG. 42 is a scatter plot of CD3-TCRγδ expression of CD3+ T lymphocytes, circling CD3+ TCRγδ -T lymphocytes (default TCRαβT lymphocytes).
FIG. 43 is a scatter plot of the expression of CD3-TRBC1 in TCRγδ -CD3+CD5dimT lymphocytes, showing that TRBC1 expression rate is 22.79% (15-85% of normal T lymphocytes) and can be determined as normal T lymphocytes.
FIG. 44 is a scatter plot of CD3-CD45RA expression of TCRγδ -CD3+CD5dimT lymphocytes, as shown by the partial positivity of this group of lymphoCD 45RA with an expression rate of 40.59%;
FIG. 45 is a scatter plot of CD3-CD45RO expression for TCRγδ -CD3+CD5dimT lymphocytes, as shown by the group of lymphoCD45 RO being partially positive, at an expression rate of 43.62%;
combining several antibody results for TRBC1, CD45RA, CD45RO, this population of 4.67% cd3+cd5dimt lymphocytes was shown to be polyclonal T lymphocytes, but not neoplastic T lymphocytes.
The results of this example show that the antibody combination designed by the invention uses CD3+CD5dim zone as main target cell group, covers various antibodies recognizing T cell large granule lymphocytes such as CD45RA, CD45RO, TRBC1, and the like, and can better recognize neoplastic T cell large granule lymphocytes by integrating logic analysis strategies, thus having better accuracy than the detection antibodies recommended in NCCN.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

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.
2. The antibody combination for detecting T cell large granule lymphocytes according to claim 1, wherein the antibody of the antibody combination is a monoclonal antibody.
3. The antibody combination for detecting T cell large particle lymphocytes according to claim 2, wherein the monoclonal antibody is a fluorescein-labeled antibody selected from the group consisting of: FITC, PE, ECD, PE-Cy TM 5.5、PE-Cy7、APC、APC-750、PB、KO。
4. The antibody combination for detecting T cell large granule lymphocytes according to claim 3, wherein the CD8 and TRBC1 antibodies label fluorescein FITC; the CD4 and CD5 antibodies label fluorescein PE; the CD16 antibody labels fluorescein ECD; the CD56 and TCRγδ antibodies label fluorescein PE-Cy TM 5.5; the CD5 and CD45RO 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.
5. 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 4.
6. Use of the antibody combination for detecting T cell large granule lymphocytes according to any one of claims 1 to 4 or the kit according to claim 5 for the assisted detection of T cell large granule lymphocytes.
7. 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;
and the data analysis module is used for analyzing the acquired data and judging whether the acquired T lymphocytes are tumor T cell large-particle lymphocytes or not according to preset analysis logic and judgment standards.
8. The system for detecting T cell large granule lymphocytes according to claim 7, 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.
9. The system for detecting T cell large granule lymphocytes according to claim 8, wherein the antibody expression pattern of the test cell and the expression pattern of the normal control population antibody are established by: and (3) clustering the nucleated cells by using the data of the detection result of the flow cytometry, circling the target cell population, and analyzing the expression condition of each fluorescent antibody pair in the target cell population.
10. The system for detecting T cell large particle lymphocytes of claim 9, wherein said fluorescent antibody pair comprises: 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.
CN202311488821.XA 2023-11-09 2023-11-09 Antibody combination and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit Pending CN117384289A (en)

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