Mature t cell clonality detection reagent composition and uses thereof

Abstract

The application provides a mature T cell clonality detection reagent composition and application thereof. The reagent composition comprises five groups of antibodies, and is used for diagnosis and subtype judgment of mature T lymphocyte tumors. In application, four tubes are used in parallel or a first tube is combined with 1-3 tubes in the second, third and fourth tubes, wherein the first group of antibodies, the second group of antibodies, the third group of antibodies and the fourth group of antibodies are respectively used for different tube samples. The reagent composition of the application can be applied to flow cytometry for mature T cell clonality detection, and diagnosis and subtype judgment of mature T lymphocyte tumors.

Description

Technical Field

[0001] This invention relates to a reagent composition for rapid and efficient detection of mature T cell clonalness and its application, belonging to the field of hematological disease detection technology. Background Technology

[0002] Mature lymphocytic tumors, commonly known as lymphomas, are a group of common malignant tumors of the lymphohematopoietic system that seriously endanger human health. Lymphomas are diverse in type, with significant phenotypic differences, disease courses, and prognoses. Since mature T lymphocytes are the most abundant immune cells in the human body, reactive or unexplained monoclonal T cell proliferation (T-CUS) caused by various viral infections also complicates diagnosis. Furthermore, diseases such as aplastic anemia often present with oligoclonal proliferation of CD57-positive TCRαβ+ or TCRγδ+ T cells, requiring differentiation from lymphomas.

[0003] Mature T-lymphocytic tumors (MTCs) are a highly heterogeneous group of diseases originating from various mature T-cell subsets. They can infiltrate in leukemic or lymphoma forms, and their clinical behavior ranges from benign to highly aggressive. Most MTCs are CD4+TCRαβ+ T-cell types, a minority are CD8+TCRαβ+ T-cell types, a small minority are TCRγδ+ T-cell types, and some are completely negative for both TCRαβ and TCRγδ. A large number of reactive T cells may appear in the presence of viral infections, autoimmune diseases, drugs, or other malignancies. Reactive T cells are often CD8+ T-cell types; therefore, for CD8+ and CD3+CD56+ NKT cell types, especially those with high CD2 expression and persistently low CD7 expression (regardless of CD4 or CD8 positivity), and considering that T-CUS typically accounts for less than 5% of lymphocytes, these cases require careful assessment. Generally, unless the clinical presentation is extremely pronounced, a six-month follow-up is recommended before confirming the diagnosis.

[0004] Flow cytometry is an important tool for detecting mature T-lymphocyte tumors. In recent years, with the refinement of research on immune cell subsets and the deepening understanding of normal T cells and reactive cells, it has played an increasingly important role in the diagnosis and subtyping of mature T-lymphocyte tumors formed from the malignant transformation of these cells. However, because there are many types of mature T-lymphocyte tumors with significant phenotypic differences, clinical detection by flow cytometry has become challenging. Therefore, although numerous articles have been published over the years, they have primarily focused on differential analyses of several immunophenotypes within a single disease, lacking a comprehensive protocol for the diagnosis and differential diagnosis of systemic diseases, especially for subtyping. While the European Flow Cytometry Consortium (Euroflow) and the Mayo Clinic in the United States published relatively standardized detection protocols for mature T-lymphocyte tumors in 2012 and 2024, respectively, neither has established a complete and standardized protocol and procedure that includes the detection of abnormal T-cell phenotypes, the diagnosis of mature T-lymphocyte tumors after excluding other differential diagnoses, and subtyping.

[0005] Therefore, there is an urgent clinical need for a complete solution that can fully address the clonal characteristics of mature T lymphocytes, including tumor diagnosis and subtype determination. Summary of the Invention

[0006] One object of the present invention is to provide a reagent composition that enables rapid and efficient diagnosis and subtyping of mature T lymphocyte tumors using flow cytometry, particularly including all common and uncommon types, and can also differentiate other types that require identification, such as monoclonal T lymphocytosis of undetermined significance (T-CUS), reactive T cells, T lymphoblastic lymphoma / leukemia (T-LBL / ALL), and other tumors or diseases, thus preventing misdiagnosis.

[0007] Another object of the present invention is to provide the application of the reagent composition described herein in the diagnosis and subtype determination of mature T lymphocyte tumors.

[0008] On one hand, the present invention provides a reagent composition for detecting the clonality of mature T cells, the reagent composition comprising a first group of antibodies, and further comprising one or more groups of a second group of antibodies, a third group of antibodies, and a fourth group of antibodies, wherein:

[0009] The first group of antibodies includes fluorescently labeled CD7, CD117, CD3, CD4, CD5, CD8, CD56, CD45, and CD2 antibodies. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are used to add to the first flow cytometer tube in which the sample to be tested is in a single-cell suspension state.

[0010] The second group of antibodies includes fluorescently labeled CD57 antibody, TRBC2 antibody, CD3 antibody, TCRVδ1 antibody, TRBC1 antibody, CD8 (or CD4) antibody, TCRVδ2 antibody, CD45 antibody, and CD5 (or CD2) antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are added to the second flow cytometer tube containing the test sample in a single-cell suspension.

[0011] The third group of antibodies includes fluorescently labeled CD7, CD26, CD3, CD10, CD279, CD4, CD25, CD45, and CD5 (or CD2) antibodies. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are used to add to the flow cytometry tubes in which the test sample is in a single-cell suspension state.

[0012] The fourth group of antibodies consists of two parts. The first part includes fluorescently labeled CD99, CD30, CD3, CD4, CD45, and CD5 (or CD2) antibodies, with the fluorescent labeling order being FITC, PE, PerCP-Cy5.5, APC-Cy7, V500, and BV605. These antibodies are added to the flow cytometry tube four, where the sample to be tested is in a single-cell suspension state. The second part includes TCL1 antibody, fluorescently labeled APC, which is added to the flow cytometry tube four, which has already been treated with the fourth group of antibodies and subjected to cell lysis.

[0013] The reagent composition of this invention can be used for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors by flow cytometry. In specific applications, a scheme of using 4 tubes in parallel or combining the first tube with 1-3 tubes from the second, third, and fourth tubes is adopted. Each tube uses CD45 and T cell markers in combination with SSC to set up lymphocyte gates, and at the same time, CD45 / SSC is used to set up control cell populations such as mature granulocytes, monocytes, and nucleated erythrocytes, thereby accurately evaluating the tumor.

[0014] According to a specific embodiment of the present invention, a four-tube parallel scheme can be adopted, or tube one can be used in addition to tubes two to three of tube four. Specifically, to determine the clonality of mature T cells and identify the number of tumor cell clones, the flow cytometry tubes used include tube one and tube two. Specifically, the selection can be based on the CD4 and CD8 expression levels listed in Table 1. For example, if the tumor cells in the sample are CD4+CD8-, tubes one through four can be selected; or if the tumor cells in the sample are CD4+CD8-, the flow cytometry tubes used initially include tube one, tube two, and tube three. If tube three shows CD10 negativity, non-strong CD279 expression, and no loss of CD26, tube four is added; or if the cells in the sample are CD4-negative, the flow cytometry tubes used include tube one and tube two; or if the cells in the sample are CD4+CD8+, or if the tumor cells are outside the lymphocyte gate set by CD45 / side-scat (SSC), or if clinically suggestive of rare T-prolymphocytic leukemia / lymphoma, the flow cytometry tubes used include tube one, tube two, and tube four.

[0015] The solution of this invention can be more economical while ensuring detection accuracy, and at the same time, it helps to form standardization, reduce the misdiagnosis rate, and facilitate the realization of automation and artificial intelligence in the future.

[0016] According to a specific embodiment of the present invention, each antibody in the reagent composition of the present invention is a monoclonal antibody.

[0017] According to a specific embodiment of the present invention, each antibody in the antibody composition of the present invention is a fluorescently labeled antibody.

[0018] In this invention, by combining different antibodies with specific fluorescein, the reagent composition of this invention can achieve excellent staining effects for all fluorescein in each channel when used for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors.

[0019] According to a specific embodiment of the present invention, all antibody components in the reagent composition of the present invention are commercially available. Each antibody should meet the requirements of relevant industry standards.

[0020] According to a specific embodiment of the present invention, in the reagent composition of the present invention, the first group of antibodies is a mixture of CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody, and CD2 antibody in a volume ratio of 5:5:5:3:2:3:3:3:3. The second group of antibodies is a mixture of CD57 antibody, TRBC2 antibody, CD3 antibody, TCRVδ1 antibody, TRBC1 antibody, CD8 (or CD4) antibody, TCRVδ2 antibody, CD45 antibody, and CD5 (or CD2) antibody in a volume ratio of 5:5:5:3:2:3:3:3:3. The third group of antibodies is a mixture of CD7 antibody, CD26 antibody, CD3 antibody, CD10 antibody, CD279 antibody, CD4 antibody, CD25 antibody, CD45 antibody, and CD5 (or CD2) antibody in a volume ratio of 5:5:5:3:2:3:3:3:3. The first part of the fourth group of antibodies is a mixture of CD99 antibody, CD30 antibody, CD3 antibody, CD4 antibody, CD45 antibody, and CD5 (or CD2) antibody in a volume ratio of 5:5:5:3:3:3. The mixing ratios of the antibodies mentioned above refer to the mixing ratios when the titers of each antibody are essentially equivalent.

[0021] Another aspect of the present invention provides a reagent kit comprising a first container, a second container, a third container, a fourth container, and a fifth container, each container containing a first portion of a first group of antibodies, a second group of antibodies, a third group of antibodies, a first portion of a fourth group of antibodies, and a second portion of a fourth group of antibodies, respectively, of the reagent composition described in the present invention.

[0022] According to a specific embodiment of the present invention, the kit may further include one or more of the following: hemolysin, membrane-permeable agent, buffer solution, and flow cytometry tubes for use with a flow cytometer. These reagents and consumables are commercially available. The membrane-permeable agent is preferably a membrane-permeable agent comprising solution A and solution B. Each reagent material may be contained in a separate container.

[0023] The kit of the present invention can be used for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors.

[0024] Another aspect of the present invention provides the use of the reagent composition in preparing flow cytometry samples for a rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors.

[0025] According to a specific embodiment of the present invention, the process for preparing flow cytometry samples for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors includes the following steps:

[0026] (1) Add the sample to be tested into flow cytometry tubes one through four (or tube A, tube B, tube C, and tube D) respectively, so that it is in a single-cell suspension state, and ensure that the cell quantity is 1×10 6 / tube-1×10 7 / Tube;

[0027] (2) Add the first group of antibodies in the reagent composition of the present invention to tube one obtained in step (1), add the second group of antibodies in the reagent composition of the present invention to tube two obtained in step (1), add the third group of antibodies in the reagent composition of the present invention to tube three obtained in step (1), and add the fourth group of antibodies in the reagent composition of the present invention to tube four obtained in step (1). Incubate each flow cytometer at room temperature in the dark.

[0028] (3) Add film-breaking agent A solution to tube 4 after incubation in step (2) and continue incubation at room temperature in the dark;

[0029] (4) Add 1×hemolysin to tubes 1, 2 and 3 after incubation in step (2) and tube 4 after incubation in step (3), and continue incubation at room temperature in the dark.

[0030] (5) After centrifuging each flow cytometer tube incubated in step (4), remove the supernatant;

[0031] (6) Add membrane-breaking agent B and the second part of the antibody in the fourth group of the reagent composition of the present invention to the tube after removing the supernatant in step (5), and incubate at room temperature in the dark;

[0032] (7) Add PBS buffer to tubes 1, 2 and 3 after removing the supernatant in step (5) and tube 4 after incubation in step (6) respectively, wash, centrifuge, remove the supernatant, and resuspend the cells with PBS buffer to obtain the flow cytometry sample.

[0033] In this invention, unless otherwise specified or clearly determined from the context, the order of the described operation steps is not intended to limit the actual order of these steps.

[0034] According to a specific embodiment of the present invention, the sample to be tested can be bone marrow or peripheral blood, or it can be any sample that can be prepared into a single live cell and is suitable for flow cytometry detection, such as tissue specimens or body fluid specimens.

[0035] According to a specific embodiment of the present invention, in step (1), the volume of each sample added does not exceed 160 μl (if the patient has a low number of peripheral blood cells, the volume can exceed 160 μl if necessary, and the sample is centrifuged to remove the supernatant and concentrated).

[0036] According to a specific embodiment of the present invention, the dosage of each reagent can be based on the conventional dosage in the art or the manufacturer's recommended dosage.

[0037] According to a specific embodiment of the present invention, in the reagent composition of the present invention, the amount of the first group of antibodies added is 16-64 μl / tube, the amount of the second group of antibodies added is 16-64 μl / tube, the amount of the third group of antibodies added is 16-64 μl / tube, the amount of the first part of the fourth group of antibodies added is 12-48 μl / tube, and the amount of the second part of the fourth group of antibodies added is 2-8 μl / tube.

[0038] According to a specific embodiment of the present invention, the incubation time in step (2) can be 10-30 minutes.

[0039] According to a specific embodiment of the present invention, in step (3), the incubation time can be 5-20 minutes. The amount of film-breaking agent A added can be according to the manufacturer's recommended dosage, usually 100 μl / tube.

[0040] According to a specific embodiment of the present invention, in step (4), the incubation time can be 5-30 minutes. The amount of 1×hemolysin added is 2-3 ml / tube.

[0041] According to a specific embodiment of the present invention, in step (5), the centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0042] According to a specific embodiment of the present invention, in step (6), the amount of film-breaking agent B added can be based on the manufacturer's recommended dosage, usually 50 μl / tube. Incubation is typically required for about 10-30 minutes.

[0043] According to a specific embodiment of the present invention, in step (7), the amount of PBS buffer added for washing is 2-3 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes. The amount of PBS buffer added for resuspension is 0.5-1 ml / tube.

[0044] According to a specific embodiment of the present invention, when resuspending cells for flow cytometry analysis, each tube is sequentially equipped with a de-adhesion cell gate P1 and a viable cell gate P2 to obtain single viable cells; within gate P2, CD45 / SSC is used to set each blood cell gate; and:

[0045] The first gate is set up as follows: Within the P2 gate, observe the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym). Perform a total lymphocyte gate including both normal and abnormal lymphocytes. (During this gate setup, if lymphocytes are observed outside the lymphocyte gate when observing the concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and lym, the gate needs to be set up in reverse using the markers; if the boundary between lymphocytes and monocytes is unclear, CD4 can be used.) Weakly positive / negative T cell markers (CD2, CD3, CD5, CD7) are used to set up a monocyte gate 2, and removed from the lymphocyte gate for precise gate setting; within the lymphocyte gate, CD3-negative / CD7-positive or CD3-negative / CD56-positive cells are NK cells, and two-dimensional dot plots are displayed for any combination of CD3 with CD5, CD2, CD7, CD4, CD8, CD56, and CD117, respectively. T cells that are different from normal T cells are selected as abnormal T cells (AT) gates (if there are more than one group, AT1 and AT2 can be set separately);

[0046] The second gate is set up as follows: Within the P2 gate, observe the degree of concordance between CD2 or CD5, CD3, CD4 or CD8 markers and the lymphocyte gate (lym). Create a total lymphocyte gate including both normal and abnormal lymphocytes. (During this gate setup, if lymphocytes are observed outside the lymphocyte gate due to the concordance between CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers and lym, the gate needs to be set up in reverse. If the boundary between lymphocytes and monocytes is unclear, a weakly positive CD4 / negative T cell marker (CD2 or CD5, CD3) gate can be set up for monocytes, and removed from the lymphocyte gate for precise gate setup). Within the lymphocyte gate, display the expression of CD57, TRBC1, TRBC2, TCRVδ1, and TCRVδ2. Select T cells different from normal T cells as the abnormal T cell (AT) gate (if there are multiple groups, AT1 and AT2 can be set up separately).

[0047] The gates in tube 3 are set up as follows: Within the P2 gate, observe the degree of concordance between CD2 or CD5, CD3, CD4, CD7 markers and the lymphocyte gate (lym). Create a total lymphocyte gate including both normal and abnormal lymphocytes. (During this gate setup, if lymphocytes are observed outside the lymphocyte gate due to the concordance between CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers and lym, the gate needs to be set up in reverse. If the boundary between lymphocytes and monocytes is unclear, a weakly positive CD4 / negative T cell marker (CD2 or CD5, CD3, CD7) gate can be set up for monocytes, and removed from the lymphocyte gate for precise gate setup). Within the lymphocyte gate, display the expression of CD26, CD10, CD279, and CD25. Select T cells different from normal T cells as the abnormal T cell (AT) gate (if there are multiple groups, AT1 and AT2 can be set up separately).

[0048] The gates in tube 4 are set up as follows: Within the P2 gate, observe the degree of concordance between CD2 or CD5, CD3, CD4 markers and the lymphocyte gate (lym). Create a total lymphocyte gate including both normal and abnormal lymphocytes. (During this gate setup, if lymphocytes are observed outside the lymphocyte gate due to the concordance between CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers and lym, the gate needs to be set up in reverse using the markers. If the boundary between lymphocytes and monocytes is unclear, use a weakly positive CD4 / negative T cell marker (CD2 or CD5, CD3) gate to set up a monocyte gate 2, and remove it from the lymphocyte gate to achieve precise gate setup). Within the lymphocyte gate, display the expression of CD99, CD30, and TCL1. Select T cells different from normal T cells as the abnormal T cell (AT) gate (if there are multiple groups, AT1 and AT2 can be set up separately). In this tube, strong expression of the abnormal marker SSC / CD99 (bri) can also be used for gating to exclude T lymphoblastic lymphoma.

[0049] In this invention, CD45 expression strength refers to the following: using normal bone marrow as the standard, mature lymphocytes show strong CD45 expression, monocytes show strong CD45 expression, granulocytes show weak CD45 expression, and nucleated erythrocytes are CD45 negative. In this invention, moderate CD99 expression means the same expression level as normal lymphocytes, while strong CD99 expression means the expression intensity exceeds that of normal lymphocytes. Unless otherwise specified, the expression strength of each antigen is based on normal lymphocytes.

[0050] In this invention, SSC size refers to the size of normal lymphocytes and nucleated erythrocytes as a standard, where the SSC of normal bone marrow is considered small (or "smaller"), and larger than normal is considered large. FSC size refers to the size of normal major lymphocyte populations and nucleated erythrocytes as a standard, where the FSC of normal bone marrow is considered small (or "smaller"), and larger than normal is considered large.

[0051] In the use of terminology, in order to distinguish the differences in the expression of markers of different types of normal lymphocytes and the differences between abnormal phenotype cells (mainly tumor cells) and normal cells, the expression intensity of normal T cells is described as high expression (high, hi) and low expression (low, lo), while the expression of abnormal phenotype cells is described as strong expression (bright, bri) and weak expression (dim).

[0052] In the detection technology of this invention, tube one (or tube 1 or tube A) is the basic detection tube, using almost all major T-cell markers: CD2, CD3, CD4, CD5, CD7, CD8, and CD56. Tubes two (or tube 2 or tube B), three (or tube 3 or tube C), and four (or tube 4 or tube D) can be selectively used in combination with tube 1 or all at once, depending on the expression of CD4 and CD8. The main function of the T-cell markers in tube 1 is to detect T cells with abnormal phenotypes. Due to the complexity of T cells and the unknown nature of tumors, especially since most mature T-lymphocyte tumors originate in extramedullary tissues and may be found in the bone marrow and peripheral blood at a relatively low proportion, it is necessary to use all major markers for detection. In addition, about 5% of lymphomas are two or more clones. Especially in recent years, with the advancement of flow cytometry technology and the use of chimeric antigen receptor T cells (CAR-T) and targeted drugs, the detection rate of tumors with two or more clones has increased, thus further necessitating the use of all T-cell markers for detection. Tube 1 used CD117 in addition to T-cell markers. This is because some T-cell lymphomas abnormally express CD117. Furthermore, aplastic anemia is a common disease characterized by a reduced proportion of CD117-positive myeloid blasts in the bone marrow. This type of disease is immune-related and prone to T-cell oligoclonal abnormalities, requiring differentiation from T-cell lymphoma. Of course, in lymphomas with a high proportion of tumor cells, normal hematopoiesis can also be suppressed, leading to a reduced proportion of CD117+ myeloid blasts. Tubes 2-4 used either CD4 or CD8 (selected based on CD4 and CD8 expression in tube 1), CD3, and either CD2 or CD5 (selected according to Table 1; CD3 subtypes that are not easily lost are more likely to show abnormalities when combined with CD5, while CD3 subtypes that are easily lost are selected based on CD5's loss rate; if easily lost, such as ALCL, CD2 is chosen; if not easily lost, such as AITL, CD5 is chosen). T cells recognize antigenic peptides through the TCR, initiating an immune response. The TCR and CD3 together constitute the core molecules of T cells. Based on whether their TCR type is TCRαβ or TCRγδ, T cells are divided into αβT cells and γδT cells. In healthy individuals, 90%-95% of T cells are αβT cells (TCRαβ+), and 5%-10% are γδT cells (TCRγδ+). αβT cells mainly include two subsets: CD4+CD8- and CD4-CD8+, with a small number of CD4-CD8- double-negative (DN) and CD4+CD8+ double-positive (DP) cells. The γδT cell subset generally does not express CD4, and is partially CD8-negative and weakly CD8-positive.CD3+CD56+ T cells are called NKT cells. Some of them overlap with CD8+αβT cells and γδT cells, so their immunophenotype is CD4 negative and CD8 partially negative and weakly positive.

[0053] CD2, CD3, CD5, CD7, CD4, CD8, and CD56 are generally used to screen for T cell abnormalities. Normally, CD3 and CD5 are found on T cells, while CD2 and CD7 are found on both T cells and CD3-CD56+ NK cells. Therefore, the expression of CD3 and CD5 is generally consistent, as are that of CD2 and CD7. However, there are slight differences in the expression intensity of CD3 and CD5 between normal CD4+ T cells and CD8+ T cells. CD3 expression is relatively highest in γδT cells (although reactive γδT cells may show weakened CD3 expression). In αβT cells, CD4+ T cells express CD3 more strongly than CD8+ T cells. CD5 expression in αβT cells is slightly weaker in CD8+ T cells than in CD4+ cells, while CD5 expression in γδT cells is relatively weakest or even negative. CD7 expression is uniform in infants and young children, but with age, corresponding to the increase in memory T cells, the proportion of CD7-CD4+ T cells increases, and these memory T cells are also accompanied by high CD2 expression. CD7-T cells are more prominent in blood and bone marrow than in lymph nodes. The expression level of CD7 in CD8+αβT cells and γδT cells is similar. CD2 expression is relatively consistent in CD8+αβT cells and γδT cells, while in CD4+ T cells, CD2 expression can be either high or low.

[0054] In other words, in most cases, CD4(+) T cells show slightly higher expression levels of CD3 and CD5 than CD8(+) T cells; γδ T cells show the strongest CD3 expression and the weakest CD5 expression. Figure 1 (Table 2) These subtle differences are the basis for gnosing and identifying abnormalities in mature T lymphocyte tumors, and also the basis for discovering two tumor clones.

[0055] Figure 1 Expression of common biomarkers of T cell subsets in bone marrow specimens from healthy individuals. Figure 1 Images 1A-1H in the image are two-dimensional dot plots. Figure 1Images 1I-1K are superimposed one-dimensional histograms. Royal blue represents NK cells, pink represents CD3(+)CD56(+)NKT cells, brown represents γδT cells, light green represents CD8(+)T cells, and dark green represents CD4(+)T cells. Images 1A-1D show the normal lymphocyte population, including the expression of CD3 and CD56 (Image 1A), γδT and CD3 (Image 1B), CD2 and CD7 (Image 1C), and CD3 and CD5 (Image 1D). The αβT cell population is predominantly CD4(+) and CD8(+) (Image 1E), with some CD8(+) cells expressing CD57 (Image 1F). NK, NKT, and γδT cells are all CD4-negative, with some weakly positive CD8 cells (Image 1G) and some expressing CD57 (Image 1H). The expression levels of CD3 and CD5 show slight differences in γδT, CD4(+), and CD8(+)αβT (Figure 1I and Figure 1J). CD57 is expressed more in NK, NKT, γδT, and CD8(+)αβT, and less in CD4(+) (Figure 1K).

[0056] The second tube used clonal markers of the T-cell receptor (TCR), TRBC1, TRBC2, TCRVδ1, and TCRVδ2. The TCR constant beta chain (TRBC) is an important component of TCR αβ discovered in recent years. TRBC includes two types, TRBC1 and TRBC2. In normal individuals, approximately 30%-70% of T cells express either TRBC1 or TRBC2, meaning they express one or the other. Furthermore, the expression of TRBC1 and TRBC2 is balanced across different subsets of αβ T cells. However, in malignant cells of mature T lymphocyte tumors, TRBC1 and TRBC2 typically show restricted positive or negative expression (<15% or >85%), or double negative expression. Therefore, in the past, when TRBC2 was not available, TRBC1 alone could effectively evaluate the clonality of αβ T cells. However, it becomes less effective when encountering tumor cells with weak TRBC1 expression, or reactive T cells with strong TRBC1 expression (e.g., ...). Figure 13 However, this can easily lead to misdiagnosis. The introduction of TRBC2 in 2024, combined with TRBC1 (e.g., tube 2), can further and more perfectly resolve the false positive and false negative issues associated with using TRBC1 alone. Of course, T cells are far more complex than B cells; oligoclonal reactive cells and T-CUS can exhibit TRBC-restrictive characteristics, making differential diagnosis difficult.

[0057] Tube 2 contains TCRVδ1 and TCRVδ2 cells used to detect the monoclonal nature of γδT cells. Human γδT cell subsets are primarily composed of approximately six γ (Vγ2-5, Vγ8, Vγ9) subtypes and approximately eight δ (primarily Vδ2, Vδ1, Vδ3) subtypes. However, these subunits are not evenly distributed; there are tissue-specific subtype differences. Peripheral blood in healthy individuals is mainly composed of two subtypes: Vδ2 (>75%) and Vδ1 (≤10%). Therefore, clinically, TCRVδ2 and TCRVδ1 (one of which is <15% or >85%) are mostly used to assess the clonality of γδT cells. Normally, TCRVδ2 is dominant, but in γδT cell lymphomas, the Vδ1 subtype is dominant.

[0058]

[0059]

[0060] In addition to T-cell markers and comprehensive TCR monoclonal markers, tube 2 also selected CD57. It primarily targets CD4-type lymphomas, such as large granular T-lymphoblastic leukemia (LGLL), which is CD57 positive, and the subtype determination of γδT-cell lymphomas (hepatosplenic γδT-cell lymphoma is CD56+CD57-, and γδT-LGLL is CD56-CD57+). CD57 is a marker of senescence and depletion, normally found mainly in CD8+ αβT cells, γδT cells, CD3-CD56+ NK cells, and CD3+CD56+ NKT cells. CD4+ T cells rarely express CD57, but it may increase in old age or various inflammatory states. The reactive CD57+CD4+ cell phenotype is characterized by a persistent decrease in CD7 and high CD2 expression. γδT cells are a weak subset, but they may proliferate after infection, autoimmune diseases, neoplastic diseases, splenectomy, and allogeneic stem cell transplantation. CD57 exhibits partially negative and partially heterogeneous expression in normal γδT cells and NKT / NK cells. Therefore, the expression pattern of CD57, especially its correlation with TRBC1, TRBC2, TCRVδ1, and TCRVδ2, as well as the abnormal expression of T cell markers, becomes a key point for diagnosis and differential diagnosis.

[0061] In addition to T-cell markers, tube 3 selected CD26, CD10, CD279, and CD25, mainly targeting the CD4+CD8- subtype, which is a common mature T-lymphocyte tumor. The subtypes mainly include angioimmunoblastic lymphoma (AITL), adult T-cell lymphoma (ATLL), mycosis fungoides / Sézary syndrome (MF / SS), anaplastic large cell lymphoma (ALCL), and nonspecific peripheral T-cell lymphoma (PTCL, NOS). Although T-prolymphoblastic leukemia (T-PLL) is also predominantly CD4+CD8-, it is rare, and CD4+CD8+ is more characteristic. Therefore, the clinical focus is on the CD4+CD8+ phenotype. Subtype determination is shown in Table 1: CD4+CD8- with strong CD279 expression and CD10 positivity (70-90% probability) suggests angioimmunoblastic lymphoma (AITL); CD4+CD8- with strong CD25 expression suggests adult T-cell lymphoma (ATLL); CD4+CD8- with loss of CD26 and / or CD7 suggests mycosis fungoides / Sézary syndrome (MF / SS). T-cell lymphomas that cannot be clearly classified into the above CD4+CD8- subtypes are considered nonspecific peripheral T-cell lymphomas (PTCL, NOS). Because ALCL and T-PLL are very rare, the first three tubes are often used for diagnosis of the CD4+CD8- subtype. However, in clinical practice, if flow cytometry is not required for subtype determination, only tubes one and two are needed. If the proportion of tumor cells is very high and the diagnosis is clear, clonal assessment in tube two can be omitted, and only tubes one and three are selected.

[0062] Tube 4 is primarily used for mature T-lymphocytic tumors of the CD4+CD8+ and rare CD4+CD8- types. Representative examples include T-PLL and ALCL. A particularly important task for this phenotype is differentiating it from T-lymphoblastic lymphoma / leukemia (T-LBL / ALL). Therefore, this tube selected CD99, CD30, and cytoplasmic TCL1. Positive CD30 with increased forward light scattering (FSC) and lateral light scattering (SSC), i.e., CD45 / SSC dotted plot outside the lymphocyte hilum, often with loss of T-cell markers, suggests anaplastic large cell lymphoma (ALCL). CD4+CD8- or CD4+CD8+ with positive cytoplasmic TCL1 and weak CD99 expression suggests T-prolymphocytic leukemia (T-PLL). Strong CD99 expression is seen in over 95% of T-LBL / ALL, but is extremely rare in mature T-lymphocytic tumors. Furthermore, CD7 expression intensity is crucial for differentiating between T-LBL / ALL and mature T lymphocyte tumors. During normal T cell development, CD7 is strongly expressed in the early stages, decreasing as cells mature. Therefore, over 95% of T-LBL / ALL cases exhibit strong CD7 expression, while mature T lymphocyte tumors show normal CD7 expression, and in many subtypes, CD7 is weakened or even absent.

[0063] On the other hand, the present invention also provides a device for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors, the device comprising a detection unit and an analysis unit, wherein:

[0064] The detection unit includes reagent materials for detecting samples from the individual to be tested by flow cytometry, and for obtaining the detection results of the samples; the reagent materials include the reagent composition described in this invention;

[0065] The analysis unit is used to analyze the detection results of the detection unit.

[0066] According to a specific embodiment of the present invention, in a rapid and efficient method for diagnosing and identifying subtypes of mature T-lymphocyte tumors, the application process of the detection unit includes: processing the sample to be tested using the reagent composition described in the present invention to prepare a flow cytometry sample; and performing flow cytometry detection. The analysis process of the analysis unit includes: analyzing the detection results to rapidly and accurately diagnose mature T-lymphocyte tumors and exclude other diseases that may cause misdiagnosis, especially reactive T cells, T-CUS, other tumors and diseases, etc.

[0067] According to a specific embodiment of the present invention, when the device of the present invention is used for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors, the gating analysis of each flow cytometer can be performed according to the aforementioned operation.

[0068] According to a specific embodiment of the present invention, in the device for rapid and efficient screening of mature T lymphocyte tumors, the analysis unit may further include a module for further determining the disease based on the gating analysis results (the determination described in this invention includes screening, suggestion, auxiliary diagnosis, and diagnosis). The present invention, through multi-marker gating, compares the displayed cell populations with corresponding normal cells to identify tumor cells, enabling rapid and accurate diagnosis and differentiation from other diseases that may be misdiagnosed or missed, especially T-CUS and reactive T cells, which are morphologically and immunophenotypically very similar, as well as other tumors. In summary, the present invention avoids these pitfalls in addressing the aforementioned challenges.

[0069] According to a specific embodiment of the present invention, the device for rapid and efficient screening of mature T lymphocyte tumors of the present invention is used for one or more of the following mature T cell clonal detection methods:

[0070] (1) T-cell clonal detection for diagnosis and differential diagnosis of mature T-cell tumors (commonly known as lymphomas): The flow cytometry tubes used include tube one and tube two;

[0071] (2) Detection of the number of clones of mature T cell tumors: The flow cytometry tubes used include tube one and tube two;

[0072] (3) Determine the mature T-cell tumor subtype:

[0073] The tumor cells in the sample to be tested are CD4+CD8-, selected from tubes one to four; or

[0074] The tumor cells in the sample to be tested were CD4+CD8-. The flow cytometry tubes used included tube one, tube two, and tube three. Tube three was CD10 negative, CD279 was not strongly expressed, and CD26 was not lost. Tube four was also added.

[0075] The tumor cells in the sample to be tested were CD4 negative, and the flow cytometry tubes used included tube one and tube two; or

[0076] The tumor cells in the sample to be tested are CD4+CD8+, or the tumor cells are outside the lymphocyte gate set by CD45 / lateral angle light scattering, or clinically suggestive of rare T prolymphocytic leukemia / lymphoma; the flow cytometry tubes used include tube one, tube two and tube four.

[0077] According to a specific embodiment of the present invention, when analyzing four tubes of samples, the CD45 / SSC used in each tube is used to initially screen whether the proportions of common cell groups (mature lymphocytes, monocytes, differentiated granulocytes, nucleated erythrocytes) in normal specimens are normal, and whether there is a high proportion of tumor cells. Further judgment is made in combination with other markers of each tube.

[0078] According to a specific embodiment of the present invention, when the analysis unit of the present invention is used to analyze the detection results of the detection unit, it can output the mature T cell clonal detection results according to one of the following judgment methods:

[0079] Mature T-lymphocyte tumors: Positive cells are present in the abnormal T-lymphocyte (AT) gate of the CD45 / SSC-gated lymphocytes or the reverse-gated T-lymphocyte gate of any flow cytometry, and the following differential diagnoses 1-3 are excluded (i.e., exclude T-lymphoblastic lymphoma, exclude myeloid or histiocytic dendritic cell tumors, exclude clonal T cells of undetermined significance (T-CUS) or reactive T cells):

[0080] Differential diagnosis 1: Tumor cells strongly express CD99 (bri), and the test result is T lymphoblastic lymphoma; or, CD7, CD5, CD3, CD8, and CD2 are all negative, cytoplasmic CD3 is positive, and TdT and / or CD34 are positive, and the test result is T lymphoblastic lymphoma.

[0081] Differential diagnosis 2: Tumor cells strongly express CD99 (bri), express CD4 (only CD4, or express CD4 and CD56), and do not express CD7, CD5, CD3, CD2, CD8, or cytoplasmic CD3. Add myeloid and tissue dendritic cell markers CD64, CD33, CD13, MPO, HLA-DR, CD14, CD42a, CD123, CD303, CD304, CD163, and CD68. Determine whether it is a myeloid or tissue dendritic cell tumor based on the test results.

[0082] Differential diagnosis 3: Abnormal phenotype: mature T cells account for <5% of lymphocytes, or the immunophenotype is high expression of CD2 / continuous weak expression of CD7 / positive CD4 or CD8. Follow up for 6 months to determine whether it is clonal T cells of undetermined significance (T-CUS) or reactive T cells.

[0083] Selectively, for samples with the above test results indicating mature T-lymphocyte tumors, further subtype testing shall be performed according to any one of the following (1)-(7):

[0084] (1) CD4+CD8- with strong expression of CD279 and positive CD10, subtype detection is angioimmunoblastic lymphoma (AITL).

[0085] (2) CD4+CD8- with strong CD25 expression, subtype detection indicates adult T-cell lymphoma (ATLL);

[0086] (3) CD4+CD8- with loss of CD26 and / or CD7, subtype detection is mycosis fungoides / Sézary syndrome (MF / SS);

[0087] (4) CD4+CD8- with CD30 positivity, and increased forward scattering (FSC) and side scattering (SSC) (i.e., tumor cells outside the lymphocyte gate set by CD45 / side scattering), loss of T cell markers, and subtype detection is anaplastic large cell lymphoma (ALCL).

[0088] (5) CD4+CD8 negative or positive with positive cytoplasmic TCL1, CD99 not strongly expressed, subtype detection is T prolymphocytic leukemia (T-PLL).

[0089] (6) CD4+CD8 negative, does not meet the AITL, ATLL, MF / SS, ALCL, T-PLL subtypes in (1)-(5) above, and the subtype test is non-specific peripheral T-cell lymphoma (PTCL, NOS).

[0090] (7) CD4-CD8+ with high expression of CD57, subtype detection showed large granular T lymphocyte leukemia (LGLL);

[0091] (8) CD4-CD8 negative or positive with TCRVδ1 or TCRVδ2, expressing CD56, and subtype detection is liver and spleen γδT cell lymphoma.

[0092] In summary, this invention provides a reagent composition and its application for rapid and efficient diagnosis and subtype determination of mature T-lymphocyte tumors using flow cytometry. This invention has the following advantages: ① Based on the understanding of the diagnosis and differential diagnosis of immunophenotypes in a large number of tumor cases, a comprehensive diagnostic and differential diagnostic scheme has been designed, greatly reducing the rate of missed diagnoses and misdiagnoses. ② In terms of application, it can quickly make accurate diagnoses. ③ This invention adopts a rapid and efficient method for diagnosing and determining the subtype of mature T-lymphocyte tumors. It allows for premixed antibodies, greatly reducing workload, improving efficiency, and accelerating reporting time. Clinical patients can obtain accurate diagnoses early and receive appropriate treatment, thereby improving remission and survival rates. ④ A significant factor that has long constrained the development of flow cytometry is the individualization of protocols and excessive manual operation, making automation, standardization, and normalization difficult. This invention can create conditions for subsequent sample preprocessing machines, automated sample loading in flow cytometers, immobilization of data analysis, and especially for the future development of artificial intelligence. ⑤ Based on the contribution, specificity, and coverage of biomarker combinations for diagnosis and differential diagnosis, this invention further develops a scoring system. Combined with computer software, this system can make accurate and rapid judgments with high sensitivity and specificity. The detection and analysis methods of this invention can meet the current needs of clinical flow cytometry diagnosis and are suitable for widespread application and promotion. Attached Figure Description

[0093] Figure 1 This shows the expression of common biomarkers of T cell subsets in bone marrow specimens from healthy individuals.

[0094] Figures 2-3 This invention shows the results of gating analysis of flow cytometry analysis of bone marrow specimens from patients with autoimmune diseases in tubes 1 and 2 according to a specific embodiment of the present invention.

[0095] Figures 4-5 This invention shows the results of gating analysis of flow cytometry analysis of bone marrow specimens from patients with aplastic anemia in tubes 1 and 2 according to a specific embodiment of the present invention.

[0096] Figures 6-8 This invention displays the results of flow cytometry analysis of bone marrow specimens from mature T-lymphocyte tumors (two clonal tumors, AITL and LGLL) in a specific embodiment of the present invention, using gating analysis tubes 1-3.

[0097] Figures 9-11 Bone marrow specimens from patients with the same mature T-lymphocyte tumor MF / SS were analyzed by flow cytometry using gating analysis of tubes 1-3.

[0098] Figures 12-15 Gating analysis was performed on bone marrow specimens from patients with the same mature T-lymphocytic ATLL (all tumors), using tubes 1-3. Figure 13 and Figure 14 Two different methods were used to analyze the same tube.

[0099] Figures 16-19 For flow cytometry analysis of bone marrow specimens from patients with the same mature T-lymphocytic ALCL, gating analysis was performed on tubes 1-4. Figure 17 The old solution differs from the optimized solution of this invention mainly in that PE is TCRγδ instead of TRBC2.

[0100] Figures 20-22 Gating analysis was performed on bone marrow specimens from patients with the same mature T-lymphocyte tumor PLL in tubes 1, 2, and 4 using flow cytometry.

[0101] Figures 23-26 This invention displays the results of flow cytometry analysis of bone marrow specimens from mature T-lymphocyte tumors (PTCL, NOS) in a specific embodiment of the present invention, using gating analysis tubes 1-4.

[0102] Figures 27-28 This display shows the gating analysis results of flow cytometry on bone marrow specimens of mature T-lymphocyte tumors (hepatic and splenic γδ T-cell lymphomas) in a specific embodiment of the present invention, using tubes 1-2. Tube 2 uses the older scheme, and its main difference from the optimized scheme of the present invention is that PE is TCRγδ instead of TRBC2. Detailed Implementation

[0103] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the following detailed description, in conjunction with specific embodiments and the technical solutions of this invention, is provided. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this invention. In the embodiments, all original reagent materials are commercially available. Experimental methods not specifically specified are conventional methods and conditions well-known in the art, or are performed according to the conditions recommended by the instrument manufacturer.

[0104] Example 1: Preparation of Reagents

[0105] The antibody combination used in this embodiment is,

[0106] The first group consists of CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody, and CD2 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. The above nine monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3:3:3 and placed in the first container.

[0107] The second group consists of: CD57 antibody, TRBC2 antibody, CD3 antibody, TCRVδ1 antibody, TRBC1 antibody, CD8 (or CD4) antibody, TCRVδ2 antibody, CD45 antibody, and CD5 (or CD2) antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. The above nine monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3:3:3 and placed in the second container.

[0108] The third group consists of: CD7 antibody, CD26 antibody, CD3 antibody, CD10 antibody, CD279 antibody, CD4 antibody, CD25 antibody, CD45 antibody, and CD5 (or CD2) antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. The above nine monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3:3:3 and placed in the third container.

[0109] The fourth group consists of: CD99 antibody, CD30 antibody, CD3 antibody, CD4 antibody, CD45 antibody, and CD5 (or CD2) antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, APC-Cy7, V500, and BV605. The above six monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:3:3 and placed in the fourth container.

[0110] The second component of the fourth group of antibodies is: cytoplasmic TCL1 monoclonal antibody, labeled with fluorescein as APC; it is placed in the fifth container.

[0111] All antibodies used in this embodiment are commercially available. Among them, TRBC1 PE, TRBC1 APC, and TRBC2 PE are products of Henan Kaipurui Biotechnology Co., Ltd., while TCRVδ1 PE-Cy7, TCRVδ2 BV421, cytoplasmic TCL1 APC, and CD30 PE are products of Becton Dickinson, Inc., USA. The remaining fluorescein-labeled antibodies are mainly products of Becton Dickinson, Inc., USA.

[0112] Optional reconstituted cell lysis buffer (hemolysin) was placed in the sixth container, membrane permeabilizer A in the seventh container, membrane permeabilizer B in the eighth container, and PBS buffer in the ninth container. Hemolysin, membrane permeabilizer, and PBS buffer are commercially available. The cell lysis buffer and membrane permeabilizer were products of Becton Dickinson (USA), and the PBS buffer was a product of Beckman Coulter (USA).

[0113] Example 2: Specimen Processing

[0114] Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples into flow cytometry tube A, ensuring that the added cell quantity is approximately 2 × 10⁶ cells / mL. 6 The cells are in a single-cell suspension state. Then, according to Table 3, 32 μl of nine different fluorescently labeled monoclonal antibody reagents for cell membranes are added to the flow cytometry tube. After thoroughly mixing with the cell suspension, the cells are incubated at room temperature in the dark for 15 minutes. Then, 3 ml of 1× hemolysin is added, and the cells are incubated in the dark for 10 minutes to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS is added and mixed. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, the cells are resuspended in 0.5 ml of PBS buffer. This is the processed sample, which is ready for flow cytometry analysis.

[0115] Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples to tube B of the flow cytometry tube, ensuring that the added cell quantity is approximately 2 × 10⁻⁶. 6The cells are in a single-cell suspension state. Then, according to Table 3, 32 μl of nine different fluorescently labeled monoclonal antibody reagents for cell membranes are added to the flow cytometry tube. After thoroughly mixing with the cell suspension, the cells are incubated at room temperature in the dark for 15 minutes. Then, 3 ml of 1× hemolysin is added, and the cells are incubated in the dark for 10 minutes to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS is added and mixed. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, the cells are resuspended in 0.5 ml of PBS buffer. This is the processed sample, which is ready for flow cytometry analysis.

[0116] Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples into the flow cytometry tube C, ensuring that the added cell quantity is approximately 2 × 10⁻⁶. 6 The cells are in a single-cell suspension state. Then, according to Table 3, 32 μl of nine different fluorescently labeled monoclonal antibody reagents for cell membranes are added to the flow cytometry tube. After thoroughly mixing with the cell suspension, the cells are incubated at room temperature in the dark for 15 minutes. Then, 3 ml of 1× hemolysin is added, and the cells are incubated in the dark for 10 minutes to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS is added and mixed. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, the cells are resuspended in 0.5 ml of PBS buffer. This is the processed sample, which is ready for flow cytometry analysis.

[0117] Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples into flow cytometry tubes, ensuring that the added cell quantity is approximately 2 × 10⁻⁶. 6 The cells are in a single-cell suspension state. Then, according to Table 3, 24 μl of six different fluorescently labeled monoclonal antibody reagents for cell membranes in the fourth container are added to the flow cytometry tube. After thorough mixing with the cell suspension, the tube is incubated at room temperature in the dark for 15 minutes. 100 μl of membrane-disrupting agent A is added, and the tube is incubated at room temperature in the dark for 5 minutes. 3 ml of 1× hemolysin is added and mixed, and the tube is incubated in the dark for 10 minutes to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 50 μl of membrane-disrupting agent B and 2 μl of the component from the fifth container (2 μl of cytoplasmic monoclonal antibody TCL1-APC) are added. The tube is incubated at room temperature in the dark for 15 minutes. 3 ml of PBS buffer is added and mixed, and the tube is centrifuged at 1500 rpm for 5 minutes. After removal of the supernatant, the cells are resuspended in 0.5 ml of PBS buffer. This is the processed sample, ready for flow cytometry analysis.

[0118]

[0119] Example 3: Specimen Detection

[0120] The specimens processed according to the method in Example 2 were analyzed on a Becton Dickinson 3-laser 10-color FACS Canto plus flow cytometer in the United States. It is preferred to obtain 1 million cells per tube (at least 300,000 is recommended). The data were then analyzed using Diva 2.8 software or other software such as Kaluza.

[0121] The gating process for flow cytometry analysis is as follows: ① Fixed gating: Adhesive cell gating, live cell gating, and blood cell gating are removed sequentially; ② Precise lymphocyte gating: The degree of concordance between T cell markers and the lymphocyte gating (lym) set by CD45 / SSC is observed to ensure no lymphocytes are outside the lymphocyte gating; otherwise, the markers are used for reverse gating. If the boundary between lymphocytes and monocytes is unclear, a monocyte gating 2 is set using weakly positive CD4 / negative T cell markers, and cells are removed from the lymphocyte gating to achieve precise gating; ③ Within the precisely set lymphocyte gating, common expression patterns and developmental patterns of various marker combinations are displayed. Based on the differences from normal cells, tumor cells (potentially more than one group) are identified. Specifically:

[0122] 1. Fixed gate: It consists of deadhesion cell gate, live cell gate, and blood cell gate, which are connected in series.

[0123] De-adhesion cell gate: First, the de-adhesion cell gate (denoted by P1) is set using the area (A) and height (H) of the forward scatter (FSC). Adhesive cells can be removed by calculating FSC - area (A) / height (H), based on the principle that cells are spherical and A and H are positively correlated. See... Figures 2-28 The first picture from left to right in the top row shows a door.

[0124] Viable cell phylogenetics: Cells in P1 are phylogenetically gorged using FSC / side scatter (SSC) (denoted as P2) to obtain single viable cells. The principle of FSC / SSC is that viable cells exhibit a near-normal distribution in size and granularity, clustering around a central point and clearly demarcated from dead cells, apoptotic cells, debris, and background noise. See [link to FSC / SSC]. Figures 2-28 The second picture from left to right in the top row shows a door.

[0125] Blood cell phylogenetic analysis: Within a single live cell phylogenetic stage (P2 phylogenetic stage), CD45 / SSCs are used to establish various blood cell phylogenetic groups, allowing for the general observation of lymphocytes, monocytes, granulocytes, nucleated erythrocytes, and the presence of obvious tumor cells or abnormal cells. CD45 / SSCs are used to roughly differentiate between blood cell groups based on differences in CD45 fluorescence intensity (mature lymphocytes > monocytes > granulocytes > nucleated erythrocytes) and SSC size (eosinophils > granulocytes > monocytes > mature lymphocytes > nucleated erythrocytes). See [link to documentation]. Figures 2-28 Image of CD45 / SSC door in the image.

[0126] 2. Precisely define lymphocyte and tumor cell gates:

[0127] In tube A: Within gate P2, observe the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) to ensure no lymphocytes are outside the lymphocyte gate. Otherwise, reverse gate setting using markers is required. If the boundary between lymphocytes and monocytes is unclear, use a weakly positive CD4 / negative T cell marker (CD2, CD3, CD5, CD7) to set monocyte gate 2, and remove them from within the lymphocyte gate to achieve precise gate setting. Within the precisely set lymphocyte gate, CD3-negative / CD7-positive or CD3-negative / CD56-positive cells represent NK cells. Create a two-dimensional dot plot of CD3, CD5, CD2, CD7, CD4, CD8, CD56, and CD117. Select T cells different from normal T cells as abnormal T cell (AT) gates. If there are multiple groups, set AT1 and AT2 separately.

[0128] Tube B: Within gate P2, observe the degree of concordance between CD2 or CD5, CD3, CD4 or CD8 markers and the lymphocyte gate (lym) to ensure that no lymphocytes are outside the lymphocyte gate. Otherwise, reverse gate setting using markers is required. If the boundary between lymphocytes and monocytes is unclear, use a weakly positive CD4 / negative T cell marker (CD2 or CD5, CD3) to set monocyte gate 2, and remove it from within the lymphocyte gate to achieve precise gate setting. Within the precisely set lymphocyte gate, display the expression of CD57, TRBC1, TRBC2, TCRVδ1, and TCRVδ2. Select T cells different from normal T cells as abnormal T cell (AT) gates. If there are multiple groups, set AT1 and AT2 separately.

[0129] In tube C; within the P2 gate, observe the degree of anastomosis between CD2 or CD5, CD3, CD4, CD7 markers and the lymphocyte gate (lym) to ensure that no lymphocytes are outside the lymphocyte gate. Otherwise, reverse gate setting using markers is required. If the boundary between lymphocytes and monocytes is unclear, use a weakly positive CD4 / negative T cell marker (CD2 or CD5, CD3, CD7) to set up monocyte gate 2, and remove it from within the lymphocyte gate to achieve precise gate setting. Within the precisely set lymphocyte gate, display the expression of CD26, CD10, CD279, and CD25. Select T cells different from normal T cells as abnormal T cell (AT) gates. If there are multiple groups, set AT1 and AT2 separately.

[0130] The main observations in tube D are as follows: Within the P2 gate, selectively, tube four is gated as follows: Within the P2 gate, observe the degree of concordance between CD2 or CD5, CD3, CD4 markers and the lymphocyte gate (lym) to ensure that no lymphocytes are outside the lymphocyte gate; otherwise, the markers need to be used to reverse the gate. If the boundary between lymphocytes and monocytes is unclear, use a weakly positive CD4 / negative T cell marker (CD2 or CD5, CD3) to set up a monocyte gate 2, and remove it from within the lymphocyte gate to achieve precise gate setting. Within the precisely set lymphocyte gate, the expression of CD99, CD30, and TCL1 is displayed. Select T cells that are different from normal T cells as the abnormal T cell (AT) gate. If there are multiple groups, set AT1 and AT2 separately.

[0131] 3. Precisely locate target cells based on observed marker combinations.

[0132] Flow cytometry is used to diagnose and subtype mature T-lymphocytic tumors, and the process is mainly divided into three steps. First, abnormal cells are identified by finding abnormally expressed cell populations, which may be multiple, and these are confirmed as monoclonal using TCR clonality markers. Second, reactive cells, T-CUS, and other tumors are excluded to confirm the diagnosis of mature T-lymphocytic tumors. Third, the subtype is determined based on the expression of characteristic markers of common subtypes.

[0133] (1) Abnormal expression

[0134] Changes in mature T cell phenotype include: ① loss of T cell antigens (such as CD2, CD3, CD5, CD7); ② altered fluorescence intensity of normally expressed antigens; ③ abnormal expression of non-T lineage antigens, such as CD10 and CD117; ④ homogeneous expression of antigens that are normally expressed in small amounts or not expressed at all, such as CD30, CD25, CD57, CD56, and CD279; and ⑤ a significant increase in the proportion of weak cell populations under normal circumstances, accompanied by abnormal expression.

[0135] (2) Monoclonal test

[0136] T cells are divided into αβ T cells and γδ T cells. αβ T cells can be identified by restricted expression of TRBC1 and TRBC2 (one of which has an expression rate >85% or <15%), while γδ T cells can be identified by restricted expression of TCRVδ1 and TCRVδ2 (one of which has an expression rate >85% or <15%). The absence of expression in either indicates an abnormality. It is important to note that T cells are the primary immune cells in humans. To prevent a large number of normal background cells from masking the monoclonal nature of a small number of tumor cells, or to identify multiple tumor cell populations, it is necessary to pinpoint the abnormally expressing cell populations based on a combination of markers such as CD2, CD3, CD4, CD5, CD7, CD8, and CD56. This involves precise gating to evaluate the restricted expression of TCRs (TCRVβ, TRBC1, TRBC2, TCRVδ1, TCRVδ2, etc.) in tumor cells.

[0137] (3) Eliminate other possibilities

[0138] Excluding reactive cells and T-CUS: Viral infections, autoimmune diseases, other tumors, old age, and T-CUS can all show changes in pan-T cell markers (CD2, CD3, CD5, CD7), a significant increase in subsets such as CD4+CD8+ T cells, TCRγδ cells, and NKT cells, an elevated or decreased CD4 / CD8 ratio, and even restricted expression of TCR. Therefore, for NKT, CD8+ T cells, CD4+CD8+ T cells (reactive expression pattern, where the expression intensity is consistent with normal CD4 or normal CD8 cells), and CD4+ T cell phenotypes with persistently weak CD7 expression and high CD2 expression, if there are no obvious clinical manifestations related to lymphocytic tumors such as hepatosplenomegaly and / or lymphadenopathy, persistent low-grade fever, etc., and the proportion of abnormal cells is low (e.g., less than 5% of lymphocytes), careful evaluation is required, as they are more likely to be reactive cells or T-CUS.

[0139] Excluding other tumors: ① If strong CD99 expression is present, along with tumor cells expressing CD7 and / or CD5 and / or CD3 and / or CD8 and / or CD2 positivity, then T-lymphoblastic lymphoma should be considered rather than mature T-lymphoblastic tumors; ② If strong CD99 expression is present, and CD7, CD5, CD3, CD8, and CD2 are all negative, cytoplasmic CD3 and TdT and / or CD34 should be tested. If cytoplasmic CD3 is positive, and the early markers TdT and / or CD34 are positive, then T-lymphoblastic lymphoma should still be considered rather than mature T-lymphoblastic lymphoma. Mature T lymphocyte tumors; ③ If strong CD99 expression is observed, only CD4 is expressed, or CD4 and CD56 are expressed, but CD7, CD5, CD3, CD2, CD8, and cytoplasmic CD3 are not expressed, then myeloid and tissue dendritic cell markers such as CD64, CD33, CD13, MPO, HLA-DR, CD14, CD42a, CD123, CD303, CD304, CD163, and CD68 need to be added to determine whether it is a myeloid and tissue dendritic cell tumor.

[0140] (4) Subtype determination

[0141] Based on the typical immunophenotypes of common mature T-cell lymphomas in Table 1, subtype identification was performed as follows: ① CD4+CD8- with strong CD279 expression and CD10 positivity (70-90% probability) suggests angioimmunoblastic lymphoma (AITL); ② CD4+CD8- with strong CD25 expression suggests adult-onset T-cell lymphoma (ATLL); ③ CD4+CD8- with loss of CD26 and / or CD7 suggests mycosis fungoides / Sézary syndrome (MF / SS); ④ CD4+CD8- with positive CD30 and increased forward scattering (FSC) and lateral scattering (SSC), often with loss of T-cell markers, suggests... ⑤ CD4+CD8 negative or positive with positive cytoplasmic TCL1 and non-strong CD99 expression, consider T-prolymphocytic leukemia (T-PLL); ⑥ CD4+CD8 negative, not meeting the above criteria for AITL, ATLL, MF / SS, ALCL, or T-PLL, consider nonspecific peripheral T-cell lymphoma (PTCL, NOS); ⑦ CD4-CD8+ with high CD57 expression, consider large granular T-cell leukemia (LGLL); ⑧ CD4-CD8 negative or positive with TCRVδ1 or TCRVδ2, often expressing CD56, consider hepatosplenic γδT-cell lymphoma.

[0142] This protocol was first used in 2022. Initially, due to the lack of TRBC2, TCRγδ PE was used. From February 2022 to September 2024, 2109 tests were conducted from 1235 patients, with 364 positive samples from 202 patients. After purchasing TRBC2 in October 2024, all tests were switched to this protocol. From October 2024 to the end of June 2025, 414 tests were completed from 259 patients, with 84 positive samples from 52 patients. To date, a total of 2523 tests have been completed from 1494 patients, with 448 positive samples from 254 patients. The median age of patients was 55 years (range 1-88 years), and the median percentage of tumor cells was 2.32% (range 0.01%-93.61%). Based on morphological, genetic, pathological, and clinical follow-up and feedback, the sensitivity, specificity, positive predictive value, and negative predictive value from February 2022 to September 2024 were 97.49%, 99.34%, 96.15%, and 99.57%, respectively. From October 2024 to June 2025, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.81%, 99.76%, 98.81%, and 99.76%, respectively. The total sensitivity, specificity, positive predictive value, and negative predictive value were 97.74%, 99.41%, 96.65%, and 99.60%, respectively.

[0143] In this invention, tube A is a universal screening tube, while tubes B, C, and D can be used together or selectively. Tube B contains a clonal marker. The use of TRBC1 solves the problem of T-cell clonal testing in most cases; however, in 9.5% of patients, TRBC1 expression is weakened, making accurate evaluation of monoclonal inconsistency difficult. Alternatively, the presence of two clones (flow cytometry can detect that 5% of mature lymphocytic tumors consist of two clones, but recent targeted therapy and gene sequencing have revealed that most patients have more than two clones, only the weaker clone is too small or has very little immunophenotypic difference to be distinguishable) may prevent TRBC1 from being restricted in expression, or... Figure 13 In reactive cells, increased TRBC1 expression can easily lead to misdiagnosis and missed diagnosis. The use of TRBC2 in this invention almost completely solves this problem. The TRBC2-included approach of this invention improves both sensitivity and specificity.

[0144] The contribution of T cell markers, such as CD2, CD3, CD4, CD5, CD7, CD8, CD56, and CD45, is twofold. Firstly, they help determine the accuracy of CD45 / SSC in lymphocyte phylogenetic analysis, preventing the omission of tumor cells outside the lymphocyte phylogenetic system due to high FSC / SSC or weakened CD45. Secondly, since 5% of lymphomas have two clones, using a combination of multiple T cell markers can accurately identify more than two clones. Furthermore, although T cell heterogeneity is significant in normal specimens, their expression is very regular. The expression of reactive cells is also very regular; for example, persistently weakened CD7 expression accompanied by enhanced CD2 expression often indicates CD4 positivity, and viral infections can also result in CD8 positivity. Double-positive CD4 and CD8 cells, as well as CD3+CD56+ cell populations, are relatively common, and the expression patterns of reactive cell populations are highly characteristic. CD4 and CD8 expression is often a "flat four, flat eight" pattern (either the expression intensity is consistent with the CD4 intensity of normal CD4+ T cells, or the CD8 expression is consistent with the CD8 expression of normal CD8+ T cells, or both CD4 and CD8, i.e., at the intersection of the centroids of CD4 and CD8 positive cells in a CD4 / CD8 two-dimensional dot plot). In contrast, mature T-lymphocyte tumors with CD4+CD8+ expression are relatively rare, primarily T-PLL, which exhibits characteristic TCL1 and restricted TCR expression. Furthermore, it is necessary to exclude T-LBL / ALL with strong CD99 expression; these markers are present in tubes four and four. Therefore, the main contribution of tube one is the discovery of T-cell markers and cell populations with different expression intensities compared to normal cells, as well as the number of abnormal clones. The tube also contains CD117, which is a marker of abnormal expression that may occur in T-cell tumors. More importantly, patients with aplastic anemia are prone to oligoclonal T-cell proliferation, and the proportion of CD117-positive myeloid blast cells in the bone marrow of these patients is extremely low. Therefore, patients with low CD117 expression rates need to be carefully considered.

[0145] Tube B contains four TCR restriction markers in addition to T cell markers. This tube can help tube A further differentiate between reactive cells and mature T lymphocyte tumors, and it is also the main tube for determining the CD4-type mature T lymphocyte subtype.

[0146] The gamma tube is primarily used to identify subtypes of common CD4+ mature T-lymphocyte tumors. In this tube, besides CD3, CD5, or CD2, CD4, CD7 as gating markers to pinpoint low proportions of tumor cells, CD26, CD25, CD10, and CD279 are characteristic markers for major CD4+CD8- mature T-lymphocyte tumor subtypes. When using strong fluorescent dyes such as PE to label CD26, over 80% of normal CD4+ T cells express CD26, while the characteristic immunophenotype of MF / SS is CD4+CD26- and / or CD7-. Although reactive cells can lose CD7 and CD26, the loss of CD7 is progressively weakened and accompanied by increased CD2 expression and mutually exclusive expression of TCRs in a balanced manner. Strong expression of CD4+CD25 is a characteristic phenotype of adult T-cell lymphoma. CD10 and CD279 are characteristic phenotypes of AITL, with CD10 showing good specificity and CD279 having high coverage.

[0147] The Ding tube primarily targets CD4+CD8+ mature T lymphocyte tumors for subtype identification and differential diagnosis. The clinical manifestations of T-LBL / ALL are very similar to those of mature T lymphocyte tumors. The expression intensity of CD99 can effectively differentiate T-LBL / ALL from mature T lymphocyte tumors. CD30 is mainly found in ALCL, a very difficult tumor to diagnose because it often loses T cell markers, sometimes leaving only CD4 or CD2. Therefore, the location of CD99 and CD45 / SSC can effectively aid in differentiation. If necessary, additional cytoplasmic CD3 and other myeloid, monocyte, and dendritic cell markers can be used for differential diagnosis. This is because CD4 is found not only in lymphocytes but also in monocytes, dendritic cells, and myeloid tumors. Similarly, CD99 is expressed at a moderate to weak level in mature T cells, while strong expression is found not only in T-LBL / ALL but also in myeloid blasts and monocytes, dendritic cells. Cytoplasmic TCL1 is a characteristic marker for identifying T-PLL. Although 65% of T-PLLs are CD4+CD8-, the diagnosis of cell type is more important than subtype determination. Therefore, the focus in clinical practice is on the CD4+CD8+ type. This is because the treatment for mature T lymphocyte tumors is relatively similar, while misdiagnosis as T-LBL / ALL would be completely wrong and would affect the treatment plan. Therefore, clinical practice pays more attention to the CD4+CD8+ type of T-PLL.

[0148] In summary, this invention employs a multi-marker gating system, using T-cell markers for precise gating and TCR clonal markers for clonal assessment, thereby excluding reactive cells, T-CUS, and other tumors. After diagnosing mature T-lymphocyte tumors, further subtype determination can be made based on several common markers with characteristic immunophenotypes.

[0149] This embodiment provides a typical example of reactive cell tubes 1 and 2. Figures 2-3 The immunophenotypes of mature T cells in tubes A and B of patients with autoimmune diseases. Figures 4-5 Immunophenotypes of mature T cells in tube A and tube B of patients with aplastic anemia. Figures 6-28 Immunophenotyping of tubes A to D in typical cases of mature T-cell lymphoma was performed, with strict gating and observation according to the above description.

[0150] Specifically, Figures 2-3 Two tubes, A and B, were used to analyze bone marrow specimens from the same patient with an autoimmune disease.

[0151] Specifically, Figure 2 This section describes the analysis of bone marrow specimens from patients with autoimmune diseases. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). In the CD117 / CD7 2D dot plot, CD117-positive and CD7-negative cells were selected as myeloid blasts (CD117+), with a proportion ranging from 0.2% to 5%. It was confirmed that there were no abnormal cells with double CD7 and CD117 positivity; ④ Within P2, the concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed, confirming that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, CD3-negative / CD7-positive cells are NK cells (orange-yellow). In addition, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are shown, with no obvious abnormal cells. However, the CD4 / CD8 ratio of CD3+CD5+ T cells (dark green cells) is high, at 77.87 / 14.14=5.51. (A CD4 / CD8 ratio greater than 10, coupled with T cell clonalness, can suggest T-cell lymphoma, but in healthy individuals, the CD4 / CD8 ratio rarely exceeds 2.5.)

[0152] Specifically, Figure 3 Display and Figure 2Bone marrow specimens from the same autoimmune disease patient were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD8 markers and the lymphocyte gate (Lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym gate, the expression of CD57, TRBC1, TRBC2, TCRVδ1, and TCRVδ2 is shown. No abnormal cells were observed in this case, but the CD4 / CD8 ratio was high. To avoid missing any small clones, the TCR expression of CD3+CD5+ T cells, CD3+CD5- T cells (pink), CD3+CD5+abT (sapphire blue and green), CD3+CD5+gdT (red), CD8-abT (i.e., CD4+abT cells, sapphire blue), and CD8+abT (green) within the Lym gate is shown separately. Within the CD3+CD5+ T cell gate, TRBC1 and TRBC2 are expressed in a normal, balanced, mutually exclusive manner. Cells expressing either TRBC1 or TRBC2 are αβT cells (CD3+CD5+abT, sapphire blue and green), while those negative for both TRBC1 and TRBC2 are γδT (CD3+CD5+gdT) cells (red), which also show a normal, balanced, mutually exclusive expression. Within the CD3+CD5- T cell phylum, γδ T cells predominate. TCRVd1 and TCRVd2 show mutually exclusive expression in a normal, balanced ratio. TRBC1 and TRBC2, although scarce, also show mutually exclusive expression in a normal, balanced ratio. CD3+CD5+abT cells are further subdivided into CD8+abT cells (green) and CD8-abT cells (i.e., CD4+abT cells, bright blue). Both cell groups show mutually exclusive expression of TRBC1 and TRBC2 in a normal, balanced ratio. Using this method, lymphoma is ruled out, and an elevated CD4 / CD8 ratio is confirmed as a reactive change in autoimmune diseases.

[0153] Specifically, Figures 4-5 Two tubes, tube A and tube B, were used for joint analysis of bone marrow specimens from the same patient with aplastic anemia.

[0154] Specifically, Figure 4Displaying bone marrow specimens from patients with aplastic anemia, tube analysis was performed. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). A CD117 / CD7 dotted plot was used to select CD117-positive and CD7-negative cells. This patient has an extremely low proportion of CD117-positive myeloid blast cells, a characteristic of aplastic anemia patients; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym gate, CD3-negative / CD7-positive cells are NK cells (orange-yellow). In addition, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are shown, with no obvious abnormal cells. However, the CD4 / CD8 ratio of CD3+CD5+ T cells (dark green cells) is high, at 57.92 / 19.14=3.03. (A CD4 / CD8 ratio greater than 10, coupled with T cell clonalness, can suggest T-cell lymphoma, but in healthy individuals, the CD4 / CD8 ratio rarely exceeds 2.5.)

[0155] Specifically, Figure 5 Display and Figure 4Analysis of bone marrow specimens from the same patient with aplastic anemia. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, the expression of CD57, TRBC1, TRBC2, TCRVδ1, and TCRVδ2 is shown. No abnormal cells were observed in this case, but the CD4 / CD8 ratio was high. To avoid missing any small clones, the TCR expression of CD3+CD5+ T cells, CD3+CD5- T cells (pink), CD3+CD5+abT (royal blue and green), CD3+CD5+gdT (red), CD4-abT (i.e., CD8+abT cells, royal blue), and CD4+abT (green) within the Lym gate is shown separately. Within the CD3+CD5+ T cell gate, TRBC1 and TRBC2 are expressed in a normal, balanced, and mutually exclusive manner. Cells expressing either TRBC1 or TRBC2 are αβT cells (CD3+CD5+abT, royal blue and green), while those negative for both TRBC1 and TRBC2 are γδT (CD3+CD5+gdT) cells (red). TCRVd2 shows clonal expression, exceeding 85%. Within the CD3+CD5- T cell phylum, γδT cells predominate, with TCRVd1 and TCRVd2 showing mutually exclusive expression in a normal, balanced ratio. No significant TRBC1 and TRBC2 expression was observed. CD3+CD5+abT cells further subdivided into CD4+abT (green) and CD4-abT (i.e., CD8+abT cells, bright blue), with both cell groups showing mutually exclusive expression of TRBC1 and TRBC2 in a normal, balanced ratio. This patient presented with clonal TCRVd2 cells. Although this cell group accounted for 15.06% (80.12% x 18.8%) of lymphocytes, exceeding 5%, it exhibited several abnormalities: first, no abnormal phenotype; second, γδT cell lymphomas are predominantly TCRVd1 with very few TCRVd2 cells, while reactive γδT cells are predominantly TCRVd2; third, the patient lacked evidence of lymphoma such as fever, hepatosplenomegaly, or lymphadenopathy; and fourth, aplastic anemia is an immune-related disease, frequently involving reactive oligoclonal cells. Therefore, a comprehensive assessment suggests reactive cells.

[0156] Specifically, Figures 6-8 Three tubes (A to C) of bone marrow specimens from a patient with the same mature T-lymphocyte tumor AITL and LGLL were analyzed together.

[0157] Specifically, Figure 6 This section describes the analysis of bone marrow specimens from a case of mature T-lymphocytic tumor AITL combined with LGLL. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocyte (lym), granulocyte (Gra), monocyte (mono), nucleated erythrocyte (NEC) cell gates, and eosinophil (eo) gates. In the CD117 / CD7 two-dimensional dot plot, no abnormal cells were observed that were double-positive for CD7 and CD117, and the proportion of CD117-positive and CD7-negative myeloid blast cells was low, at 0.14%; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym phylum, CD3-negative / CD7-positive cells are NK cells (orange-yellow). Additionally, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are displayed. In the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) represent normal T cells, and two abnormal cell populations are visible: CD3+CD5- represents abnormal T cell 1 (AT1, red), and CD3-CD5+ represents abnormal T cell 2 (AT2, bright blue). AT1 expresses CD3 and CD2, with a small amount of CD7, but does not express CD4, CD8, CD5, CD117, or CD56, and its FSC and SSC are both large, classifying it as CD4-. AT2 expresses CD4, CD5, and CD2, but does not express CD7, CD8, CD3, CD117, or CD56, and its FSC and SSC are both small, classifying it as CD4+CD8-.

[0158] Specifically, Figure 7 Display and Figure 6Analysis of bone marrow specimens from patients with AITL (AI-LTL) and LGLL (LGLL) of the same mature T-lymphocytic tumor. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym's phylum, in the two-dimensional dot plot of CD3 / CD5, CD3+CD5+ (dark green) represent normal T cells, while two abnormal cell populations are visible: CD3+CD5- represent abnormal T cell 1 (AT1, red), and CD3-CD5+ represent abnormal T cell 2 (AT2, bright blue). AT1 expresses CD3, CD57, and TRBC1, but does not express CD4, CD5, TRBC2, TCRVd1, or TCRVd2, and both FSC and SSC are enlarged. AT2 expresses CD4, CD5, and TRBC1, but does not express CD57, CD3, TRBC2, TCRVd1, or TCRVd2, and both FSC and SSC are small. This tube of AT1 was diagnosed as LGLL, but the common type is CD8+CD4- αβ T cell type; this patient has the second most common type, CD8-CD4- αβ T cell type.

[0159] Specifically, Figure 8 Display and Figure 6 and Figure 7Bone marrow specimens from patients with the same mature T-lymphocytic tumor AITL combined with LGLL were analyzed using a gamma globulin. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, CD4, and CD7 markers and the lymphocyte gate (Lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym phylum, in the 2D dot plot of CD3 / CD5, CD3+CD5+ (dark green) represent normal T cells. Two abnormal cell populations are visible: CD3+CD5- represent abnormal T cell 1 (AT1, red), and CD3-CD5+ represent abnormal T cell 2 (AT2, bright blue). AT1 expresses CD3, with limited expression of CD279 and CD7, but does not express CD4, CD5, CD26, CD10, or CD25, and both its FSC and SSC are relatively large. AT2 expresses CD4 and CD5, strongly expresses (bri) CD279, partially strongly expresses CD10, but does not express CD7, CD3, CD26, or CD25, and both its FSC and SSC are small. Figure 7 AT1 has been diagnosed as LGLL. Figure 8 The AT2 diagnosis was AITL, therefore this case is a rare AITL combined with LGLL.

[0160] Specifically, Figures 9-11 Three tubes (A to C) of bone marrow specimens from the same mature T-lymphocyte tumor MF / SS patient were analyzed together.

[0161] Specifically, Figure 9Bone marrow specimens from patients with mature T-lymphocyte tumors (MF / SS) were displayed, and tube analysis was performed. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). In the CD117 / CD7 two-dimensional dot plot, no abnormal cells were observed that were double-positive for CD7 and CD117. The proportion of CD117-positive and CD7-negative myeloid blast cells was low, at 0.05%; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, CD3-negative / CD7-positive cells represent NK cells (orange-yellow). Additionally, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are displayed. In the CD3 / CD4 two-dimensional dot plot, partially positive and partially negative CD3+CD4 cells (dark green) represent normal T cells, while weakly positive CD3 (dim) and CD4dim represent abnormal T cells (AT, red), belonging to the CD4+CD8- type. AT cells express CD5, CD3dim, CD4dim, and CD2dim, but do not express CD7, CD8, CD117, or CD56, and both FSC and SSC are relatively small.

[0162] Specifically, Figure 10 Display and Figure 9Bone marrow specimens from patients with the same mature T-lymphocytic tumor MF / SS were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym phylum, in the two-dimensional dot plot of CD3 / CD4, CD3+CD4 positive and negative cells (dark green) represent normal T cells. Normal T cells express TRBC1 and TRBC2 in a balanced and mutually exclusive manner. In TRBC1 and TRBC2 negative γδT cells (TCRgd, pink), TCRVd1 and TCRVd2 are expressed in a balanced and mutually exclusive manner. CD3dim and CD4dim represent abnormal T cells (AT, red). AT cells express CD5, CD3dim, CD4dim, and TRBC2, but do not express CD57, TRBC1, TCRVd1, or TCRVd2, and their FSC and SSC are both smaller. Up to this point... Figure 9 A tube and Figure 10 Tube B confirmed the presence of mature T-lymphocyte tumor cells in the specimen and demonstrated monoclonal activity. Normally, flow cytometry in the laboratory does not require subtype differentiation and can stop here. If further subtyping of CD4+ / CD8- tumor cells is desired, tube C should be tested.

[0163] Specifically, Figure 11 Display and Figure 9 and Figure 10Bone marrow specimens from the same mature T-lymphocytic tumor MF / SS case were analyzed using a gamma globulin. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym phylum, in the CD3 / CD4 2D dot plot, CD3+CD4 partially positive and partially negative (dark green) represent normal T cells, while CD3dim and CD4dim represent abnormal T cells (AT, red). AT cells express CD5, CD3dim, and CD4dim, but do not express CD10, CD279, CD26, CD25, or CD7, and both FSC and SSC are small. The subtype is MF / SS.

[0164] Specifically, Figures 12-15 These are bone marrow specimens from the same mature T-cell lymphoma ATLL patient, among which Figure 13 and Figure 14 Different methods were used to analyze the same tube B, therefore Figures 12-15 The analysis is performed on all three pipes, from pipe A to pipe C.

[0165] Specifically, Figure 12This image shows a bone marrow specimen from a patient with mature T-lymphocytic angina pectoris (ATLL), analyzed in tube A. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). The boundary between lymphocytes and monocytes was unclear, therefore, Lym and some monocytes were obtained at this stage. In the CD117 / CD7 two-dimensional dot plot, no abnormal cells were observed that were double-positive for both CD7 and CD117. The proportion of CD117-positive and CD7-negative myeloid blasts was 0.02%, a significantly reduced percentage. ④ Within the P2 gate, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and Lym and some monocyte gates was observed to confirm that no lymphocytes were outside Lym and some monocyte gates. Using the CD5 and CD4 two-dimensional dot plot, monocyte 2 (dark brown) was set as CD5-negative CD4dim. Through the CD2 / CD7 and CD3 / CD5 two-dimensional dot plots, it was confirmed that monocyte 2 did not express CD2, CD7, CD5, or CD3. CD5-positive and CD4-partially positive and negative cells were normal lymphocytes (dark green), and CD5dimCD4bri cells were abnormal cells (AT, red). The logic gate "Total Lymphocytes" is set with the instruction "not Monocytes 2 and Lym and Some Monocytes," meaning monocytes within the Lym and Some Monocytes gate are excluded. Within the Total Lymphocytes gate, CD3-negative / CD56-positive cells are NK cells (orange-yellow). AT cells with low SSC / moderate CD5 intensity (int) are between AT and normal cells; this gate is set to AT2* (sapphire blue). Both AT and AT2* belong to the CD4+CD8- type. AT cells express CD5dim and CD4, partially express CD3dim and CD7dim, but do not express CD8, CD117, CD56, or CD2, and their FSC and SSC are of medium size. AT2* cells express CD2, CD3, CD4, CD5int, and CD7, partially express CD117, but do not express CD8 or CD56, and their FSC and SSC are small.

[0166] Specifically, Figure 13 and Figure 14 Display and Figure 12 Bone marrow specimens from patients with the same mature T-lymphocytic tumor ATLL, analyzed in tube sigmoid, because... Figure 12 In the study, a cell population with AT2* was found. To better demonstrate how to use tube B to differentiate between reactive and neoplastic cells with abnormal phenotypes, and why the old regimen without TRBC2 was changed to the new regimen containing TRBC2, data from the same tube were analyzed separately. Figure 13 and Figure 14 .

[0167] Specifically, Figure 13 Display and Figure 12 Bone marrow specimens from patients with the same mature T-cell lymphocytic tumor (ATLL) were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the viable cell gate, resulting in single viable cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). The boundary between lymphocytes and monocytes was unclear, therefore, a Lym and some monocyte gates were obtained at this stage; ④ Within P2, the concordance between CD3, CD4, and CD5 markers and the Lym and some monocyte gates was observed to ensure no lymphocytes were outside the Lym and some monocyte gates. Two-dimensional dot plots of CD5 and CD4 were then used. Figure 12 The same method was used to set up total lymphocyte gates, normal lymphocyte gates, and AT gates. Within the AT gate, TRBC1bri was gated as AT2* (royal blue), appearing as small CD5ints in SSC, and two-dimensional dotted plots of CD45 / SSC and CD4 / CD5. Figure 12 The results were consistent with AT2*. If the old protocol was followed, i.e., only TRBC1 was analyzed, omitting TRBC2, it might be mistakenly identified as monoclonal cells. Because of this, the TRBC1 / TRBC2 combination was subsequently used, improving detection sensitivity. Within the total lymphocyte phylum, the expression of CD57, TRBC1, TRBC2, TCRVd1, and TCRVd2 was observed in lymphocytes. AT cells expressed CD5dim, CD4, and TRBC2, partially expressed CD3dim, and did not express CD57, TRBC1, TCRVd1, or TCRVd2. The AT2* cell population expressed CD5int, CD4, CD3, and TRBC1bri, but did not express CD57, TRBC2, TCRVd1, or TCRVd2. Partially CD3+CD4 positive and partially negative (dark green) cells were normal T cells, expressing TRBC1 and TRBC2 in a balanced and mutually exclusive manner. Up to this point... Figure 12 A tube and Figure 13 Tube B confirmed the presence of mature T-lymphocyte tumor AT in the specimen and confirmed its monoclonal nature. AT2* was suspicious, but the proportion was extremely low. Normal laboratory flow cytometry does not require subtype differentiation and can stop here. If further determination of the CD4+ / CD8- tumor cell subtype is desired, tube C should be tested.

[0168] Specifically, Figure 14 Display and Figure 12 Bone marrow specimens from patients with the same mature T-lymphocytic tumor ATLL, analyzed in tube sigmoid. Figure 13 The difference lies in the use of SSC small CD5int gating for AT2* (royal blue), displaying a two-dimensional dot plot of TRBC1 / TRBC2 for AT2*. Although TRBC1bri is less prominent compared to normal T cells, the proportion of TRBC1bri and TRBC2bri is balanced, indicating reactive cells. Furthermore, because αβT cells predominate in mature T lymphocyte tumors, the new protocol targets two clonal markers for both αβT and γδT, enhancing the detection of αβT cells.

[0169] Specifically, Figure 15 Display and Figures 12 to 14 Bone marrow specimens from the same mature T-lymphocytic ALL case were analyzed using a gamma globulin. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the viable cell gate, again resulting in single viable cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). The boundary between lymphocytes and monocytes was unclear, thus a Lym and some monocyte gates were obtained at this stage; ④ Within P2, the concordance between CD3, CD4, and CD5 markers and the Lym and some monocyte gates was observed to ensure no lymphocytes were outside the Lym and some monocyte gates. A two-dimensional dot plot of CD5 and CD4 was used to compare... Figures 12-14 Similarly, a normal lymphocyte gate (dark green), an abnormal cell gate (AT, red), and a "total lymphocytes" logic gate were set up. AT cells express CD5dim, CD4, and CD25, partially express CD3dim, CD7dim, and CD26, and do not express CD10 and CD279. Therefore, the subtype is ATLL. At this point, note the AT2* cell population; compared to normal T cells, it highly expresses CD279, further confirming that AT2* is an activated and exhausted reactive cell.

[0170] Specifically, Figures 16-19 Four tubes (tubes A through D) of bone marrow specimens from the same mature T-cell lymphoblastic ALCL patient were analyzed together. Figure 17 and Figure 19 This is the old solution, as the new solution has only been used for a short time and no rare cases like ALCL have been encountered. The main difference between the old solution and the optimized solution of this invention is that the PE in pipe B is TCRγδ instead of TRBC2, and BV421 is CD7 instead of TCRVd2. The FITC in pipe D is ki67 instead of CD99.

[0171] Specifically, Figure 16Bone marrow specimens from patients with mature T-lymphocyte tumor ALCL, and thyroid tube analysis. The following steps were performed sequentially: ① FSC-A / H set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in the gates for lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). In the CD117 / CD7 two-dimensional dot plot, no abnormal cells with double positive CD7 and CD117 were observed. The percentage of CD117-positive and CD7-negative myeloid blast cells was 1.98%, which is within the normal range; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed. At this point, CD2bri / CD7-negative AT2 (bright blue) cells were observed outside Lym. The total Lym gate was then set, defined as Lym and AT2 cells. Within the total Lym gate, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are displayed. In the CD3 / CD7 dot plot, CD3-negative / CD7-positive cells are represented by NK cells (orange-yellow), CD3+CD7- by AT1 cells (red), and CD3+CD7+ by normal T cells (dark green). AT1 cells express CD5, CD3dim, CD4dim, and CD2, but do not express CD7, CD8, CD117, or CD56. Their FSC and SSC values ​​are both small, indicating a CD4+CD8- type. AT2 cells express CD2 and CD4, but do not express CD3, CD5, CD7, CD8, CD56, or CD117. Their FSC and SSC values ​​are both large, indicating a CD4+CD8- type.

[0172] Specifically, Figure 17 Display and Figure 16Bone marrow specimens from patients with the same mature T-lymphocytic leukemia (ALCL) were analyzed in tube B. Because ALCL is a rare case and the new protocol has only been used for a short time, this tube was analyzed using the old protocol. The difference is that PE was TCRγδ instead of TRBC2, and BV421 was CD7, not TCRVd2. The following steps were performed sequentially: ① FSC-A / H was set as the de-adhesion cell gate in P1, resulting in single cells within P1; ② FSC / SSC was set as the live cell gate in P1, resulting in single live cells within P2; ③ CD45 / SSC was used to set the blood cell gate in P2, resulting in lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ In P2, the degree of concordance between CD2, CD3, CD4, and CD7 markers and the lymphocyte gate (Lym) was observed. It was found that CD2briCD7-negative AT2 cells were located outside the lymphocyte gate. A total Lym gate was then set, defined as Lym and AT2 cells. Within the total Lym phylum, in the CD3 / CD7 2D dot plot, CD3+CD7+ (dark green) represent normal T cells, showing CD4 and TRBC1 in normal T cells. Regardless of whether CD4 is positive or negative (i.e., CD8+), TRBC1 is expressed in a balanced proportion. CD3+TCRgd+ represents gdT (pink), partially expressing TCRVd1. CD3+CD7- represents abnormal T cells (AT1, red). AT1 expresses CD3dim, CD4dim, and CD2, but does not express CD57, TRBC1, TCRVd1, TCRgd, or CD7, and both FSC and SSC are small. AT2 expresses CD4 and CD2, but does not express CD3, CD57, TRBC1, TCRVd1, TCRgd, or CD7, and both FSC and SSC are large. Up to this point... Figure 16 A tube and Figure 17 Tube B confirmed the presence of two groups of mature T lymphocyte tumors in the specimen and demonstrated monoclonal activity. Normally, flow cytometry in the laboratory does not require subtype differentiation and can stop here. If further determination of the CD4+ / CD8- tumor cell subtype is desired, tube C should be tested.

[0173] Specifically, Figure 18 Display and Figure 16 and Figure 17Bone marrow specimens from the same mature T-lymphocytic leukemia (ALCL) case were analyzed using a gamma globulin. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the concordance between CD2, CD3, CD4, and CD7 markers and the lymphocyte gate (Lym) was observed. It was found that CD2briCD7-negative AT2 cells were located outside the lymphocyte gate, thus a total Lym gate was set, defined as Lym and AT2 cells. Within the total Lym phylum, in the CD3 / CD7 2D dot plot, CD3+CD7+ (dark green) represent normal T cells, while CD3+CD7- represent abnormal T cells (AT1, red). AT1 expresses CD3dim, CD4dim, and CD2, partially expresses CD26dim and CD279dim, but does not express CD7, CD10, or CD25, and both its FSC and SSC are relatively small. AT2 expresses CD25, CD4, and CD2, but does not express CD3, CD7, CD10, CD26, or CD279, and both its FSC and SSC are relatively large.

[0174] Specifically, Figure 19 Display and Figures 16 to 18Bone marrow specimens from the same mature T-lymphocytic leukemia (ALCL) case were analyzed using the old protocol with two tubes. The difference was that FITC was Ki67 instead of CD99. The following settings were implemented sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the concordance between CD2, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed. It was found that CD2briCD4+ AT2 cells were located outside the lymphocyte gate. A total Lym gate was then set, defined as Lym and AT2 cells. Within the total Lym phylum, in the CD3 / CD4 2D dot plot, CD3+CD4 partially positive and partially negative (dark green) represent normal T cells, while CD3dim and CD4dim represent abnormal T cells (AT1, red). AT1 expresses CD3dim, CD4dim, and CD2, but does not express ki67, CD30, or cTCL1, and its FSC and SSC are both small. AT2 expresses CD4 and CD2, partially expresses ki67 and CD30, but does not express CD3 or cTCL1, and its FSC and SSC are both large. Therefore, AT2 is ALCL. If AT1 did not have AT2, it would be considered PTCL or NOS, but because of the clearly identifiable AT2, the clinical and pathological diagnosis is ALCL. Flow cytometry revealed two clones.

[0175] Specifically, Figures 20-22 Three tubes (tubes A, B, and D) of bone marrow specimens from the same mature T-lymphocyte tumor PLL patient were analyzed together.

[0176] Specifically, Figure 20Bone marrow specimens from patients with mature T-lymphocytic tumors (PLL) were displayed, and tube analysis was performed. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). In the CD117 / CD7 two-dimensional dot plot, no abnormal cells were observed that were double-positive for CD7 and CD117. The proportion of CD117-positive and CD7-negative myeloid blast cells was 0.03%, an extremely low percentage; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym phylum, in the CD3 / CD5 2D dot plot, CD3+CD5+ (darkened in dark green) represent normal T cells, CD3-CD5bri represent abnormal T cells (AT, red), and CD3-CD5- represent non-T cells. Within the non-T cell phylum, CD56+CD4- represent NK cells (darkened in black). AT cells express CD5bri, CD4bri, CD7bri, and CD2dim, partially express CD8, but do not express CD3, CD117, or CD56. Furthermore, their FSC and SSC values ​​are relatively small, indicating a CD4+CD8+ type.

[0177] Specifically, Figure 21 Display and Figure 20 Bone marrow specimens from patients with the same mature T-lymphocytic tumor PLL were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the concordance between CD5, CD3, and CD8 markers and the lymphocyte gate (Lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym phylum, in the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (darkened in dark green) represent normal T cells, which express TRBC1 and TRBC2 in a balanced and mutually exclusive manner; CD3-CD5bri represent abnormal T cells (AT, red), which express CD5bri, partially express CD8, and do not express CD3, CD57, TRBC1, TRBC2, TCRVd1, and TCRVd2, and both FSC and SSC are smaller.

[0178] Specifically, Figure 22Display and Figure 20 and Figure 21 Bone marrow specimens from the same mature T-lymphocytic tumor (PLL) case were analyzed in tube D. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm the absence of lymphocytes outside the lymphocyte gate. Within the Lym phylum, in the CD3 / CD5 2D dot plot, CD3+CD5+ (darkened in dark green) represent normal T cells, while CD3-CD5bri represent abnormal T cells (AT, red). AT cells express CD5bri and CD4bri, partially express cTCL1, and do not express CD99, CD30, or CD3. Furthermore, both FSC and SSC are smaller than normal. The subtype is PLL.

[0179] Specifically, Figures 23-26 Four tubes (A to D) of bone marrow specimens from patients with mature T-lymphocyte tumors PTCL and NOS were analyzed together.

[0180] Specifically, Figure 23Bone marrow specimens from patients with mature T-lymphocyte tumors PTCL and NOS were analyzed. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). In the CD117 / CD7 two-dimensional dot plot, no abnormal cells were observed that were double-positive for CD7 and CD117. The percentage of CD117-positive and CD7-negative myeloid blasts was 1.25%, within the normal range; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, CD3-negative / CD7-positive cells are NK cells (orange-yellow). Additionally, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are displayed. In the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) represent normal T cells, while CD3dimCD5bri represents abnormal T cells (AT, red), belonging to the CD4+CD8- type. AT cells express CD5bri, CD3dim, CD4bri, CD7bri, and CD2dim, but do not express CD8, CD117, or CD56, and both FSC and SSC are relatively small.

[0181] Specifically, Figure 24 Display and Figure 23Bone marrow specimens from patients with the same mature T-lymphocyte tumor PTCL and NOS were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the anastomosis between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to ensure no lymphocytes were outside the lymphocyte gate. Within the Lym gate, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive and partially negative (dark green) represented normal T cells. Normal T cells expressed TRBC1 and TRBC2 in a balanced and mutually exclusive manner. CD3dimCD4bri represents abnormal T cells (AT, red). AT cells express CD5bri, CD3dim, CD4bri, and TRBC1, but do not express CD57, TRBC2, TCRVd1, or TCRVd2. Furthermore, both FSC and SSC are smaller than normal. Up to this point... Figure 23 A tube and Figure 24 Tube B confirmed the presence of mature T-lymphocyte tumor cells in the specimen and demonstrated monoclonal activity. Normally, flow cytometry in the laboratory does not require subtype differentiation and can stop here. If further subtyping of CD4+ / CD8- tumor cells is desired, tube C should be tested.

[0182] Specifically, Figure 25 Display and Figure 23 and Figure 24Bone marrow specimens from the same mature T-lymphocyte tumor PTCL and NOS case were analyzed using a plasmapheresis tube. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive and partially negative (dark green) represent normal T cells, while CD3dimCD4bri represent abnormal T cells (AT, red). AT cells express CD5bri, CD3dim, CD4bri, and CD7bri, partially express CD279, CD26, and CD25, but do not express CD10, and both FSC and SSC are small. This is not the aforementioned AITL, ATLL, or MF / SS, therefore, a second tube was added.

[0183] Specifically, Figure 26 Display and Figures 23 to 25 Bone marrow specimens from the same mature T-lymphocyte tumor PTCL and NOS case were analyzed in tube D. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the degree of concordance between CD5, CD3, and CD4 markers and the lymphocyte gate (Lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, in the two-dimensional dot plot of CD3 / CD4, CD3+CD4 partially positive and partially negative (dark green) represent normal T cells, while CD3dimCD4bri represent abnormal T cells (AT, red). AT cells express CD5bri, CD3dim, and CD4bri, partially express CD99, but do not express CD30 or cTCL1, and both FSC and SSC are relatively small. Figure 25 and Figure 26 Consider PTCL, NOS.

[0184] Specifically, Figures 27-28 For the joint analysis of two tubes (A and B) of bone marrow specimens from patients with mature T-cell lymphoma of the liver and spleen γδT-cell lymphoma, the following was performed: Figure 28The old solution differs from the optimized solution of this invention in that PE is TCRγδ instead of TRBC2.

[0185] Specifically, Figure 27 This section displays bone marrow specimens from patients with mature T-cell lymphoma, specifically liver and spleen γδT-cell lymphoma, analyzed in tube A. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo). In the CD117 / CD7 two-dimensional dot plot, no abnormal cells were observed that were double-positive for CD7 and CD117. The percentage of CD117-positive and CD7-negative myeloid blasts was 1.04%, within the normal range; ④ Within P2, the degree of concordance between CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers and the lymphocyte gate (lym) was observed to confirm that no lymphocytes were outside the lymphocyte gate. Within the Lym gate, CD3-negative / CD56-positive cells are NK cells (orange-yellow). Additionally, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 are displayed. In the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) represent normal T cells, while CD3+CD5- represent abnormal T cells (AT, red), belonging to the CD4-CD8- type. AT cells express CD3bri, CD7bri, CD56, and CD2, but do not express CD4, CD8, CD117, or CD5. Both FSCs and SSCs are of medium size.

[0186] Specifically, Figure 28 Display and Figure 27Bone marrow specimens from patients with hepatosplenic γδT-cell lymphoma of the same mature T-cell type were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, the anastomosis between CD5, CD3, and CD8 markers and the lymphocyte gate (Lym) was observed to ensure no lymphocytes were outside the lymphocyte gate. Within the Lym gate, CD3+TCRgd- showed normal abT (dark green), and CD8+ and CD8- (mainly CD4+) showed balanced partial expression of TRBC1. In the CD3 / CD5 2D dotted map, CD3+CD5- represent abnormal T cells (AT, red). AT cells express CD3bri, CD4bri, TCRgd, and TCRVd1, but do not express CD5bri, CD57, TRBC1, and TCRVd2. Up to this point... Figure 27 A tube and Figure 28 The second tube confirmed the presence of a mature T-cell lymphoma in the specimen and confirmed its monoclonal nature, with the subtype being hepatosplenic γδT-cell lymphoma.

[0187] Clinical validation was conducted using the method described in this embodiment: Hebei Yanda Lu Daopei Hospital began using this protocol in 2022. Initially, TRBC2 was not available, and the channel used at that time was TCRγδ PE. From February 2022 to September 2024, 2109 tests were performed on 1235 patients, of which 364 tests were positive from 202 patients. From October 2024 onwards, all tests were changed to the protocol of this invention, which includes TRBC2. From October 2024 to the end of June 2025, 414 tests were completed on 259 patients, of which 84 tests were positive from 52 patients. To date, a total of 2523 tests have been completed on 1494 patients, of which 448 tests were positive from 254 patients. The median age of the patients was 55 years (range 1-88 years), and the median percentage of tumor cells was 2.32% (range 0.01%-93.61%). Based on morphological, genetic, pathological, and clinical follow-up and feedback, the sensitivity, specificity, positive predictive value, and negative predictive value were 97.49%, 99.34%, 96.15%, and 99.57% from February 2022 to September 2024, respectively. From October 2024 to June 2025, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.81%, 99.76%, 98.81%, and 99.76%, respectively. The total sensitivity, specificity, positive predictive value, and negative predictive value were 97.74%, 99.41%, 96.65%, and 99.60%, respectively. Using this method, especially the improved method, the sensitivity, specificity, positive predictive value, and negative predictive value are all above approximately 98%. Therefore, this invention can improve efficiency, save costs, and reduce the misdiagnosis rate.

Claims

1. A reagent composition for detecting the clonality of mature T cells, the reagent composition comprising a first group of antibodies, a second group of antibodies, a third group of antibodies, and a fourth group of antibodies, wherein: The first group of antibodies includes fluorescently labeled CD7, CD117, CD3, CD4, CD5, CD8, CD56, CD45, and CD2 antibodies. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are used to add to the first flow cytometer tube in which the sample to be tested is in a single-cell suspension state. The second group of antibodies includes fluorescently labeled CD57 antibody, TRBC2 antibody, CD3 antibody, TCRVδ1 antibody, TRBC1 antibody, CD8 or CD4 antibody, TCRVδ2 antibody, CD45 antibody, and CD5 or CD2 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are added to the second flow cytometer tube containing the test sample in a single-cell suspension. The third group of antibodies includes fluorescently labeled CD7, CD26, CD3, CD10, CD279, CD4, CD25, CD45, and CD5 or CD2 antibodies. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are used to add to the flow cytometry tubes in which the test sample is in a single-cell suspension state. The fourth group of antibodies consists of two parts. The first part includes fluorescently labeled CD99 antibody, CD30 antibody, CD3 antibody, CD4 antibody, CD45 antibody, and CD5 or CD2 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, APC-Cy7, V500, and BV605. This is used to add the sample to be tested in a single-cell suspension in the flow cytometry tube. The second part includes TCL1 antibody, which is fluorescently labeled APC. This is used to add the sample to the flow cytometry tube after the fourth group of antibodies has been added and the cells have been lysed.

2. The reagent composition according to claim 1, wherein, All antibodies are monoclonal antibodies.

3. The reagent composition according to claim 1, wherein: The first group of antibodies is a mixture of CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody in a volume ratio of 5:5:5:3:2:3:3:3:3; The second group of antibodies is a mixture of CD57 antibody, TRBC2 antibody, CD3 antibody, TCRVδ1 antibody, TRBC1 antibody, CD8 or CD4 antibody, TCRVδ2 antibody, CD45 antibody and CD5 or CD2 antibody in a volume ratio of 5:5:5:3:2:3:3:3:

3. The third group of antibodies is a mixture of CD7 antibody, CD26 antibody, CD3 antibody, CD10 antibody, CD279 antibody, CD4 antibody, CD25 antibody, CD45 antibody and CD5 or CD2 antibody in a volume ratio of 5:5:5:3:2:3:3:3:3; The first part of the fourth group of antibodies is a mixture of CD99 antibody, CD30 antibody, CD3 antibody, CD4 antibody, CD45 antibody and CD5 or CD2 antibody in a volume ratio of 5:5:5:3:3:

3.

4. A kit for detecting the clonality of mature T cells by flow cytometry, the kit comprising a first container, a second container, a third container, a fourth container and a fifth container, each container containing a first group antibody, a second group antibody, a third group antibody, a first portion of a fourth group antibody and a second portion of a fourth group antibody of the reagent composition according to any one of claims 1-3; The kit also includes: One or more of the following: cell lysis buffer, membrane breaking agent, buffer solution, and flow cytometry tubes used with a flow cytometer.

5. The use of the reagent composition according to any one of claims 1-3 in the preparation of flow cytometry samples for the detection of mature T cell clonality.

6. The application according to claim 5, wherein, The process for preparing flow cytometry samples for mature T cell clonality detection includes the following steps: (1) Prepare flow cytometry tube 1, flow cytometry tube 2, flow cytometry tube 3 and flow cytometry tube 4. Add the sample to be tested into each flow cytometry tube to make it into a single cell suspension and ensure that the cell count is 1×10⁻⁶. 6 / tube-1×10 7 / Tube; (2) Add the first group of antibodies in the reagent composition of any one of claims 1-3 to the flow cytometer tube one obtained in step (1), add the second group of antibodies in the reagent composition of any one of claims 1-3 to the flow cytometer tube two obtained in step (1), add the third group of antibodies in the reagent composition of any one of claims 1-3 to the flow cytometer tube three obtained in step (1), and add the first part of the fourth group of antibodies in the reagent composition of any one of claims 1-3 to the flow cytometer tube four obtained in step (1). Incubate each flow cytometer tube at room temperature in the dark. (3) Add film-breaking agent A solution to the flow cytometer tube after incubation in step (2) and continue incubation at room temperature in the dark; (4) Add 1×hemolysin to flow cytometer tube 1, flow cytometer tube 2, flow cytometer tube 3 after incubation in step (2) and flow cytometer tube 4 after incubation in step (3), and continue incubation at room temperature in the dark. (5) After centrifuging each flow cytometer tube incubated in step (4), remove the supernatant; (6) Add membrane-breaking agent B and the second part of the fourth group of antibodies in the reagent composition of any one of claims 1-3 to the flow cytometer tube after removing the supernatant in step (5), and incubate at room temperature in the dark; (7) Add PBS buffer to flow cytometry tubes 1, 2, and 3 after removing the supernatant in step (5) and flow cytometry tube 4 after incubation in step (6), respectively, wash, centrifuge, remove the supernatant, and resuspend the cells with PBS buffer to obtain flow cytometry samples.

7. A flow cytometry apparatus for detecting the clonality of mature T cells, the apparatus comprising a detection unit and an analysis unit, wherein: The detection unit includes reagent materials for detecting samples from the individual to be tested by flow cytometry, and obtaining the test results of the samples; the reagent materials include the reagent composition according to any one of claims 1-3; The analysis unit is used to analyze the detection results of the detection unit.

8. The apparatus according to claim 7, wherein, when used for detecting the clonality of mature T cells, The process of detecting samples from the individual being tested using flow cytometry includes: The sample to be tested is treated with the reagent composition according to any one of claims 1-3 to prepare a flow cytometry sample; Perform flow cytometry analysis; During flow cytometry analysis, each flow cytometry tube was sequentially configured with an anti-adhesion cell gate (P1) and a viable cell gate (P2) to obtain single viable cells. Within gate P2, CD45 / side-angle light scattering (SSC) was used to configure each blood cell gate, and: The flow cytometry tube is gated as follows: Within the P2 gate, observe the degree of anastomosis between the markers CD2, CD3, CD4, CD5, CD7, CD8, and CD56 and the lymphocyte gate, and construct a total lymphocyte gate including normal and abnormal lymphocytes; within the lymphocyte gate, CD3-negative / CD7-positive or CD3-negative / CD56-positive cells are NK cells, and display two-dimensional dot plots of any combination of CD3 with CD5, CD2, CD7, CD4, CD8, CD56, and CD117, respectively, and select T cells that are different from normal T cells as abnormal T cell gates; The flow cytometry tube 2 is gated as follows: Within the P2 gate, observe the degree of anastomosis between CD2 or CD5, CD3, CD4 or CD8 markers and the lymphocyte gate, and construct a total lymphocyte gate including normal lymphocytes and abnormal lymphocytes; within the lymphocyte gate, display the expression of CD57, TRBC1, TRBC2, TCRVδ1, and TCRVδ2, and select T cells that are different from normal T cells as the abnormal T cell gate; In flow cytometry, gating is performed as follows: Within the P2 gate, the degree of anastomosis between CD2 or CD5, CD3, CD4, and CD7 markers and the lymphocyte gate is observed, and a total lymphocyte gate including normal and abnormal lymphocytes is constructed; within the lymphocyte gate, the expression of CD26, CD10, CD279, and CD25 is displayed, and T cells that differ from normal T cells are selected as abnormal T cell gates; The flow cytometry tube was gated as follows: within the P2 gate, the degree of anastomosis between CD2 or CD5, CD3, CD4 markers and the lymphocyte gate was observed, and a total lymphocyte gate including normal lymphocytes and abnormal lymphocytes was constructed; within the lymphocyte gate, the expression of CD99, CD30, and cytoplasmic TCL1 was displayed, and T cells that differed from normal T cells were selected as abnormal T cell gates.

9. The apparatus according to claim 7 or 8, used for one or more of the following mature T cell clonality assays: (1) T cell clonal detection for diagnosis and differential diagnosis of mature T cell tumors: The flow cytometers used include flow cytometer 1 and flow cytometer 2. (2) Detection of the number of clones of mature T cell tumors: The flow cytometry tubes used include flow cytometry tube one and flow cytometry tube two; (3) Determine the mature T-cell tumor subtype: The tumor cells in the sample to be tested are CD4+CD8-. Flow cytometry tubes one through four can be selected; or The tumor cells in the test sample were CD4+CD8-. Flow cytometry tubes used included flow cytometry tube one, flow cytometry tube two, and flow cytometry tube three. Flow cytometry tube three was used to detect CD10 negativity, non-strong CD279 expression, and no loss of CD26. Flow cytometry tube four was then added. The tumor cells in the sample to be tested were CD4 negative, and the flow cytometry tubes used included flow cytometry tube one and flow cytometry tube two; or The tumor cells in the sample to be tested are CD4+CD8+, or the tumor cells are outside the lymphocyte gate set by CD45 / lateral angle light scattering, or clinically suggestive of rare T prolymphocytic leukemia / lymphoma; the flow cytometers used include flow cytometer I, flow cytometer II, and flow cytometer IV.

10. The apparatus according to any one of claims 7-9, wherein, When the analysis unit analyzes the detection results of the detection unit, it outputs the mature T cell clonal detection results according to one of the following judgment methods: Mature T-lymphocyte tumors: Positive cells are present in the abnormal T-lymphocyte gates of CD45 / SSC-gated lymphocytes or in any flow cytometry-gated T-lymphocyte gates, and the following differential diagnoses 1-3 are excluded: Differential diagnosis 1: Tumor cells strongly express CD99, and the test result is T lymphoblastic lymphoma; or, CD7, CD5, CD3, CD8, and CD2 are all negative, cytoplasmic CD3 is positive, and TdT and / or CD34 are positive, and the test result is T lymphoblastic lymphoma. Differential diagnosis 2: Tumor cells strongly express CD99 and CD4, but do not express CD7, CD5, CD3, CD2, CD8, or cytoplasmic CD3. They also express myeloid and tissue dendritic cell markers CD64, CD33, CD13, MPO, HLA-DR, CD14, CD42a, CD123, CD303, CD304, CD163, and CD68. The results are used to determine whether the tumor is a myeloid or tissue dendritic cell tumor. Differential diagnosis 3: Abnormal phenotype: mature T cells account for <5% of lymphocytes, or the immunophenotype is high expression of CD2 / continuous weak expression of CD7 / positive CD4 or CD8. Follow up for 6 months to determine whether they are clonal T cells of undetermined significance or reactive T cells. For samples with the above test results indicating mature T-lymphocyte tumors, further subtype testing shall be performed according to any one of the following (1)-(8): (1) CD4+CD8- with strong expression of CD279 and positive CD10, subtype detection indicates angioimmunoblastic lymphoma; (2) CD4+CD8- with strong CD25 expression, subtype detection indicates adult T-cell lymphoma; (3) CD4+CD8- with loss of CD26 and / or CD7, subtype detection indicates mycosis fungoides / Sézary syndrome; (4) CD4+CD8- with CD30 positivity, increased forward and lateral light scattering, loss of T cell markers, and subtype detection indicated anaplastic large cell lymphoma; (5) CD4+CD8 negative or positive with positive cytoplasmic TCL1, CD99 not strongly expressed, subtype detection is T prolymphocytic leukemia; (6) CD4+CD8-, which does not meet any of the above (1)-(5), and the subtype detection is non-specific peripheral T-cell lymphoma; (7) CD4-CD8+ with high expression of CD57, subtype detection indicates large granular T-lymphocytic leukemia; (8) CD4-CD8 negative or positive with TCRVδ1, rarely TCRVδ2, expressing CD56, subtype detection is γδ type liver and spleen T cell lymphoma.

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