Mature T cell clonality detection reagent composition and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to a reagent composition for rapid and efficient detection of mature T cell clonality and its application, belonging to the technical field of blood disease detection. BACKGROUND

[0002] Mature lymphocyte tumors, commonly known as lymphoma, are a group of common malignant lymphoid hematopoietic system tumors that seriously endanger human health. Lymphomas are diverse in species, have great differences in phenotype, and have great differences in disease course and prognosis. Since mature T lymphocytes in the human body are the most important immune cells, reactive or unknown significance monoclonal T cell proliferation (T-CUS) caused by various viral infections also brings difficulties to diagnosis. In addition, diseases such as aplastic anemia are prone to CD57-positive TCRαβ+ or TCRγδ+ T cell oligoclonal proliferation, which needs to be differentiated from lymphoma.

[0003] Mature T lymphocyte tumors are a group of highly heterogeneous diseases, derived from multiple mature T cell subgroups. There are leukemia forms and lymphoma forms, and the clinical behavior varies from benign to highly invasive. Most mature T lymphocyte tumors are CD4+ TCRαβ+ T cell types, a few are CD8+ TCRαβ+ T cell types, and a few are TCRγδ+ T cell types, as well as TCRαβ and TCRγδ completely negative types. A large number of reactive T cells appear in the case of viral infection, autoimmune disease, drug or other malignant tumors. Reactive T cells are often CD8+ T cell types, so for CD8+ and CD3+ CD56+ NKT cell types, especially T cells with high expression of CD2 and low expression of CD7 continuity (whether CD4 positive or CD8 positive), and the characteristics of T-CUS are that the proportion of lymphocytes is less than 5%, so these cases need to be judged carefully. In general, unless the clinical manifestations are extremely obvious, it is generally recommended to follow up for half a year to determine the diagnosis.

[0004] Flow cytometry is an important tool for detecting mature T lymphocyte tumors. In recent years, with the fine study of immune cell subgroups, the understanding of normal T cells and reactive cells has gradually deepened. The diagnosis and subtype judgment of tumors formed by the malignant transformation of these cells, mature T lymphocyte tumors, also play an increasingly important role. However, because there are many types of mature T lymphocyte tumors and great differences in phenotypes, it brings difficulties to clinical detection by flow cytometry. Therefore, although articles have been published for many years, they are basically differential analyses of the differences of several immune phenotypes of a certain disease, and lack of systematic disease detection diagnosis and differential diagnosis, especially the complete scheme of subtype judgment. Although the European Flow Cytometry Union (euroflow) and the Mayo Clinic in the United States have successively published relatively standardized detection schemes for mature T lymphocyte tumors in 2012 and 2024, they have not formed a complete standard scheme and process including the discovery of abnormal phenotype T cells, the diagnosis of mature T lymphocyte tumors after excluding other differential diagnoses, and the subtype judgment.

[0005] Therefore, there is an urgent need in the clinic for a complete solution to the diagnosis and subtype judgment of mature T lymphocyte clonality, including tumors. SUMMARY

[0006] An object of the present application is to provide a reagent composition capable of quickly and efficiently using flow cytometry to diagnose and subtype judge mature T lymphocyte tumors, especially including all common and uncommon types, and capable of making differential diagnosis for other types that need to be identified, such as T-cell clonal syndrome (T-CUS), reactive T cells, T lymphoblastic lymphoma / leukemia (T-LBL / ALL), and other tumors or diseases, to prevent misdiagnosis.

[0007] Another object of the present application is to provide the use of the reagent composition in the diagnosis and subtype judgment of mature T lymphocyte tumors.

[0008] In one aspect, the present application provides a reagent composition for mature T cell clonality detection, which comprises a first group of antibodies, and further comprises one or more of a second group of antibodies, a third group of antibodies, and a fourth group of antibodies, wherein:

[0009] The first group of antibodies comprises CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody labeled with fluorescein, and each antibody is labeled with fluorescein in the order of FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500 and BV605, and is added to a flow tube in a single cell suspension state of the sample to be tested;

[0010] The second group of antibodies includes CD57 antibody, TRBC2 antibody, CD3 antibody, TCRVd1 antibody, TRBC1 antibody, CD8 (or CD4) antibody, TCRVd2 antibody, CD45 antibody and CD5 (or CD2) antibody, each of which is labeled with fluorescein in the order of FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500 and BV605, and is added to the flow tube two in a single cell suspension state of the sample to be tested;

[0011] The third group of antibodies includes CD7 antibody, CD26 antibody, CD3 antibody, CD10 antibody, CD279 antibody, CD4 antibody, CD25 antibody, CD45 antibody and CD5 (or CD2) antibody, each of which is labeled with fluorescein in the order of FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500 and BV605, and is added to the flow tube three in a single cell suspension state of the sample to be tested;

[0012] The fourth group of antibodies includes two parts, the first part includes CD99 antibody, CD30 antibody, CD3 antibody, CD4 antibody, CD45 antibody and CD5 (or CD2) antibody, each of which is labeled with fluorescein in the order of FITC, PE, PerCP-Cy5.5, APC-Cy7, V500 and BV605, and is added to the flow tube four in a single cell suspension state of the sample to be tested; the second part includes TCL1 antibody, which is labeled with APC, and is added to the flow tube four after the fourth group of antibodies is added and the cells are lysed.

[0013] The reagent composition of the present application can be applied to rapid and efficient mature T lymphocyte tumor diagnosis and subtype judgment by flow cytometry. In specific application, the scheme of using 4 tubes in parallel or combining the first tube with 1-3 tubes in the second, third and fourth tubes is used, CD45 and T cell markers are used in each tube to set lymphocyte gate in combination with SSC, and control cell groups such as mature granulocytes, monocytes and nucleated red blood cells are set in combination with CD45 / SSC, so as to accurately evaluate.

[0014] According to the specific embodiments of the present application, four tubes can be used in parallel, or 1-3 tubes among tube one plus tube two to tube four can be used. Specifically, to determine the maturity of T cells and identify the number of tumor cell clones, the selected flow tubes include tube one and tube two; specifically, the CD4 and CD8 expressions listed in Table 1 can be selected, for example, the tumor cells of the sample to be tested are CD4+CD8-, and tubes one to four are selected; or the tumor cells of the sample to be tested are CD4+CD8-, and the selected flow tubes include tube one, tube two and tube three, and if tube three detects CD10 negative, CD279 non-strong expression, and CD26 is not lost, tube four is added; or the cells of the sample to be tested are CD4 negative, and the selected flow tubes include tube one and tube two; or the cells of the sample to be tested are CD4+CD8+, or the tumor cells are outside the lymphocyte gate set by CD45 / side angle light scattering (SSC), or the clinical indication is rare T prolymphocytic leukemia / lymphoma, and the selected flow tubes include tube one, tube two and tube four.

[0015] The scheme of the present application can be more economical while ensuring the accuracy of detection, and can help to form standardization, reduce misdiagnosis rate, and provide convenience for future automation and artificial intelligence.

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

[0017] According to the specific embodiments of the present application, each antibody in the antibody composition of the present application is a fluorescein-labeled antibody.

[0018] In the present application, by matching specific fluorescein with different antibodies, the reagent composition of the present application can be used for rapid and efficient diagnosis and subtype determination of mature T lymphocyte tumors, and all fluoresceins in each channel can achieve excellent staining effect.

[0019] According to the specific embodiments of the present application, each antibody component in the reagent composition of the present application can be commercially available. Each antibody should meet the requirements of relevant industry standards.

[0020] According to a specific embodiment of the present application, in the reagent composition of the present application, 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 ratio of each antibody is based on the case where the titer of each antibody is substantially equivalent.

[0021] Another aspect of the present application provides a kit comprising a first container, a second container, a third container, a fourth container and a fifth container, each of which contains the first group of antibodies, the second group of antibodies, the third group of antibodies, the first part of the fourth group of antibodies and the second part of the fourth group of antibodies of the reagent composition of the present application.

[0022] According to a specific embodiment of the present application, the kit of the present application can further comprise one or more of hemolysin, membrane disrupter, buffer and flow tube for use with flow cytometer. These reagents and consumables are commercially available. The membrane disrupter preferably comprises solution A and solution B. Each reagent material can be contained in a different container.

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

[0024] Another aspect of the present application also provides the use of the reagent composition in the preparation of a flow cytometer sample for rapid and efficient mature T lymphocyte tumor diagnosis and subtype determination.

[0025] According to a specific embodiment of the present application, the process of preparing a flow cytometer sample for rapid and efficient mature T lymphocyte tumor diagnosis and subtype determination comprises the following steps:

[0026] (1) Add the sample to be tested into flow tube 1 to flow tube 4 (or referred to as tube A, tube B, tube C and tube D) respectively, so as to be in a single cell suspension state, and ensure that the cell amount is 1×10 6 / tube-1×10 7 / tube;

[0027] (2) Add the first group of antibodies in the reagent composition to tube 1 obtained in step (1), add the second group of antibodies in the reagent composition to tube 2 obtained in step (1), add the third group of antibodies in the reagent composition to tube 3 obtained in step (1), and add the fourth group of antibodies in the reagent composition to tube 4 obtained in step (1), and incubate each flow tube at room temperature in the dark;

[0028] (3) Add membrane breaker A solution to tube 4 after incubation in step (2), and continue to incubate at room temperature in the dark;

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

[0030] (5) Centrifuge each flow tube after incubation in step (4) and remove the supernatant;

[0031] (6) Add membrane breaker B solution and the second part of antibodies in the fourth group of antibodies in the reagent composition to tube 4 after removing the supernatant in step (5), and incubate at room temperature in the dark;

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

[0033] In the present application, the description of the operation steps is not used to limit the actual operation sequence of the steps, unless it is specifically indicated or it can be obviously determined from the context that there is a sequence relationship.

[0034] According to a specific embodiment of the present application, in the present application, the sample to be tested can be bone marrow or peripheral blood, and can also be used for tissue specimens, body fluid specimens and all samples which can prepare single living cells and are suitable for flow cytometry detection.

[0035] According to a specific embodiment of the present application, in step (1), the sample addition amount of each tube is not more than 160 μl (if the peripheral blood cell amount of the patient is small, a volume exceeding 160 μl can be added according to the need, and then concentrated by centrifugation and removal of the supernatant).

[0036] According to the specific embodiments of the present application, the amount of each reagent can refer to the conventional amount in the prior art or the recommended dosage of the manufacturer.

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

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

[0039] According to the specific embodiments of the present application, the incubation time in step (3) can be 5-20 minutes. The amount of the membrane breaker A solution can be added according to the recommended dosage of the manufacturer, usually 100 μl / tube.

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

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

[0042] According to the specific embodiments of the present application, the amount of the membrane breaker B solution in step (6) can be added according to the recommended dosage of the manufacturer, usually 50 μl / tube. Usually, incubation is required for about 10-30 minutes.

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

[0044] According to the specific embodiments of the present application, when the resuspended cells are subjected to flow cytometry detection, each tube is sequentially set with a non-adherent cell gate P1 and a live cell gate P2 to obtain single live cells; each blood cell gate is set using CD45 / SSC within the P2 gate; and:

[0045] Tube 1 is set as follows: P2 gate, observe the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with the lymphocyte gate (lym), make a total lymphocyte gate including normal lymphocytes and abnormal lymphocytes (in the process of setting this cell gate, if the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with the lymphocyte gate (lym) has lymphocytes outside the lymphocyte gate, the marker reverse gate needs to be used; if the lymphocytes and monocytes are not clear, CD4 weak positive / T cell markers (CD2, CD3, CD5, CD7) negative monocyte gate 2 can be used to remove from the lymphocyte gate to achieve accurate gating); CD3 negative / CD7 positive or CD3 negative / CD56 positive in the lymphocyte gate are NK cells, and any combination of CD3 and CD5, CD2, CD7, CD4, CD8, CD56, CD117 two-dimensional dot plots are displayed, and T cells different from normal T cells are selected as abnormal T cell (AT) gate (if there is more than one group, AT1 and AT2 can be set respectively);

[0046] Tube 2 is set as follows: P2 gate, observe the degree of coincidence of CD2 or CD5, CD3, CD4 or CD8 markers with the lymphocyte gate (lym), make a total lymphocyte gate including normal lymphocytes and abnormal lymphocytes (in the process of setting this cell gate, if the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with the lymphocyte gate (lym) has lymphocytes outside the lymphocyte gate, the marker reverse gate needs to be used; if the lymphocytes and monocytes are not clear, CD4 weak positive / T cell markers (CD2 or CD5, CD3) negative monocyte gate 2 can be used to remove from the lymphocyte gate to achieve accurate gating); display the expression of CD57, TRBC1, TRBC2, TCRVδ1, TCRVδ2 in the lymphocyte gate, and select T cells different from normal T cells as abnormal T cell (AT) gate (if there is more than one group, AT1 and AT2 can be set respectively);

[0047] Tube 3: P2 gate, observe the degree of coincidence of CD2 or CD5, CD3, CD4, CD7 markers with lymphocyte gate (lym), make total lymphocyte gate including normal lymphocytes and abnormal lymphocytes (during the setting of this cell gate, if the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with lymphocyte gate (lym) has lymphocytes outside the lymphocyte gate, the marker reverse gate needs to be used; if the lymphocytes and monocytes are not clear, CD4 weak positive / T cell marker (CD2 or CD5, CD3) negative monocyte gate 2 is used and removed from the lymphocyte gate to achieve accurate gating); in the lymphocyte gate, the expression of CD26, CD10, CD279, CD25 is displayed, and the T cells different from normal T cells are selected as abnormal T cell (AT) gate (if there are more than one group, AT1 and AT2 can be set respectively);

[0048] Tube 4: P2 gate, observe the degree of coincidence of CD2 or CD5, CD3, CD4 markers with lymphocyte gate (lym), make total lymphocyte gate including normal lymphocytes and abnormal lymphocytes (during the setting of this cell gate, if the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with lymphocyte gate (lym) has lymphocytes outside the lymphocyte gate, the marker reverse gate needs to be used; if the lymphocytes and monocytes are not clear, CD4 weak positive / T cell marker (CD2 or CD5, CD3) negative monocyte gate 2 is used and removed from the lymphocyte gate to achieve accurate gating); in the lymphocyte gate, the expression of CD99, CD30, TCL1 is displayed, and the T cells different from normal T cells are selected as abnormal T cell (AT) gate (if there are more than one group, AT1 and AT2 can be set respectively). In this tube, abnormal marker SSC / CD99 strong expression (bri) gate can also be used to exclude T lymphoblastic lymphoma.

[0049] In the present application, the expression strength of CD45 refers to that the CD45 expression of mature lymphocytes is strong, the CD45 expression of monocytes is strong, the CD45 expression of granulocytes is weak, and the CD45 expression of nucleated red blood cells is negative, with normal bone marrow as the standard. In the present application, the medium intensity expression of CD99 refers to the same expression as normal lymphocytes, and the strong expression of CD99 refers to the expression intensity exceeding that of normal lymphocytes. Unless otherwise specified, the expression strength of each antigen is based on normal lymphocytes.

[0050] In the present invention, SSC size refers to the SSC of normal lymphocytes and nucleated red blood cells being small (or "smaller") compared to normal bone marrow, and greater than normal. FSC size refers to the FSC of normal lymphocyte populations and nucleated red blood cells being small (or "smaller") compared to normal bone marrow, and greater than normal.

[0051] In the use of the terms, in order to distinguish between the differences in marker expression of different kinds of normal lymphocytes and the differences between abnormal phenotype cells (mainly tumor cells) and normal cells, the differences in expression intensity of normal T cells are referred to as high (hi) and low (lo), and the expression of abnormal phenotype cells is referred to as bright (bri) and dim.

[0052] In the detection technology of the application, tube one (or tube 1 or pipe A) is the basic detection tube, which uses almost all the main T cell markers, CD2, CD3, CD4, CD5, CD7, CD8, CD56, tube two (or tube 2 or pipe B), tube three (or tube 3 or pipe C), tube four (or tube 4 or pipe D) can be selectively used in combination with tube 1 or all together according to the expression of CD4 and CD8. The main role of the T cell markers of tube 1 is to find abnormal phenotype T cells. Because of the complexity of T cells and the unknown nature of tumors, especially most of the primary sites of mature T lymphocyte tumors are in extramedullary tissues, which may be in bone marrow and peripheral blood, and the proportion is not high, so it is necessary to use all the main markers for detection. In addition, about 5% of lymphomas are more than two clones, especially in recent years, with the progress of flow cytometry technology, as well as the use of chimeric antigen receptor T cells (CAR-T) and targeted drugs, the detection rate of more than two clone tumors is increasing, so it is more necessary to use all T cell markers for detection. In addition to T cell markers, CD117 is also used in tube 1. On the one hand, part of T cell lymphoma abnormally expresses CD117, on the other hand, aplastic anemia is a common disease, mainly manifested as a decrease in the proportion of CD117 positive myeloid blast cells in bone marrow, such diseases are related to immunity, and are prone to T cell oligoclonality, which needs to be differentiated from T cell lymphoma. Of course, in the case of high proportion of tumor cells, normal hematopoiesis is also inhibited, resulting in a decrease in the proportion of CD117+ myeloid blast cells. In tubes 2-4, CD4 or CD8 (which can be selected according to the expression of CD4 and CD8 in the first tube), CD3 and CD2 or CD5 (the selection basis is according to table 1, CD3 is not easy to lose subtype, combined with CD5 to easily find abnormalities, CD3 is easy to lose subtype, according to whether CD5 is easy to lose, if it is easy to lose, for example ALCL, select CD2, not easy to lose such as AITL, select CD5). T cells recognize antigen peptides through TCR to start immune response, and TCR and CD3 together constitute the core molecules of T cells. According to the type of TCR, T cells are divided into αβT and γδT cells. In healthy people, 90%-95% of T cells are TCRαβ+ αβT cells, and 5%-10% are TCRγδ+ γδT cells. αβT cells mainly include CD4+CD8- and CD4-CD8+ two groups, as well as a small amount of CD4-CD8- double negative (double negative, DN) and CD4+CD8+ double positive (double positive, DP) cells. The γδT cell subgroup generally does not express CD4, and CD8 is partially negative and partially weakly positive.T cells that are CD3+CD56+ are called NKT cells, and partially overlap with CD8+ αβ T cells and γδ T cells, thus are CD4 negative, and CD8 partially negative and partially weakly positive.

[0053] CD2, CD3, CD5, CD7, CD4, CD8, and CD56 are generally used to screen for abnormalities in T cells. Normally, CD3 and CD5 are found on T cells, CD2 and CD7 are found on T cells and CD3-CD56+ NK cells, so the expression of CD3 and CD5 is substantially uniform, and the expression of CD2 and CD7 is substantially uniform. However, there are slight differences in the intensity of CD3 and CD5 expression between normal CD4+ T cells and CD8+ T cells. The intensity of CD3 expression is relatively highest in γδ T cells (but reactive γδ T cells can appear to have reduced CD3 expression), and the intensity of CD3 expression is higher in CD4+ T cells than in CD8+ T cells in αβ T cells. The expression of CD5 in αβ T cells is slightly weaker in CD8+ T cells than in CD4+ cells, and the expression of CD5 is relatively weakest or even negative in γδ T cells. CD7 is uniformly expressed in infants, but as age increases, the proportion of CD7-CD4+ T cells increases, corresponding to the increase in memory T cells, and these memory T cells are accompanied by high expression of CD2. CD7-T cells are more obvious in blood and bone marrow than in lymph nodes, and the expression of CD7 is similar in CD8+ αβ T cells and γδ T cells. CD2 is expressed relatively uniformly in CD8+ αβ T cells and γδ T cells, but in CD4+ T cells, CD2 expression has both high expression and low expression.

[0054] In other words, in most cases, the intensity of CD3 and CD5 expression in CD4 (+) T cells is slightly higher than that in CD8 (+) T cells; the expression of CD3 is relatively strongest in γδ T cells, and the expression of CD5 is relatively weakest in γδ T cells. Figure 1 Table 2, because these subtle differences are the basis for setting the gate and judging abnormalities of mature T lymphocyte tumors, and are the basis for finding two tumor clones.

[0055] Figure 1 Common marker expression of T cell subsets in healthy human bone marrow samples. Figure 1 Figures 1A-1H in the drawings are two-dimensional dot plots, Figure 1Figure 1I-1K are superimposed one-dimensional histograms. Royal blue is NK cells, pink is CD3(+)CD56(+)NKT cells, brown is γδT cells, light green is CD8(+)T cells, and dark green is CD4(+)T cells. Figure 1A-1D are the expression of CD3 and CD56 (Figure 1A), γδT and CD3 (Figure 1B), CD2 and CD7 (Figure 1C), and CD3 and CD5 (Figure 1D) in normal lymphocyte groups. In the αβT cell group, CD4(+) and CD8(+) are dominant (Figure 1E), CD8(+) cells partially express CD57 (Figure 1F), NK, NKT, and γδT are CD4 negative and partially weakly positive for CD8 (Figure 1G), and partially express CD57 (Figure 1H). The expression intensity of CD3 and CD5 is slightly different in γδT, CD4(+), and CD8(+) αβT (Figure 1I and Figure 1J), CD57 is more expressed in NK, NKT, γδT, and CD8(+) αβT, and less expressed in CD4(+) (Figure 1K).

[0056] The T cell receptor (TCR) clonality markers TRBC1, TRBC2, TCRVδ1, and TCRVδ2 are used in tube two. The T-cell receptor constant beta chain (TRBC) is an important component of TCRαβ discovered in recent years. TRBC includes TRBC1 and TRBC2. About 30%-70% of T cells in normal people express TRBC1 or TRBC2, i.e., one of the two, and the expression of TRBC1 and TRBC2 in each subgroup of αβT cells is balanced. However, the malignant cells of mature T lymphocyte tumors usually exhibit limited positive or negative expression (<15% or >85%) of TRBC1 and TRBC2, or double negative. Therefore, in the early years when there was no TRBC2, the use of TRBC1 alone could efficiently evaluate the clonality of αβT cells. However, when the tumor cells exhibit weak expression of TRBC1, or the reactive T cells exhibit strong expression of TRBC1 (for example Figure 13 ), it is easy to cause misjudgment. TRBC2 was introduced in 2024. The use of TRBC1 in combination with TRBC2 (for example, tube 2) can further perfectly solve the false positive and false negative problems of TRBC1 alone. Of course, T cells are much more complex than B cells. The oligoclonal reactive cells and T-CUS will exhibit limited TRBC, leading to difficulties in differential diagnosis.

[0057] TCRVδ1 and TCRVδ2 of tube 2 are used to detect the monoclonality of γδT cells. Human γδT cell subgroups are mainly assembled from about 6 γ (Vγ2-5, Vγ8, Vγ9) types and about 8 δ (mainly Vδ2, Vδ1, Vδ3) types, but these subunits are not evenly distributed, but have organized site subtype differences. The peripheral blood of healthy people is mainly composed of Vδ2 (>75%) and Vδ1 (≤10%) two subtypes, so clinically most of them use TCRVδ2 and TCRVδ1 (one of them <15% or >85%) to judge the clonality of γδT cells, and TCRVδ2 is dominant under normal circumstances, but in γδT cell lymphoma, the Vδ1 subtype is dominant.

[0058]

[0059]

[0060] In addition to T cell markers and comprehensive TCR monoclonality markers, tube 2 also selects CD57. It is mainly aimed at CD4-type lymphoma, such as large granular T lymphocyte leukemia (LGLL) is CD57 positive, and the subtype judgment of γδT cell lymphoma (hepatosplenic γδT cell lymphoma is CD56+CD57-, γδT-LGLL is CD56-CD57+). CD57 is a marker of aging and exhaustion, which is mainly seen in CD8+αβT, γδT cells and CD3-CD56+ NK cells and CD3+CD56+ NKT cells under normal circumstances, and CD4+T cells rarely express CD57, but it may increase in old age or various inflammatory states, and the phenotype of reactive CD57+CD4+ cells is CD7 persistence weakened CD2 highly expressed. γδT cells are a weak subgroup, but they can expand after infection, autoimmune diseases, tumor diseases, splenectomy and allogeneic stem cell transplantation. CD57 is partially negative and partially heterogeneous on normal γδT cells and NKT, NK cells. Therefore, the expression pattern of CD57, especially the abnormal expression of TRBC1, TRBC2, TCRVδ1 and TCRVδ2, and T cell markers, becomes a key point for diagnosis and differential diagnosis.

[0061] In addition to T cell markers, CD26, CD10, CD279, CD25 are selected for CD4+CD8- subtype, which is common mature T lymphoma, including angioimmunoblastic T-cell lymphoma (AITL), adult T-cell lymphoma (ATLL), mycosis fungoides / Sézary syndrome (MF / SS), anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS), etc. T-prolymphocytic leukemia (T-PLL) is also mainly CD4+CD8-, but it is rare, and CD4+CD8+ is more characteristic, so the clinical concern is CD4+CD8+ phenotype. Subtype judgment is shown in Table 1, i.e., CD4+CD8- with strong expression of CD279 and positive CD10 (70-90% probability), considering angioimmunoblastic T-cell lymphoma (AITL), CD4+CD8- with strong expression of CD25, considering adult T-cell lymphoma (ATLL), CD4+CD8- with loss of CD26 and / or CD7, considering mycosis fungoides / Sézary syndrome (MF / SS). T-cell lymphoma that cannot be clearly classified into the above CD4+CD8- subtype is considered peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). Because ALCL and T-PLL are very rare, the first three tubes of CD4+CD8- subtype are often used for diagnosis, and in clinical work, if subtype judgment by flow cytometry is not required, only tube 1 and tube 2 need to be performed. If the proportion of tumor cells is very high and the diagnosis is very clear, the clonality judgment of tube 2 can not be performed, and only tube 1 and tube 3 are selected.

[0062] Tube 4 is mainly used for CD4+CD8+and rare CD4+CD8-mature T lymphocyte tumors. Representative is T-PLL and ALCL. The tumor of this phenotype, especially important task is needed to distinguish from T lymphoblastic lymphoma / leukemia (T-LBL / ALL). Therefore the tube selects CD99, CD30, cytoplasmic TCL1. With CD30 positive, and forward angle light scattering (FSC) and side angle light scattering (SSC) increase, that is, CD45 / SSC two-dimensional dot map outside the lymphocyte gate, often with the loss of T cell markers, considering anaplastic large cell lymphoma (ALCL), CD4+CD8- or CD4+CD8+with cytoplasmic TCL1 positive, CD99 non-strong expression, consider T prolymphocytic leukemia (T-PLL). CD99 strong expression is found in more than 95% of T-LBL / ALL, and rarely in mature T lymphocyte tumors. In addition, the expression intensity of CD7 is also crucial for the differentiation of T-LBL / ALL and mature T lymphocyte tumors. Because during the development of normal T cells, CD7 is strongly expressed in the early stage, and the expression is weakened with maturation. Therefore, more than 95% of T-LBL / ALL show strong expression of CD7, while mature T lymphocyte tumors, CD7 normal intensity or even many subtypes are CD7 weakened or even lost.

[0063] In another aspect, the present application also provides a device for quickly and efficiently diagnosing mature T lymphocyte tumors and judging subtypes, which comprises a detection unit and an analysis unit, wherein:

[0064] The detection unit comprises reagent materials for detecting samples from the individual to be tested by flow cytometry, for obtaining the detection results of the samples; the reagent materials comprise the reagent composition of the present application;

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

[0066] According to a specific embodiment of the present application, in the device for quickly and efficiently diagnosing mature T lymphocyte tumors and judging subtypes of the present application, the application process of the detection unit comprises: using the reagent composition of the present application to process the sample to be tested to prepare the flow cytometry sample; and performing flow cytometry detection. The analysis process of the analysis unit comprises: analyzing the detection results to quickly 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 application, when the device of the present application is used for quickly and efficiently diagnosing mature T lymphocyte tumors and judging subtypes, the gate analysis of each flow tube can be performed according to the foregoing operation.

[0068] According to the specific embodiments of the present application, the device for rapid and efficient screening of mature T lymphocyte tumors of the present application can further comprise a module for further determining the disease according to the gating analysis of the detection results (the determination of the present application includes screening, prompting, auxiliary diagnosis, and diagnosis). The present application compares each group of cells displayed with the corresponding normal cells in the gating of the combination of multiple markers, finds out the tumor cells, and performs rapid and accurate diagnosis, and distinguishes from other diseases that can be misdiagnosed or missed, especially T-CUS and reactive T cells which are similar in morphology and immunophenotype, and other tumors. In summary, the above-mentioned problems can avoid these pitfalls using the present application.

[0069] According to the specific embodiments of the present application, the device for rapid and efficient screening of mature T lymphocyte tumors of the present application is used for one or more of the following mature T cell clonality detection:

[0070] (1) T cell clonality detection for diagnosis and differential diagnosis of mature T cell tumors (commonly known as lymphoma): wherein the selected flow tubes include tube one and tube two;

[0071] (2) Detection of the number of clonality of mature T cell tumors: wherein the selected flow tubes include tube one and tube two;

[0072] (3) Subtype determination of mature T cell tumors:

[0073] The tumor cells of the sample to be tested are CD4+CD8-, and tube one to tube four are selected; or

[0074] The tumor cells of the sample to be tested are CD4+CD8-, and the selected flow tubes include tube one, tube two, and tube three; wherein tube three detects CD10 negative, non-strong expression of CD279, and CD26 not lost, and tube four is additionally used; or

[0075] The tumor cells of the sample to be tested are CD4 negative, and the selected flow tubes include tube one and tube two; or

[0076] The tumor cells of the sample to be tested are CD4+CD8+, or the tumor cells are outside the lymphocyte gate set by CD45 / side angle light scattering, or the clinical indication is rare T prolymphocytic leukemia / lymphoma; and the selected flow tubes include tube one, tube two, and tube four.

[0077] According to the specific embodiments of the present application, when analyzing the four tubes of samples, the CD45 / SSC commonly used in each tube is used to preliminarily screen whether the proportions of each group of cells (mature lymphocytes, monocytes, differentiated stage granulocytes, and nucleated red blood cells) in the normal sample are normal, and whether there is a high proportion of tumor cells, and further combined with other markers of each tube for determination.

[0078] According to a specific embodiment of the present application, the analysis unit of the present application can output the mature T cell clonality detection result according to one of the following judgment modes when analyzing the detection result of the detection unit:

[0079] Mature T lymphocyte tumor: there are positive cells in the abnormal T lymphocyte (AT) gate in the lymphocyte gate set by CD45 / SSC or the T lymphocyte reverse gate of any flow tube, and the differential diagnosis 1-3 below is excluded (i.e., T lymphoblastic lymphoma is excluded, myeloid or histiocytic dendritic cell tumor is excluded, and clonal T cells of unknown significance (T-CUS) or reactive T cells are excluded):

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

[0081] Differential diagnosis 2: tumor cells express CD99 strongly (bri), express CD4 (express CD4 only, or express CD4 and CD56), do not express CD7, CD5, CD3, CD2, CD8, and additional cytoplasmic CD3, and additional myeloid and histiocytic dendritic cell markers CD64, CD33, CD13, MPO, HLA-DR, CD14, CD42a, CD123, CD303, CD304, CD163, CD68, and whether it is a myeloid or histiocytic dendritic cell tumor is determined according to the detection result;

[0082] Differential diagnosis 3: the proportion of abnormal phenotype mature T cells in lymphocytes is less than 5%, or the immunophenotype is CD2 high expression / CD7 weak expression / CD4 positive or CD8 positive, and whether it is a clonal T cell of unknown significance (T-CUS) or a reactive T cell is determined by follow-up for half a year;

[0083] Optionally, for the sample with the above detection result of mature T lymphocyte tumor, the following (1)-(7) are further performed for subtype detection:

[0084] (1) CD4+CD8- accompanied by strong expression of CD279 and positive CD10, and the subtype detection is angioimmunoblastic T-cell lymphoma (AITL);

[0085] (2) CD4+CD8- accompanied by strong expression of CD25, and the subtype detection is adult T-cell lymphoma (ATLL);

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

[0087] (4) CD4+CD8- accompanied by CD30 positive, and forward angle light scattering (FSC) and side angle light scattering (SSC) increase (i.e. tumor cells outside the lymphocyte gate of CD45 / side angle light scattering settings), loss of T cell markers, subtype detection is anaplastic large cell lymphoma (ALCL);

[0088] (5) CD4+CD8 negative or positive accompanied by cytoplasmic TCL1 positive, CD99 non-strong expression, subtype detection is T prolymphocytic leukemia (T-PLL);

[0089] (6) CD4+CD8 negative, not in accordance with the AITL, ATLL, MF / SS, ALCL, T-PLL subtype in (1)-(5) above, subtype detection is non-specific peripheral T cell lymphoma (PTCL, NOS);

[0090] (7) CD4-CD8+ accompanied by CD57 high expression, subtype detection is large granular T lymphocyte leukemia (LGLL);

[0091] (8) CD4-CD8 negative or positive accompanied by TCRVδ1 or TCRVδ2, expressing CD56, subtype detection is hepatosplenic γδ T cell lymphoma.

[0092] In summary, the present application provides a reagent composition for rapid and efficient mature T lymphocyte tumor diagnosis and subtype judgment by flow cytometry and its application. The present application has the following advantages: ① Based on the understanding of the diagnosis and differential diagnosis of a large number of tumor cases, a comprehensive diagnosis and differential diagnosis scheme is designed, which greatly reduces the misdiagnosis rate. ② In terms of application, accurate diagnosis can be made quickly. ③ The present application adopts a method for rapid and efficient mature T lymphocyte tumor diagnosis and subtype judgment, which can pre-mix antibodies, greatly reducing the workload, improving the work efficiency, speeding up the report time, and enabling clinical patients to obtain accurate diagnosis early to take appropriate treatment, thereby improving the remission rate and survival rate. ④ For a long time, one of the important reasons restricting the development of flow cytometry is individualization of the scheme, too much manual operation, which leads to difficulty in automation, standardization and standardization, and the present application can create conditions for the development of artificial intelligence in the future. ⑤ The present application further develops a scoring system according to the contribution, specificity and coverage of the marker combination for diagnosis and differential diagnosis, which can accurately and quickly make judgments with high sensitivity and good specificity. The detection and analysis method of the present application can meet the current clinical flow cytometry diagnosis needs and is suitable for wide application and promotion. BRIEF DESCRIPTION OF DRAWINGS

[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] In order to have a clearer understanding of the technical features, objectives and benefits of the present application, the following detailed description of the technical solutions of the present application will be given in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not limit the scope of the present application. In the examples, each of the original reagent materials can be commercially available, and the experimental methods not specified with specific conditions are the conventional methods and conventional conditions well known in the art, or the conditions recommended by the instrument manufacturers.

[0104] Example 1, preparation of reagents

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

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

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

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

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

[0110] The second part of the fourth group of antibodies is: cytoplasmic TCL1 monoclonal antibody, and the fluorescent label is APC; and the fifth container is used.

[0111] Each antibody in the embodiment can be commercially available, wherein the TRBC1 PE, TRBC1 APC and TRBC2 PE are products of Henan Kaiper Biological Technology Co., Ltd., the TCRVδ1 PE-Cy7, TCRVδ2 BV421, cytoplasmic TCL1 APC and CD30 PE are products of Becton Dickinson, USA, and the remaining directly labeled fluorescent antibodies are mainly products of Becton Dickinson, USA.

[0112] Optionally, a cell lysis solution (hemolysin) is arranged in a sixth container, a membrane breaker A solution is arranged in a seventh container, a membrane breaker B solution is arranged in an eighth container, and a PBS buffer is arranged in a ninth container. The hemolysin, membrane breaker and PBS buffer can be commercially available, wherein the cell lysis solution and membrane breaker are products of Becton Dickinson, USA, and the PBS buffer is a product of Beckman Coulter.

[0113] Example 2, processing of the specimen

[0114] According to the cell counting result, the heparin or EDTA anticoagulated bone marrow or peripheral blood sample is added to the flow tube B tube, so that the amount of added cells is about 2×10 6 μl of nine kinds of cell membrane monoclonal antibody reagents with different fluorescent labels are added to the flow tube according to Table 3, and the cell suspension is mixed well, and then incubated at room temperature for 15 minutes in the dark. 3ml 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 1500rpm for 5 minutes to remove the supernatant, 3ml of PBS is added and mixed, and then centrifuged at 1500rpm for 5 minutes to remove the supernatant. 0.5ml of PBS buffer is added to resuspend the cells, and the treated specimen is ready for machine detection.

[0115] According to the cell counting result, the heparin or EDTA anticoagulated bone marrow or peripheral blood sample is added to the flow tube B tube, so that the amount of added cells is about 2×10 6cells; then 32 μl of nine different fluorescein-labeled monoclonal antibodies reagents for cell membrane were added to the flow tube according to Table 3, and the cell suspension was mixed thoroughly, and then incubated at room temperature for 15 minutes in the dark. Then 3 ml of 1 x hemolysin was added, and the mixture was incubated for 10 minutes in the dark to lyse the red blood cells. The mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then 3 ml of PBS was added, and the mixture was mixed thoroughly, and then centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then 0.5 ml of PBS buffer was added to resuspend the cells, and the treated sample was ready for machine detection.

[0116] According to the cell counting results, the heparin or EDTA-anticoagulated bone marrow or peripheral blood sample was added to the flow tube, and the amount of the added cells was about 2 x 10 6 cells; then 32 μl of nine different fluorescein-labeled monoclonal antibodies reagents for cell membrane were added to the flow tube according to Table 3, and the cell suspension was mixed thoroughly, and then incubated at room temperature for 15 minutes in the dark. Then 3 ml of 1 x hemolysin was added, and the mixture was incubated for 10 minutes in the dark to lyse the red blood cells. The mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then 3 ml of PBS was added, and the mixture was mixed thoroughly, and then centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then 0.5 ml of PBS buffer was added to resuspend the cells, and the treated sample was ready for machine detection.

[0117] According to the cell counting results, the heparin or EDTA-anticoagulated bone marrow or peripheral blood sample was added to the flow tube, and the amount of the added cells was about 2 x 10 6 cells; then 32 μl of nine different fluorescein-labeled monoclonal antibodies reagents for cell membrane were added to the flow tube according to Table 3, and the cell suspension was mixed thoroughly, and then incubated at room temperature for 15 minutes in the dark. Then 3 ml of 1 x hemolysin was added, and the mixture was incubated for 10 minutes in the dark to lyse the red blood cells. The mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then 3 ml of PBS was added, and the mixture was mixed thoroughly, and then centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then 0.5 ml of PBS buffer was added to resuspend the cells, and the treated sample was ready for machine detection.

[0118]

[0119] Example 3, Detection of the Sample

[0120] The samples processed according to the method of Example 2 are detected on a Becton Dickinson 3 laser 10 color FACS Canto plus flow cytometer, preferably 1 million cells per tube (at least 300,000 recommended) are obtained, and the data are analyzed using diva 2.8 software or other software such as kaluza.

[0121] Wherein, the flow cytometry detection is set according to the following method: ① fixed gate setting: sequentially remove the adhesion cell gate, live cell gate, blood cell gate; ② accurately set the lymphocyte gate: observe the coincidence degree of T cell marker and CD45 / SSC set lymphocyte gate (lym), determine that there is no lymphocyte outside the lymphocyte gate, otherwise the marker reverse gate needs to be set. If the boundary between lymphocytes and mononuclear cells is not clear, use CD4 weak positive / T cell marker negative to set the mononuclear cell gate 2, and remove it from the lymphocyte gate to achieve accurate gate setting; ③ in the accurately set lymphocyte gate, display the common expression pattern and development pattern of various marker combinations, find out the tumor cells according to the difference from normal cells, and there may be more than one group. Specifically:

[0122] 1. Fixed gate setting: composed of de-adhesion cell gate, live cell gate, blood cell gate, in series.

[0123] De-adhesion cell gate: first use forward angle light scattering (FSC) area (A) and height (H) to set the de-adhesion cell gate (represented by P1), which can remove the adherent cells by FSC-area (A) / height (H), the principle is that the cell is spherical, A is positively correlated with H. See Figures 2-28 The first gate setting picture from left to right in the top row of

[0124] Live cell gate: P1 cells, use FSC / SSC to set the live cell gate (represented by P2), get single live cells. The principle of FSC / SSC is that the size and granularity of live cells are normally distributed, which are clustered around a center, and have obvious boundaries with dead cells, apoptotic cells, debris and background noise. See Figures 2-28 The second gate setting picture from left to right in the top row of

[0125] Blood cell gate: within the single live cell gate (P2 gate), using CD45 / SSC setting, roughly observe lymphocytes, monocytes, granulocytes, nucleated red blood cells, and whether there are obvious tumor cells or abnormal cells. Roughly distinguish each group of blood cells by CD45 / SSC, the principle is that according to the different CD45 expression fluorescence intensity of hematopoietic cells (mature lymphocytes > monocytes > granulocytes > nucleated red blood cells), and the size difference of SSC (eosinophils > granulocytes > monocytes > mature lymphocytes > nucleated red blood cells). See the CD45 / SSC gate picture in Figures 2-28

[0126] 2. Precise setting of lymphocyte gate and tumor cell gate:

[0127] A pipe: within the P2 gate, observe the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with the lymphocyte gate (lym), and determine that there are no lymphocytes outside the lymphocyte gate, otherwise the marker reverse gate needs to be used. If the boundary between lymphocytes and monocytes is not clear, set monocyte gate 2 using CD4 weak positive / T cell markers (CD2, CD3, CD5, CD7) negative, and remove from the lymphocyte gate to achieve precise gating. Within the precisely set lymphocyte gate, CD3 negative / CD7 positive or CD3 negative / CD56 positive are NK cells, and two-dimensional dot plots of CD3, CD5, CD2, CD7, CD4, CD8, CD56, CD117 are made to select abnormal T cells (AT) gate that are different from normal T cells, if there are more than one group, set AT1 and AT2 respectively;

[0128] B pipe: within the P2 gate, observe the degree of coincidence of CD2 or CD5, CD3, CD4 or CD8 markers with the lymphocyte gate (lym), and determine that there are no lymphocytes outside the lymphocyte gate, otherwise the marker reverse gate needs to be used. If the boundary between lymphocytes and monocytes is not clear, set monocyte gate 2 using CD4 weak positive / T cell markers (CD2 or CD5, CD3) negative, and remove from the lymphocyte gate to achieve precise gating. Within the precisely set lymphocyte gate, display the expression of CD57, TRBC1, TRBC2, TCRVδ1, TCRVδ2, and select abnormal T cells (AT) gate that are different from normal T cells, if there are more than one group, set AT1 and AT2 respectively;

[0129] ​P2 gate, observe the degree of CD2 or CD5, CD3, CD4, CD7 marker and lymphocyte gate (lym) match, determine that there is no lymphocyte outside the lymphocyte gate, otherwise need to use the marker reverse gate, if the lymphocyte and monocyte boundary is not clear, use CD4 weak positive / T cell marker (CD2 or CD5, CD3) negative to set monocyte gate 2, and remove from the lymphocyte gate, to achieve accurate gate setting. In the accurately set lymphocyte gate, display the expression of CD26, CD10, CD279, CD25, and select T cells different from normal T cells as abnormal T cells (AT) gate, if there is more than one group, set AT1 and AT2 respectively;

[0130] D tube, mainly observe: P2 gate, selectively, tube four gate setting according to the following way: P2 gate, observe the degree of CD2 or CD5, CD3, CD4 marker and lymphocyte gate (lym) match, determine that there is no lymphocyte outside the lymphocyte gate, otherwise need to use the marker reverse gate, if the lymphocyte and monocyte boundary is not clear, use CD4 weak positive / T cell marker (CD2 or CD5, CD3) negative to set monocyte gate 2, and remove from the lymphocyte gate, to achieve accurate gate setting. In the accurately set lymphocyte gate, display the expression of CD99, CD30, TCL1, and select T cells different from normal T cells as abnormal T cells (AT) gate, if there is more than one group, set AT1 and AT2 respectively.

[0131] 3. According to the observation of the combination of markers to accurately lock the target cells

[0132] Flow cytometry is used for the diagnosis and subtype judgment of mature T lymphocyte tumors, which is mainly divided into three steps. First, lock abnormal cells, find abnormal expression of cell groups, which may be more than one group, and determine monoclonality by TCR clonality index; second, exclude reactive cells and T-CUS and other tumors, and determine as mature T lymphocyte tumor; third, judge the subtype according to the expression of characteristic markers of common subtypes.

[0133] (1) Abnormal expression

[0134] Changes in mature T cell phenotype: ① loss of T cell antigens (such as CD2, CD3, CD5, CD7); ② change in fluorescence intensity of normally expressed antigens; ③ abnormal expression of non-T lineage antigens, such as CD10, CD117, etc.; ④ consistent expression of antigens that are normally expressed in small amounts or not expressed: CD30, CD25, CD57, CD56, CD279, etc.; ⑤ significant increase in the proportion of weak cell population and abnormal expression;

[0135] (2) Monoclonality judgment

[0136] T cells are divided into αβ T cells and γδ T cells. αβ T cells can be expressed by the restriction of TRBC1 and TRBC2 (one of them is expressed at a rate of > 85%, or < 15%), and γδ T cells are expressed by the restriction of TCRVδ1 and TCRVδ2 (one of them is expressed at a rate of > 85%, or < 15%). If none of them is expressed, it also indicates abnormality. It should be noted that T cells are the main immune cells of humans. In order to prevent a large number of normal background cells from masking the monoclonality of a small number of tumor cells, or more than one group of tumor cells, it is necessary to lock the abnormally expressed cell group according to the combination of markers such as CD2, CD3, CD4, CD5, CD7, CD8, CD56, etc. That is, after accurate gating, the TCR (TCRVβ, TRBC1, TRBC2, TCRVδ1, TCRVδ2, etc.) restriction expression of tumor cells is evaluated.

[0137] (3) Exclude other possibilities

[0138] Exclude reactive cells and T-CUS: viral infection, autoimmune disease, other tumors, old age, T-CUS, etc. can all appear changes in pan-T cell markers (CD2, CD3, CD5, CD7), significant increase in CD4+CD8+ T cells, TCRγδ cells, NKT cells, etc. Subgroups, CD4 / CD8 ratio increased or decreased, and even TCR restriction expression. Therefore, for NKT, CD8+ T cells, CD4+CD8+ T cells (reactive expression pattern, the expression intensity is consistent with the intensity of normal CD4 or normal CD8 cells), CD4+ T cells with weak CD7 persistence and high CD2 expression, if they do not have obvious hepatosplenomegaly and / or lymphadenopathy, persistent low fever and other clinical manifestations related to lymphocyte tumors, and the proportion of abnormal cells is low (for example, less than 5% of lymphocytes), careful evaluation is needed, and most of them are reactive cells or T-CUS.

[0139] Exclusion of other tumors: ① If CD99 strongly expressed, and tumor cells positive for CD7 and / or CD5 and / or CD3 and / or CD8 and / or CD2, consider T lymphoblastic lymphoma rather than mature T lymphocyte tumor; ② If CD99 strongly expressed, CD7, CD5, CD3, CD8, CD2 all negative, plus cytoplasmic CD3 and TdT and / or CD34, if cytoplasmic CD3 positive, early marker TdT and / or CD34 positive, still consider T lymphoblastic lymphoma rather than mature T lymphocyte tumor; ③ If CD99 strongly expressed, only express CD4, or express CD4 and CD56, do not express CD7, CD5, CD3, CD2, CD8, and plus cytoplasmic CD3, need to do myeloid and histiocytic dendritic cell markers CD64, CD33, CD13, MPO, HLA-DR, CD14, CD42a, CD123, CD303, CD304, CD163, CD68, etc., to determine whether it is a myeloid and histiocytic dendritic cell tumor.

[0140] (4) Subtype judgment

[0141] According to the typical immunophenotype of common mature T lymphocyte tumors in Table 1, the subtype is judged, that is: ① CD4+CD8- with strong expression of CD279, CD10 positive (70-90% probability), consider angioimmunoblastic T cell lymphoma (AITL), ② CD4+CD8- with strong expression of CD25, consider adult T cell lymphoma (ATLL), ③ CD4+CD8- with loss of CD26 and / or CD7, consider mycosis fungoides / Sézary syndrome (MF / SS), ④ CD4+CD8- with CD30 positive, and forward angle light scattering (FSC) and side angle light scattering (SSC) increased, often with loss of T cell markers, consider anaplastic large cell lymphoma (ALCL), ⑤ CD4+CD8 negative or positive with cytoplasmic TCL1 positive, CD99 non-strong expression, consider T prolymphocytic leukemia (T-PLL), ⑥ CD4+CD8 negative, not AITL, ATLL, MF / SS, ALCL, T-PLL, consider non-specific peripheral T cell lymphoma (PTCL, NOS), ⑦ CD4-CD8+ with high expression of CD57, consider large granular T lymphocyte leukemia (LGLL), ⑧ CD4-CD8 negative or positive with TCRVδ1 or TCRVδ2, often express CD56, consider hepatosplenic γδ T cell lymphoma.

[0142] From 2022, this scheme was used, initially because there was no TRBC2, the channel used TCRγδ PE at that time, from February 2022 to September 2024, 2109 detections from 1235 patients were detected, of which 364 detections from 202 positive samples were detected, after purchasing TRBC2 from October 2024, all were changed to this scheme, from October 2024 to the end of June 2025, 414 detections from 259 patients were completed, of which 84 detections from 52 positive samples were detected. So far, a total of 2523 detections from 1494 people have been completed, of which 448 detections from 254 positive samples have been completed. The median age of the patients was 55 years (1-88 years), and the median proportion of tumor cells was 2.32% (0.01%-93.61%). After morphology, genetics, pathology, 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 were 98.81%, 99.76%, 98.81%, and 99.76%, respectively, and the total sensitivity, specificity, positive predictive value, and negative predictive value were 97.74%, 99.41%, 96.65%, and 99.60%, respectively.

[0143] In the present application, the A tube is a general screening tube, and the B tube, the C tube, and the D tube can be used together or selectively. The B tube contains a clonal marker. The use of TRBC1 solves the problem of most T cell clonal examination, but 9.5% of patients have weak TRBC1 expression, making it difficult to accurately evaluate monoclonality. Or because of the existence of two clones (flow cytometry can find that 5% of mature lymphocyte tumors are composed of two clones, but in recent years, due to targeted therapy and gene sequencing work, it has been found that most patients have more than two clones, only that the weak clone is too small, or the immunophenotypic difference is extremely small, and cannot be distinguished) Figure 13 , the expression of TRBC1 in reactive cells is enhanced, which can easily lead to misdiagnosis and missed diagnosis. The use of TRBC2 in the present application can almost solve this problem by 100%. The scheme of the present application including TRBC2 improves the sensitivity and specificity.

[0144] The contribution of the first tube, using CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD45 markers, on the one hand to help determine the accuracy of the lymphocyte gate CD45 / SSC settings, to prevent missing tumor cells because of the cell FSC and SSC is large or CD45 weakened, not in the lymphocyte gate. On the other hand, 5% of lymphoma exists two clones, using multiple T cell marker combination can accurately find more than two clones. In addition, in normal specimens, although there is a great heterogeneity of T cells, but the expression is very regular, reactive cells, also very regular expression, such as CD7 extension of the weakening accompanied by CD2 expression enhancement, mostly CD4 positive, viral infection can also be CD8 positive cell group. CD4 and CD8 double positive, and CD3+CD56+ cell group, more common reactive, and the expression pattern of reactive cell group is very characteristic, CD4 and CD8 expression, often flat flat eight (either the expression intensity of normal CD4+T cells CD4 consistent with the intensity of normal CD8+T cells CD8 expression consistent, or consistent with CD4 and CD8, that is, in the CD4 / CD8 two-dimensional point graph of CD4 and CD8 positive cell center intersection position), and in the tumor cells, CD4+CD8+ mature T lymphocyte tumor is rare, mainly T-PLL, its phenotype has characteristic TCL1, and limited expression of TCR. In addition, this need to exclude CD99 strong expression of T-LBL / ALL, these markers in the fourth tube Ding tube. Therefore, the main contribution of the first tube is to find T cell markers and combined expression intensity different from normal cell groups and the number of abnormal clones. At the same time, the tube also has a CD117, on the one hand, T cell tumors may appear abnormal expression of the mark, more importantly, patients with aplastic anemia prone to oligoclonal T cell proliferation, such patients with CD117 positive myeloid blast cell proportion of bone marrow is extremely low, so the expression rate of CD117 in patients with low need to be careful.

[0145] The second tube is in addition to T cell markers, including four TCR restriction indicators, the tube can help the first tube to further identify reactive cells and mature T lymphocyte tumors, but also the main tube for CD4-type mature T lymphocyte subtype judgment.

[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] Briefly, the present application uses T cell markers to precisely gate in the multiple marker combination gating, and uses TCR clonality markers to determine clonality, so as to exclude reactive cells, T-CUS and other tumors. After diagnosing mature T lymphocyte tumors, further subtype determination can be made according to several common markers with characteristic immunophenotypes.

[0149] This embodiment provides a typical case of reactive cell tube 1 and tube 2. Figures 2-3 Immune phenotypes of mature T cells of tube A and tube B for autoimmune disease patients, Figures 4-5 Immune phenotypes of mature T cells of tube A and tube B for aplastic anemia patients. Figures 6-28 Immune phenotyping of tube A to tube D for a typical case of mature T cell lymphoma, which is strictly gated and observed according to the above description, respectively.

[0150] Specifically, Figures 2-3 For the same autoimmune disease patient bone marrow sample, tube A and tube B 2 tubes are analyzed together.

[0151] Specifically, Figure 2 Analysis of tube A of the autoimmune disease patient bone marrow sample is shown. The settings are as follows: ① FSC-A / H setting P1 is a non-adherent cell gate, obtaining single cells in P1; ② FSC / SSC setting P2 is a live cell gate in P1, obtaining single live cells in P2; ③ blood cell gate is used in P2 gate using CD45 / SSC, obtaining lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gate (eo), respectively. In the CD117 / CD7 two-dimensional dot plot, CD117 positive CD7 negative cells are selected as myeloid blast cells (CD117+), the proportion is in the range of 0.2-5%. And it is determined that there is no abnormal cell with double positive of CD7 and CD117; ④ In P2 gate, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with lymphocyte gate (lym) is observed, and it is determined that there is no lymphocyte outside the lymphocyte gate. In the Lym gate, CD3 negative / CD7 positive is NK cell (orange yellow), in addition, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, CD3 / CD56, etc. are shown, and no obvious abnormal cells are found, but the CD3+CD5+T cell (dark green cell) CD4 / CD8 ratio is high, which is 77.87 / 14.14=5.51, (CD4 / CD8 greater than 10 plus T cell clonality can be considered as T cell lymphoma, but the CD4 / CD8 of healthy people rarely exceeds 2.5).

[0152] Specifically, Figure 3 Analysis of tube A and tube B of the same autoimmune disease patient bone marrow sample is shown. The settings are as follows: ① FSC-A / H setting P1 is a non-adherent cell gate, obtaining single cells in P1; ② FSC / SSC setting P2 is a live cell gate in P1, obtaining single live cells in P2; ③ blood cell gate is used in P2 gate using CD45 / SSC, obtaining lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gate (eo), respectively. In the CD117 / CD7 two-dimensional dot plot, CD117 positive CD7 negative cells are selected as myeloid blast cells (CD117+), the proportion is in the range of 0.2-5%. And it is determined that there is no abnormal cell with double positive of CD7 and CD117; ④ In P2 gate, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, CD56 markers with lymphocyte gate (lym) is observed, and it is determined that there is no lymphocyte outside the lymphocyte gate. In the Lym gate, CD3 negative / CD7 positive is NK cell (orange yellow), in addition, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, CD3 / CD56, etc. are shown, and no obvious abnormal cells are found, but the CD3+CD5+T cell (dark green cell) CD4 / CD8 ratio is high, which is 77.87 / 14.14=5.51, (CD4 / CD8 greater than 10 plus T cell clonality can be considered as T cell lymphoma, but the CD4 / CD8 of healthy people rarely exceeds 2.5). Figure 2Bone marrow sample of the same autoimmune disease patient, pipe B analysis. In turn set: ① FSC-A / H set P1 as a non-adherent cell gate, and get single cells in P1; ② In P1, show FSC / SSC set P2 as a live cell gate, and get single live cells in P2; ③ In P2 gate, use CD45 / SSC to set a blood cell gate, and get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo); ④ In P2 gate, observe the degree of coincidence of CD5, CD3, and CD8 markers with the lymphocyte gate (Lym) to determine that there are no lymphocytes outside the lymphocyte gate. In the Lym gate, show the expression of CD57, TRBC1, TRBC2, TCRVd1, and TCRVd2. In this case, there are no abnormal cells, but the CD4 / CD8 ratio is high. In order not to miss any small clones, show the TCR expression of CD3+CD5+T cells, CD3+CD5-T cells (pink), CD3+CD5+abT (blue plus green), CD3+CD5+gdT (red), CD8-abT (CD4+abT cells, blue), and CD8+abT (green) in the Lym gate. In the CD3+CD5+T cell gate, TRBC1 and TRBC2 are normally balanced and mutually exclusive, and those expressing TRBC1 or TRBC2 are αβT cells (CD3+CD5+abT, blue plus green), and those negative for TRBC1 and TRBC2 are γδT (CD3+CD5+gdT) cells (red), also normally balanced and mutually exclusive. In the CD3+CD5-T cell gate, γδT cells are dominant, and TCRVd1 and TCRVd2 are normally balanced and mutually exclusive, and TRBC1 and TRBC2, although rare, are also normally balanced and mutually exclusive. CD3+CD5+abT is further split into CD8+abT (green) and CD8-abT (CD4+abT cells, blue), and both groups of cells have normally balanced and mutually exclusive TRBC1 and TRBC2. Through this method, lymphoma is ruled out, and the high CD4 / CD8 ratio is determined to be a reactive change in autoimmune disease.

[0153] Specifically, Figures 4-5 Bone marrow sample of the same aplastic anemia patient, pipe A and pipe B 2-pipe joint analysis.

[0154] Specifically, Figure 4The bone marrow sample of aplastic anemia patient is displayed, and the tube analysis is carried out. The settings are as follows: ① FSC-A / H is set as P1 to be a cell adhesion gate, and single cells in P1 are obtained; ② FSC / SSC is set as P2 to be a live cell gate in P1, and single live cells in P2 are obtained; ③ blood cell gates are set using CD45 / SSC in P2, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) are obtained, respectively; CD117 / CD7 two-dimensional dot plot is selected to be CD117 positive CD7 negative cells, the proportion of which is extremely low in this patient, and almost no CD117 positive myeloid blast cells, which is a characteristic of aplastic anemia patients; ④ in P2 gate, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym) is observed, and it is determined that there is no lymphocyte outside the lymphocyte gate. In the Lym gate, CD3 negative / CD7 positive is NK cells (orange yellow), in addition, CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, CD3 / CD56, and the like are displayed in two-dimensional dot plots, and no obvious abnormal cells are found, but the CD3+CD5+T cell (dark green cell) CD4 / CD8 ratio is high, which is 57.92 / 19.14=3.03, (CD4 / CD8 greater than 10 plus T cell clonality can be considered T cell lymphoma, but the CD4 / CD8 of healthy people rarely exceeds 2.5).

[0155] Specifically, Figure 5 displayed with Figure 4Bone marrow sample of the same aplastic anemia patient was analyzed by using the same method. The settings were as follows: ① FSC-A / H was set as P1 to remove the adherent cells, and P1 was single cells; ② FSC / SSC was set as P2 to show the live cells in P1, and P2 was single live cells; ③ CD45 / SSC was used to set the blood cell gate in P2, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophilic granulocyte gate (eo) were obtained; ④ In P2, the degree of coincidence of CD5, CD3, and CD4 markers with the lymphocyte gate (Lym) was observed to determine that there were no lymphocytes outside the lymphocyte gate. In the Lym gate, the expression of CD57, TRBC1, TRBC2, TCRVd1, and TCRVd2 was shown. In this case, there were no abnormal cells, but the CD4 / CD8 ratio was high. In order to not miss any small clones, the TCR expression of CD3+CD5+T cells, CD3+CD5-T cells (pink), CD3+CD5+abT (blue plus green), CD3+CD5+gdT (red), CD4-abT (CD8+abT cells, blue), and CD4+abT (green) in the Lym gate was shown. In the CD3+CD5+T cell gate, TRBC1 and TRBC2 were normally proportionally balanced and mutually exclusive, and the expression of TRBC1 or TRBC2 was αβT cells (CD3+CD5+abT, blue plus green), and TRBC1 and TRBC2 were negative γδT (CD3+CD5+gdT) cells (red), and TCRVd2 showed clonal expression, with a proportion of more than 85%. In the CD3+CD5-T cell gate, γδT cells were dominant, and TCRVd1 and TCRVd2 showed normally proportionally balanced and mutually exclusive expression, and no obvious TRBC1 and TRBC2 expression was observed. CD3+CD5+abT was further split into CD4+abT (green) and CD4-abT (CD8+abT cells, blue), and both groups of cells showed normally proportionally balanced and mutually exclusive expression of TRBC1 and TRBC2. This patient had clonal TCRVd2 cells, and although the proportion of this group of cells accounted for 15.06% of lymphocytes (80.12% x 18.8%), it was more than 5%, but first, there was no abnormal phenotype, second, γδT cell lymphoma was mostly TCRVd1, and rarely TCRVd2, and third, the patient had no fever, hepatosplenomegaly, and lymphadenopathy related to lymphoma, and fourth, aplastic anemia was an immune-related disease, and reactive oligoclonal cells were often seen, so the reactive cells were considered comprehensively.

[0156] Specifically, Figures 6-8 Bone marrow sample of the same mature T lymphocyte tumor AITL combined with LGLL patient was analyzed by using the same method.

[0157] In particular, Figure 6 The bone marrow sample of the AITL combined with LGLL case was analyzed by the aorta tube. The settings were as follows: ① FSC-A / H was set as P1 to remove the adherent cells, and the single cells in P1 were obtained; ② FSC / SSC was set as P2 to show the live cells in P2, and the single live cells in P2 were obtained; ③ CD45 / SSC was used to set the blood cell gate in P2, and the lymphocyte (lym), granulocyte (Gra), monocyte (mono), nucleated erythrocyte (NEC), and eosinophil cell gates were obtained, respectively; in the CD117 / CD7 two-dimensional dot plot, no abnormal cells with CD7 and CD117 double positivity were observed, and the proportion of CD117 positive CD7 negative myeloid blast cells was low, which was 0.14%; ④ In P2, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym) was observed, and it was determined that there were no lymphocytes outside the lymphocyte gate. In the Lym gate, CD3 negative / CD7 positive was NK cells (orange yellow), and in addition, the two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 were displayed, respectively. In the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) was normal T cells, and two groups of abnormal cells were observed, CD3+CD5- was abnormal T cell 1 (AT1, red), and CD3-CD5+ was abnormal T cell 2 (AT2, sapphire blue). AT1 expressed CD3 and CD2, expressed a small amount of CD7, did not express CD4, CD8, CD5, CD117, and CD56, and FSC and SSC were large, which belonged to the CD4- type. AT2 expressed CD4, CD5, and CD2, did not express CD7, CD8, CD3, CD117, and CD56, and FSC and SSC were small, which belonged to the CD4+CD8- type.

[0158] In particular, Figure 7 The bone marrow sample of the AITL combined with LGLL case was analyzed by the aorta tube. The settings were as follows: ① FSC-A / H was set as P1 to remove the adherent cells, and the single cells in P1 were obtained; ② FSC / SSC was set as P2 to show the live cells in P2, and the single live cells in P2 were obtained; ③ CD45 / SSC was used to set the blood cell gate in P2, and the lymphocyte (lym), granulocyte (Gra), monocyte (mono), nucleated erythrocyte (NEC), and eosinophil cell gates were obtained, respectively; in the CD117 / CD7 two-dimensional dot plot, no abnormal cells with CD7 and CD117 double positivity were observed, and the proportion of CD117 positive CD7 negative myeloid blast cells was low, which was 0.14%; ④ In P2, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym) was observed, and it was determined that there were no lymphocytes outside the lymphocyte gate. In the Lym gate, CD3 negative / CD7 positive was NK cells (orange yellow), and in addition, the two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 were displayed, respectively. In the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) was normal T cells, and two groups of abnormal cells were observed, CD3+CD5- was abnormal T cell 1 (AT1, red), and CD3-CD5+ was abnormal T cell 2 (AT2, sapphire blue). AT1 expressed CD3 and CD2, expressed a small amount of CD7, did not express CD4, CD8, CD5, CD117, and CD56, and FSC and SSC were large, which belonged to the CD4- type. AT2 expressed CD4, CD5, and CD2, did not express CD7, CD8, CD3, CD117, and CD56, and FSC and SSC were small, which belonged to the CD4+CD8- type. Figure 6Bone marrow sample of the same mature T lymphocyte tumor AITL combined with LGLL case was analyzed by the following steps: ① FSC-A / H was set as P1 to remove the adherent cells, and single cells were obtained in P1; ② FSC / SSC was set as P2 to show the live cells in P1, and single live cells were obtained in P2; ③ CD45 / SSC was set as blood cell gate in P2 to obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophils (eo) gates, respectively; ④ In P2, the degree of coincidence of CD5, CD3, and CD4 markers with the lymphocyte gate (Lym) was observed, and it was determined that there were no lymphocytes outside the lymphocyte gate. In the Lym gate, in the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) was normal T cells, and two groups of abnormal cells were observed, CD3+CD5- (red) was abnormal T cell 1 (AT1), and CD3-CD5+ (royal blue) was abnormal T cell 2 (AT2). AT1 expressed CD3, CD57, and TRBC1, but did not express CD4, CD5, TRBC2, TCRVd1, and TCRVd2, and FSC and SSC were both large. AT2 expressed CD4, CD5, and TRBC1, but did not express CD57, CD3, TRBC2, TCRVd1, and TCRVd2, and FSC and SSC were both small. AT1 in this tube was diagnosed as LGLL, but the common type was CD8+CD4- αβ T cell type, and the patient was of the less common type, CD8-CD4- αβ T cell type.

[0159] In particular, Figure 8 showing a correlation with Figure 6 and Figure 7Bone marrow sample of the same mature T lymphocyte tumor AITL combined with LGLL case, propylene tube analysis. Set in order: ① FSC-A / H set P1 as a non-adherent cell gate, and get single cells in P1; ② In P1, show FSC / SSC set P2 as a live cell gate, and get single live cells in P2; ③ In P2 gate, use CD45 / SSC to set a blood cell gate, and get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophils (eo) gates, respectively; ④ In P2 gate, observe the degree of coincidence of CD5, CD3, CD4, and CD7 markers with the lymphocyte gate (Lym), and determine that there are no lymphocytes outside the lymphocyte gate. In the Lym gate, in the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) is normal T cells, and two groups of abnormal cells can be seen, CD3+CD5- is abnormal T cell 1 (AT1, red), and CD3-CD5+ is abnormal T cell 2 (AT2, sapphire blue). AT1 expresses CD3, a small amount of CD279 and CD7, does not express CD4, CD5, CD26, CD10, and CD25, and FSC and SSC are both large. AT2 expresses CD4 and CD5, strongly expresses (bri) CD279, partially strongly expresses CD10, does not express CD7, CD3, CD26, and CD25, and FSC and SSC are both small. Figure 7 AT1 has been determined to be diagnosed as LGLL, Figure 8 AT2 is diagnosed as AITL, so this case is a rare AITL combined with LGLL.

[0160] Specifically, Figures 9-11 Bone marrow sample of the same mature T lymphocyte tumor MF / SS patient is analyzed in three tubes A to C.

[0161] Specifically, Figure 9The bone marrow sample of MF / SS patient showing mature T lymphocyte tumor was analyzed by flow cytometry. The settings were as follows: ① FSC-A / H was set as P1 to remove the adherent cells, and the single cells in P1 were obtained; ② FSC / SSC was set as P2 to show the live cells in P1, and the single live cells in P2 were obtained; ③ CD45 / SSC was used to set the blood cell gate in P2, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophils (eo) were obtained, respectively; in the CD117 / CD7 two-dimensional dot plot, no abnormal cells with CD7 and CD117 double positivity were observed, and the proportion of CD117 positive CD7 negative myeloid blast cells was low, which was 0.05%; ④ P2 gate was used to observe the coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym), and it was determined that there was no lymphocyte outside the lymphocyte gate. In the Lym gate, CD3 negative / CD7 positive was NK cells (orange yellow), in addition, the two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 were displayed, respectively, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive partially negative (dark green) was normal T cells, CD3 weakly positive (dim) CD4dim was abnormal T cells (AT, red), which belonged to the CD4+CD8-type. AT expressed CD5, CD3dim, CD4dim, and CD2dim, did not express CD7, CD8, CD117, and CD56, and the FSC and SSC were both small.

[0162] Specifically, Figure 10 showing that Figure 9The same mature T lymphocyte tumor MF / SS patient bone marrow sample, the B tube analysis. In turn set: ① FSC-A / H set P1 as a non-adherent cell gate, P1 inside the single cell; ② FSC / SSC settings P2 in P1 show live cell gate, P2 inside the single live cell; ③ P2 gate using CD45 / SSC settings blood cell gate, lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), eosinophil gate (eo) are obtained respectively; ④ P2 gate, observe the degree of coincidence of CD5, CD3, CD4 markers and lymphocyte gate (Lym), to determine that there is no lymphocyte outside the lymphocyte gate. In Lym gate, in CD3 / CD4 two-dimensional dot plot, CD3+CD4 part positive part negative (dark green) is normal T cells, normal T cells balancedly and mutually exclusive expression of TRBC1 and TRBC2, TRBC1 and TRBC2 negative γδT (TCRgd, pink) cells, balancedly and mutually exclusive expression of TCRVd1 and TCRVd2; CD3dimCD4dim is abnormal T cells (AT, red), AT expression CD5, CD3dim, CD4dim and TRBC2, not expression CD57, TRBC1, TCRVd1 and TCRVd2, and FSC and SSC are both small. So far, Figure 9 A tube and Figure 10 B tube determines the presence of mature T lymphocyte tumor in the sample, and confirms the monoclonality. General laboratory flow cytometry does not need to distinguish subtypes, can stop here. If you want to further determine the subtype of CD4+ / CD8- tumor cells, further detect C tube.

[0163] Specifically, Figure 11 Show and Figure 9 And Figure 10Bone marrow sample of the same mature T lymphocyte tumor MF / SS case, prop tube analysis. Set in order: ① FSC-A / H set P1 as a cell adhesion gate, and get single cells in P1; ② FSC / SSC in P1 shows live cell gate P2, and get single live cells in P2; ③ blood cell gate is used in P2 gate using CD45 / SSC, and get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gate (eo); ④ P2 gate, observe the degree of coincidence of CD5, CD3, CD4 markers and lymphocyte gate (Lym), and determine that there are no lymphocytes outside the lymphocyte gate. In Lym gate, in CD3 / CD4 two-dimensional dot plot, CD3+CD4 part positive part negative (dark green) is normal T cells, CD3dimCD4dim is abnormal T cells (AT, red), AT expresses CD5, CD3dimand CD4dim, does not express CD10, CD279, CD26, CD25 and CD7, and FSC and SSC are both small. Subtype is MF / SS.

[0164] Specifically, Figures 12-15 Bone marrow sample of the same mature T lymphocyte tumor ATLL patient, wherein Figure 13 and Figure 14 Same ethyl tube, different method analysis, therefore Figures 12-15 A to C three tube common analysis.

[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 andFigure 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 SSCs, 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 is that SSC small CD5int gated AT2* (royal blue) is used to show the TRBC1 / TRBC2 two-dimensional dot plot of AT2*. Although TRBC1bri compared with normal T cells, but the proportion of TRBC1bri and TRBC2bri is balanced, so it is a reactive cell. In addition, because the mature T lymphocyte tumor is dominated by αβT type, the new scheme targets two clonal markers of αβT and γδT, which enhances the detection of αβT type.

[0169] Specifically, Figure 15 Showed that Figures 12 to 14 The same mature T lymphocyte tumor ATLL case bone marrow specimen, prop tube analysis. In turn: ① FSC-A / H set P1 as a de-adhesion cell gate, obtaining single cells in P1; ② In P1, show FSC / SSC set P2 as a live cell gate, obtaining single live cells in P2; ③ In P2 gate, use CD45 / SSC to set a blood cell gate, respectively, obtaining granulocytes (Gra), mononuclear cells (mono), nucleated red blood cells (NEC), and eosinophil gates (eo). Lymphocytes and mononuclear cells are not clearly separated, so Lym and part of the mononuclear cell gate are obtained at this time; ④ In P2 gate, observe the degree of coincidence of CD3, CD4, CD5 markers with Lym and part of the mononuclear cell gate, determine that there are no lymphocytes outside the Lym and part of the mononuclear cell gate, use CD5 and CD4 two-dimensional dot plot, and Figures 12-14 The same method is used to set the normal lymphocyte gate (dark green), the abnormal cell gate (AT, red), and the "total lymphocyte" logical gate. AT cells AT express CD5dim, CD4 and CD25, and part of them express CD3dim, CD7dim, CD26, but do not express CD10 and CD279. Therefore, the subtype is ATLL. At this time, pay attention to the AT2* cell group, which expresses CD279 more than normal T cells, further confirming that AT2* is an activated and exhausted reactive cell.

[0170] Specifically, Figures 16-19 The same mature T lymphocyte tumor ALCL patient bone marrow specimen is analyzed in four tubes from A to D. Among them Figure 17 And Figure 19 The old scheme, because the new scheme has been used for a short time, has not encountered such a rare case of ALCL. The main difference between the old scheme and the optimized scheme of the present application is that the PE of the B tube is TCRγδ, not TRBC2, and the BV421 is CD7, not TCRVd2. The FITC of the D tube is ki67, not CD99.

[0171] Specifically, Figure 16Bone marrow samples of patients with mature T lymphocyte tumor ALCL were displayed for analysis. The settings were as follows: ① FSC-A / H was set as P1 to be a non-adherent cell gate, and single cells were obtained in P1; ② FSC / SSC was displayed in P1 to set P2 as a live cell gate, and single live cells were obtained in P2; ③ blood cell gates were set using CD45 / SSC in P2 to obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo), respectively; in the CD117 / CD7 two-dimensional dot plot, no abnormal cells with CD7 and CD117 double positivity were observed, and CD117-positive CD7-negative myeloid blast cells were 1.98%, which was within the normal range; ④ P2 gate was used to observe the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym), and at this time, CD2bri / CD7-negative AT2 (blue) cells were observed outside the Lym, and a total Lym gate was set, which was defined as Lym and AT2 cells. In the total Lym gate, the two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 were displayed, respectively, and the CD3 / CD7 two-dimensional dot plot was set as CD3-negative / CD7-positive NK cells (orange yellow), CD3+CD7- AT1 (red), and CD3+CD7+ normal T cells (dark green). AT1 expressed CD5, CD3dim, CD4dim, and CD2, did not express CD7, CD8, CD117, and CD56, and FSC and SSC were both small, which belonged to the CD4+CD8-type. AT2 expressed CD2 and CD4, did not express CD3, CD5, CD7, CD8, CD56, and CD117, and FSC and SSC were both large, which belonged to the CD4+CD8-type.

[0172] In particular, Figure 17 Displaying and Figure 16The same mature T lymphoma ALCL patient bone marrow sample, the B tube analysis. Because ALCL is a rare case, the new program is short in time, and ALCL has not been encountered, so this tube is analyzed by the old program, the difference is that PE is TCRγδ instead of TRBC2, and BV421 is CD7 instead of TCRVd2. In turn: ①FSC-A / H set P1 as a de-adhesion cell gate, and get single cells in P1; ②In P1, show FSC / SSC set P2 as a live cell gate, get single live cells in P2; ③In P2 gate, use CD45 / SSC to set a blood cell gate, get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo), respectively; ④In P2 gate, observe the degree of coincidence of CD2, CD3, CD4 and CD7 markers with the lymphocyte gate (Lym), and find that CD2briCD7 negative AT2 is located outside the lymphocyte gate, set the total Lym gate, defined as Lym and AT2 cells. In the total Lym gate, in the CD3 / CD7 two-dimensional dot plot, CD3+CD7+ (dark green) is normal T cells, showing CD4 and TRBC1 of normal T cells, whether CD4 positive or CD4 negative (i.e. CD8+) all proportionally express TRBC1, CD3+TCRgd+ set gdT (pink), partially express TCRVd1. CD3+CD7- is abnormal T cells (AT1, red), AT1 expresses CD3dim, CD4dim and CD2, does not express CD57, TRBC1, TCRVd1, TCRgd and CD7, and FSC and SSC are both small. AT2 expresses CD4 and CD2, does not express CD3, CD57, TRBC1, TCRVd1, TCRgd and CD7, and FSC and SSC are both large. So far, Figure 16 The A tube and Figure 17 The B tube determines that there are 2 groups of mature T lymphoma cells in the sample, and confirms the monoclonality. General laboratory flow cytometry does not need to distinguish subtypes, and can stop here. If you want to further determine the subtype of CD4+ / CD8- tumor cells, further detect the C tube.

[0173] Specifically, Figure 18 Show with Figure 16 And Figure 17The same mature T lymphocyte tumor ALCL case bone marrow specimen, propylene tube analysis. In turn set: ①FSC-A / H set P1 as a de-adhesion cell gate, get P1 inside as single cells; ②In P1, show FSC / SSC set P2 as a live cell gate, get P2 inside as single live cells; ③P2 gate, use CD45 / SSC set blood cell gate, get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gate (eo) respectively; ④In P2 gate, observe the degree of coincidence of CD2, CD3, CD4 and CD7 markers with lymphocyte gate (Lym), find that CD2briCD7 negative AT2 is located outside the lymphocyte gate, set the total Lym gate, defined as Lym and AT2 cells. In the total Lym gate, in the CD3 / CD7 two-dimensional dot plot, CD3+CD7+ (dark green) is normal T cells, CD3+CD7- is abnormal T cells (AT1, red), AT1 expresses CD3dim, CD4dim and CD2, part expresses CD26dim and CD279dim, does not express CD7, CD10 and CD25, and FSC and SSC are both small. AT2 expresses CD25, CD4 and CD2, does not express CD3, CD7, CD10, CD26 and CD279, and FSC and SSC are both large.

[0174] In particular, Figure 19 showing Figures 16 to 18The same mature T lymphocyte tumor ALCL case bone marrow specimen, old scheme D tube analysis, the difference is that FITC is ki67, not CD99. In turn: ① FSC-A / H set P1 as a de-adhesion cell gate, obtaining single cells in P1; ② FSC / SSC in P1 shows P2 as a live cell gate, obtaining single live cells in P2; ③ P2 gate uses CD45 / SSC to set a blood cell gate, respectively obtaining lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo); ④ P2 gate, observe the degree of coincidence of CD2, CD3 and CD4 markers with the lymphocyte gate (Lym), find that CD2briCD4+ AT2 is outside the lymphocyte gate, set the total Lym gate, defined as Lym and AT2 cells. In the total Lym gate, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive partially negative (dark green) is normal T cells, CD3dimCD4dim is abnormal T cells (AT1, red), AT1 expresses CD3dim, CD4dim and CD2, does not express ki67, CD30 and cTCL1, and FSC and SSC are both small. AT2 expresses CD4 and CD2, partially expresses ki67 and CD30, does not express CD3 and cTCL1, and FSC and SSC are both large. Therefore, AT2 is ALCL. AT1 is considered as PTCL, NOS if there is no AT2, but because there is AT2 that can be clearly typed, the clinical and pathological diagnosis is ALCL. Flow cytometry found two clones.

[0175] In particular, Figures 20-22 The same mature T lymphocyte tumor PLL patient bone marrow specimen A tube, B tube, and D tube are analyzed together.

[0176] In particular, 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 22shows that Figure 20 and Figure 21 Bone marrow sample of the same mature T lymphocyte tumor PLL case, analysis of tubes A to D. In order: ① FSC-A / H setting P1 as a non-adherent cell gate, obtaining single cells within P1; ② within P1, FSC / SSC setting P2 as a live cell gate, obtaining single live cells within P2; ③ within P2 gate, using CD45 / SSC setting blood cell gate, obtaining lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo) gates; ④ within P2 gate, observing the degree of coincidence of CD5, CD3, and CD4 markers with the lymphocyte gate (Lym), and determining that there are no lymphocytes outside the lymphocyte gate. Within the Lym gate, in the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green accentuation) are normal T cells, and CD3-CD5bri are abnormal T cells (AT, red). AT expresses CD5bri and CD4bri, partially expresses cTCL1, does not express CD99, CD30, and CD3, and both FSC and SSC are small. The subtype is PLL.

[0179] Specifically, Figures 23-26 Bone marrow sample of the same mature T lymphocyte tumor PTCL, NOS patient, four tubes A to D for joint analysis.

[0180] Specifically, Figure 23The bone marrow sample of a patient with mature T lymphocyte tumor PTCL, NOS was displayed, and the tube analysis was performed. The settings were as follows: ① FSC-A / H was set as P1 to be a cell gate, and single cells in P1 were obtained; ② FSC / SSC was displayed in P1, and P2 was set as a live cell gate, and single live cells in P2 were obtained; ③ blood cell gates were set using CD45 / SSC in P2, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) were obtained, respectively; in a CD117 / CD7 two-dimensional dot plot, no abnormal cells with CD7 and CD117 double positivity were observed, and CD117-positive CD7-negative myeloid blast cells were 1.25%, which was within the normal range; ④ in P2, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym) was observed, and it was determined that no lymphocytes were outside the lymphocyte gate. In the Lym gate, CD3-negative / CD7-positive was NK cells (orange yellow), and in addition, two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 were displayed, respectively. In the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) was a normal T cell, and CD3dimCD5bri was an abnormal T cell (AT, red), which belonged to the CD4+CD8-type. AT expressed CD5bri, CD3dim, CD4bri, CD7bri, and CD2dim, did not express CD8, CD117, and CD56, and FSC and SSC were both small.

[0181] Specifically, Figure 24 displayed with Figure 23The same mature T lymphocyte tumor PTCL, NOS patient bone marrow specimen, the B tube analysis. In turn set: ① FSC-A / H set P1 as a de-adhesion cell door, get P1 inside as a single cell; ② FSC / SSC in P1 show P2 as a live cell door, get P2 inside as a single live cell; ③ P2 door using CD45 / SSC set blood cell door, respectively get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), eosinophil door (eo); ④ P2 door, observe the degree of coincidence of CD5, CD3, CD4 markers and lymphocyte door (Lym), determine that there is no lymphocyte outside the lymphocyte door. In Lym door, in CD3 / CD4 two-dimensional dot plot, CD3+CD4 part positive part negative (dark green) is normal T cells, normal T cells proportion balanced mutually exclusive expression TRBC1 and TRBC2. CD3dimCD4bri is abnormal T cells (AT, red), AT expresses CD5bri, CD3dim, CD4bri and TRBC1, does not express CD57, TRBC2, TCRVd1 and TCRVd2, and FSC and SSC are both small. So far, Figure 23 A tube and Figure 24 B tube determines the presence of mature T lymphocyte tumor in the specimen, and confirms the monoclonality. General laboratory flow cytometry does not need to distinguish subtypes, can stop here. If you want to further determine the subtype of CD4+ / CD8- tumor cells, further detect C tube.

[0182] Specifically, Figure 25 Show and Figure 23 And Figure 24The same mature T lymphocyte tumor PTCL, NOS case bone marrow specimen, prop tube analysis. In turn set: ①FSC-A / H set P1 as a non-adherent cell gate, get P1 inside as single cells; ②In P1, show FSC / SSC set P2 as a live cell gate, get P2 inside as single live cells; ③In P2 gate, use CD45 / SSC to set a blood cell gate, get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) respectively; ④In P2 gate, observe the degree of coincidence of CD5, CD3, and CD4 markers with the lymphocyte gate (Lym), and determine that there are no lymphocytes outside the lymphocyte gate. In the Lym gate, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive partially negative (dark green) is normal T cells, and CD3dimCD4bri is abnormal T cells (AT, red). AT expresses CD5bri, CD3dim, CD4bri, and CD7bri, partially expresses CD279, CD26, and CD25, does not express CD10, and both FSC and SSC are small. Not the aforementioned AITL, ATLL, MF / SS, so further do D tube.

[0183] Specifically, Figure 26 showing that Figures 23 to 25 The same mature T lymphocyte tumor PTCL, NOS case bone marrow specimen, prop tube analysis. In turn set: ①FSC-A / H set P1 as a non-adherent cell gate, get P1 inside as single cells; ②In P1, show FSC / SSC set P2 as a live cell gate, get P2 inside as single live cells; ③In P2 gate, use CD45 / SSC to set a blood cell gate, get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) respectively; ④In P2 gate, observe the degree of coincidence of CD5, CD3, and CD4 markers with the lymphocyte gate (Lym), and determine that there are no lymphocytes outside the lymphocyte gate. In the Lym gate, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive partially negative (dark green) is normal T cells, and CD3dimCD4bri is abnormal T cells (AT, red). AT expresses CD5bri, CD3dim, and CD4bri, partially expresses CD99, does not express CD30 and cTCL1, and both FSC and SSC are small. Combined with Figure 25 and Figure 26 , consider PTCL, NOS.

[0184] Specifically, Figures 27-28 The same mature T lymphocyte tumor PTCL, NOS case bone marrow specimen, prop tube analysis. In turn set: ①FSC-A / H set P1 as a non-adherent cell gate, get P1 inside as single cells; ②In P1, show FSC / SSC set P2 as a live cell gate, get P2 inside as single live cells; ③In P2 gate, use CD45 / SSC to set a blood cell gate, get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) respectively; ④In P2 gate, observe the degree of coincidence of CD5, CD3, and CD4 markers with the lymphocyte gate (Lym), and determine that there are no lymphocytes outside the lymphocyte gate. In the Lym gate, in the CD3 / CD4 two-dimensional dot plot, CD3+CD4 partially positive partially negative (dark green) is normal T cells, and CD3dimCD4bri is abnormal T cells (AT, red). AT expresses CD5bri, CD3dim, CD4bri, and CD7bri, partially expresses CD279, CD26, and CD25, does not express CD10, and both FSC and SSC are small. Not the aforementioned AITL, ATLL, MF / SS, so further do D tube. Figure 28For the old scheme, the difference from the optimized scheme of the present application is that PE is TCRy5, not TRBC2.

[0185] Specifically, Figure 27 The bone marrow sample of a patient with mature T lymphocyte tumor hepatosplenic gd T cell lymphoma was analyzed by flow cytometry. The settings were as follows: ① FSC-A / H was set as P1 to obtain single cells in P1; ② FSC / SSC was set as P2 to obtain single live cells in P2; ③ CD45 / SSC was set to obtain blood cell gates, respectively, to obtain lymphocyte (lym), granulocyte (Gra), monocyte (mono), nucleated red blood cell (NEC), and eosinophil gate (eo) gates; in the CD117 / CD7 two-dimensional dot plot, no abnormal cells with double positive CD7 and CD117 were observed, and the CD117 positive CD7 negative myeloid blast cells were 1.04%, which was within the normal range; ④ In P2 gate, the degree of coincidence of CD2, CD3, CD4, CD5, CD7, CD8, and CD56 markers with the lymphocyte gate (lym) was observed to determine that there were no lymphocytes outside the lymphocyte gate. In the Lym gate, CD3 negative / CD56 positive was NK cells (orange yellow), in addition, the two-dimensional dot plots of CD3 / CD5, CD2 / CD7, CD4 / CD8, CD4 / CD3, and CD3 / CD56 were displayed, respectively, in the CD3 / CD5 two-dimensional dot plot, CD3+CD5+ (dark green) was normal T cells, and CD3+CD5- was abnormal T cells (AT, red), which belonged to the CD4-CD8-type. AT expressed CD3bri, CD7bri, CD56, and CD2, but did not express CD4, CD8, CD117, and CD5, and both FSC and SSC were medium size.

[0186] Specifically, Figure 28 Displaying and Figure 27The same mature T lymphocyte tumor hepatosplenic gd T cell lymphoma patient bone marrow specimen, B tube analysis. In turn set: ① FSC-A / H set P1 for de-adhesion cell door, get P1 for single cell; ② In P1 show FSC / SSC set P2 for live cell door, get P2 for single live cell; ③ P2 door inside blood cell door using CD45 / SSC set, get lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), eosinophil door (eo) respectively; ④ P2 door, observe the degree of coincidence of CD5, CD3, CD8 markers and lymphocyte door (Lym), determine that there is no lymphocyte outside the lymphocyte door. In Lym door, CD3+TCRgd- is normal abT (dark green), CD8+ and CD8- (mainly CD4+) balancedly partially express TRBC1. CD3 / CD5 two-dimensional dot plot CD3+CD5- is abnormal T cell (AT, red), AT expresses CD3bri, CD4bri, TCRgd and TCRVd1, does not express CD5bri, CD57, TRBC1 and TCRVd2. So far, Figure 27 A tube and Figure 28 B tube determines the presence of mature T lymphocyte tumor in the specimen, and confirms monoclonality, subtype is hepatosplenic gd T cell lymphoma.

[0187] Clinical verification was carried out by using the method of the embodiment: Hebei Yanda Lu Dao Pei Hospital has used the scheme since 2022, and initially there was no TRBC2, and the channel used TCRy5PE at that time, from February 2022 to September 2024, 2109 detection times from 1235 patients were detected, among which 364 detection times of positive samples were from 202 people, and from October 2024, all were changed to the scheme including TRBC2 of the application, from October 2024 to the end of June 2025, 414 detection times from 259 patients were completed, among which 84 detection times of positive samples were from 52 people. So far, a total of 2523 detection times from 1494 people have been completed, among which 448 detection times of positive samples were from 254 people. The median age of the patients was 55 years old (1-88 years old), and the median proportion of tumor cells was 2.32% (0.01%-93.61%). After 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, the sensitivity, specificity, positive predictive value and negative predictive value from October 2024 to June 2025 were 98.81%, 99.76%, 98.81% and 99.76% respectively, and the total sensitivity, specificity, positive predictive value and negative predictive value were 97.74%, 99.41%, 96.65% and 99.60% respectively. By using the method, especially the improved method, the sensitivity, specificity, positive predictive value and negative predictive value are all above about 98%. It can be seen that the application can improve the efficiency, save the cost 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, 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: 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; Preferably, the kit further 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 one, and at least one of flow cytometry tube two, flow cytometry tube three, and flow cytometry tube four. 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 tube one obtained in step (1), selectively add the second group of antibodies in the reagent composition of any one of claims 1-3 to tube two obtained in step (1), add the third group of antibodies in the reagent composition of any one of claims 1-3 to 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 tube four obtained in step (1). Incubate each flow cytometer at room temperature in the dark. (3) Selectively, add film-breaking agent A solution to tube 4 after incubation in step (2) and continue incubation at room temperature in the dark; (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. (5) After centrifuging each flow cytometer tube incubated in step (4), remove the supernatant; (6) Selectively, add membrane-breaking agent B and the second part of the fourth group of antibodies in the reagent composition according to any one of claims 1-3 to tube four after the supernatant is removed in step (5), and incubate at room temperature in the dark; (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.

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; Preferably, during 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 / side-scat (SSC) is used to set each blood cell gate, and: The first 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 lymphocytes 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; Selectively, tube 2 is gated in the following manner: within the P2 gate, the degree of anastomosis between CD2 or CD5, CD3, CD4 or CD8 markers and the lymphocyte gate is observed, and a total lymphocyte gate including normal lymphocytes and abnormal lymphocytes is constructed; within the lymphocyte gate, the expression of CD57, TRBC1, TRBC2, TCRVδ1, and TCRVδ2 is displayed, and T cells different from normal T cells are selected as abnormal T cell gates; Selectively, tube 3 is gated in the following manner: within the P2 gate, the degree of anastomosis between CD2 or CD5, CD3, CD4, CD7 markers and lymphocyte gates is observed, and a total lymphocyte gate including normal lymphocytes 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; Selectively, tube four is gated in the following manner: within the P2 gate, the degree of anastomosis between CD2 or CD5, CD3, CD4 markers and lymphocyte gates is observed, and a total lymphocyte gate including normal lymphocytes and abnormal lymphocytes is constructed; within the lymphocyte gate, the expression of CD99, CD30, and cytoplasmic TCL1 is displayed, and T cells that are different from normal T cells are 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 cytometry tubes used include tube one and tube two; (2) Detection of the number of clones of mature T cell tumors: The flow cytometry tubes used include tube one and tube two; (3) Determine the mature T-cell tumor subtype: The tumor cells in the sample to be tested are CD4+CD8-, selected from tubes one to four; or 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. 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 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.

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. 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)-(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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