Antibody composition and kit for detecting early precursor acute T-cell lymphocytes and application of antibody composition and kit

Abstract

The invention belongs to the technical field of immunological detection, and discloses an antibody composition for detecting early precursor acute T-cell lymphocytes, which comprises a first group of antibodies, a second group of antibodies and a third group of antibodies, the first group of antibodies comprises a CD99 antibody, a CD4 antibody, a CD34 antibody, a CD56 antibody, a CD5 antibody, a CD3 antibody, a CD8 antibody, a CD2 antibody, a CD7 antibody and a CD45 antibody; the second group of antibodies comprises an HLA-DR (Human Leukocyte Antigen-DR) antibody, a CD33 antibody, a CD34 antibody, a CD117 antibody, a CD13 antibody, a CD11b antibody, a CD7 antibody, a CD64 antibody, a CD38 antibody and a CD45 antibody; the third group of antibodies comprises a TdT antibody, an MPO antibody, a CD10 antibody, a CD1a antibody, a cCD3 antibody, a CD7 antibody, a CD19 antibody, a cCD22 antibody and a CD45 antibody; each antibody is a monoclonal antibody marked with a detection marker. The antibody composition can be used for rapidly, accurately and comprehensively detecting early-stage precursor T cell acute lymphocytes at one time.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection, and in particular relates to an antibody composition for detecting early precursor T cell acute lymphocytes, a kit and applications thereof. Background Art

[0002] T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive lymphocyte malignancy, accounting for 20% to 25% of adult ALL. Early T-cell precursor ALL (ETP-ALL) is a recently identified subtype of T-ALL. Compared with other T-ALLs, ETP-ALL exhibits distinct immunophenotypes and molecular features. It accounts for 12.6% to 47.6% of adult T-ALL cases, is more common in males than in females, and has a median age of onset of 29 to 40 years. The reported proportion of ETP-ALL in adult T-ALL in China ranges from 17.3% to 47.6%, slightly higher than the 12.6% to 32% reported internationally, possibly due to ethnic differences. Compared with classic T-ALL, ETP-ALL patients tend to develop at an older age, have lower white blood cell counts at onset, and have a higher incidence of central nervous system infiltration (16% vs. 3%). The clinical prognosis is poorer, and timely and accurate diagnosis is particularly important for patient treatment.

[0003] The diagnostic criteria for ETP-ALL follow the WHO 2022 Classification of Neoplasms of Lymphoid and Hematopoietic Tissue: Based on a diagnosis of T-ALL, immunophenotyping of blasts must meet the following criteria: ① CD7 positive, CD1a and CD8 negative. ② CD5 negative or a positivity rate of <75%. ③ Positive for at least one myeloid / stem cell antigen marker, including CD34, CD117, HLA-DR, CD13, CD33, CD11b, or CD65; MPO negative. ④ Typical ETP-ALL may express CD2 and cytoplasmic CD3; CD4 expression is possible but is not a diagnostic requirement. The Chinese Expert Consensus on the Diagnosis and Treatment of Early Precursor T-Cell Acute Lymphoblastic Leukemia in Adults (2023 edition) proposes 11 more specific immune markers for the diagnosis of ETP-ALL, including CD1a, sCD3 (cytoplasmic CD3), CD5, CD8, CD10, CD13, CD33, CD34, CD117, TdT, and MPO. MPO expression ≥3% was considered MPO-positive, CD5 expression ≥75% was considered CD5-positive, and all other markers were considered positive if their expression was ≥20%. A score of 8 or more was considered ETP-ALL, and a score of <8 was considered other types of T-ALL.

[0004] However, due to the uneven technical capabilities of the flow cytometry platforms in different hospitals in China, the antibody composition matching, experimental operations and data analysis processes for screening ETP-ALL diseases are different, which is not conducive to the accuracy of the results of early precursor T-cell acute lymphoblastic leukemia (ETP-ALL) detection. Summary of the Invention

[0005] The object of the present invention is to provide an antibody composition that can rapidly, accurately and comprehensively detect early precursor T cells and acute lymphocytes at one time.

[0006] The above-mentioned objectives are achieved by the following technical solutions.

[0007] A first aspect of the present invention provides an antibody composition for detecting early precursor T cell acute lymphocytes, the antibody composition comprising a first group of antibodies, a second group of antibodies, and a third group of antibodies;

[0008] The first panel of antibodies includes CD99, CD4, CD34, CD56, CD5, CD3, CD8, CD2, CD7, and CD45 antibodies;

[0009] The second group of antibodies includes: HLA-DR, CD33, CD34, CD117, CD13, CD11b, CD7, CD64, CD38 and CD45 antibodies;

[0010] The third group of antibodies includes: TdT, MPO, CD10, CD1a, cCD3, CD7, CD19, cCD22 and CD45 antibodies;

[0011] Each of the antibodies is a monoclonal antibody labeled with a detection marker.

[0012] In some embodiments, the CD99, HLA-DR and TdT antibodies are labeled with the same fluorescein;

[0013] The CD4, CD33 and MPO antibodies are labeled with the same fluorescein;

[0014] The CD34 and CD10 antibodies are labeled with the same fluorescein;

[0015] The CD56, CD117 and CD1a antibodies are labeled with the same fluorescein;

[0016] The CD5 and CD13 antibodies are labeled with the same fluorescein;

[0017] The CD3, CD11b and cCD3 antibodies are labeled with the same fluorescein;

[0018] The CD8, the CD7 antibody in the second group of antibodies, and the CD7 antibody in the third group of antibodies are labeled with the same fluorescent dye;

[0019] The CD2, CD64 and CD19 antibodies are labeled with the same fluorescein;

[0020] The CD38, cCD22 and CD7 antibodies in the first group of antibodies are labeled with the same fluorescent dye;

[0021] The CD45 antibody is labeled with a fluorescent dye;

[0022] The fluorescein is selected from the group consisting of FITC, PE, ECD, PECY5.5, PECY7, APC, APC-700, APC-750, PB, and KO.

[0023] In some embodiments, the CD99, HLA-DR and TdT antibodies are labeled with fluorescein FITC;

[0024] The CD4, CD33 and MPO antibodies are labeled with fluorescein PE;

[0025] The CD34 and CD10 antibodies are labeled with fluorescein ECD;

[0026] The CD56, CD117 and CD1a antibodies are labeled with fluorescein PECY5.5;

[0027] The CD5 and CD13 antibodies are labeled with fluorescein PECY7;

[0028] The CD3, CD11b and cCD3 antibodies are labeled with fluorescein APC;

[0029] The CD8, the CD7 antibody in the second group of antibodies, and the CD7 antibody in the third group of antibodies are labeled with fluorescein APC-700;

[0030] The CD2, CD64 and CD19 antibodies are labeled with fluorescein APC-750;

[0031] The CD38, cCD22 and CD7 antibodies in the first group of antibodies are labeled with fluorescein PB;

[0032] The CD45 antibody was labeled with fluorescein KO.

[0033] A second aspect of the present invention provides a use of the antibody composition described above for detecting early precursor T cells in acute lymphocytes for non-diagnostic purposes.

[0034] The third aspect of the present invention provides a kit for detecting early precursor T cell acute lymphocytes, wherein the kit comprises the antibody composition for detecting early precursor T cell acute lymphocytes as described above.

[0035] In some embodiments, the kit further comprises reagents for flow cytometry detection.

[0036] In some embodiments, in the flow cytometry detection, the single cell suspension of the sample to be tested is divided into three parts, and the first group of antibodies, the second group of antibodies and the third group of antibodies are added to the three parts for incubation, and then the flow cytometry detection is performed.

[0037] In some embodiments, the amount of the first group of antibodies added per 100 μl of the sample to be tested is 40 μl to 50 μl, wherein the amount of the CD99 antibody added is 15 to 25 μl, and the amount of the remaining antibodies added is 1 μl to 5 μl, and the titer of each antibody in the first group of antibodies is 1:1 to 7; specifically, the titers of CD99, CD4, CD34, CD56, CD5, CD3, CD8, CD2, CD7 and CD45 antibodies in the first group of antibodies are 1:1 to 2, 1:3 to 5, 1:1 to 3, 1:5 to 7, 1:4 to 6, 1:4 to 6, 1:4 to 6, 1:4 to 6, and 1:4 to 6, respectively.

[0038] In some embodiments, the amount of the second group of antibodies added per 100 μl of the sample to be tested is 15 μl to 20 μl, wherein the amount of each antibody in the second group of antibodies added is 1 μl to 5 μl, and the titer of each antibody in the second group of antibodies is 1:1 to 5; specifically, the titers of HLA-DR, CD33, CD34, CD117, CD13, CD11b, CD7, CD64, CD38 and CD45 antibodies in the second group of antibodies are 1:4 to 5, 1:4 to 5, 1:1 to 3, 1:4 to 5, 1:4 to 5, 1:4 to 5, 1:4 to 5, 1:4 to 5, 1:4 to 5, and 1:4 to 5, respectively.

[0039] In some embodiments, the amount of the third group of antibodies added per 100 μl of the sample to be tested is 40 μl to 50 μl, wherein the amount of the cCD22 antibody added is 15 to 25 μl, and the amount of the remaining antibodies added is 1 μl to 5 μl, and the titer of each antibody in the third group of antibodies is 1:1 to 5; specifically, the titers of TdT, MPO, CD10, CD1a, cCD3, CD7, CD19, cCD22 and CD45 antibodies in the third group of antibodies are 1:4 to 5, 1:4 to 5, 1:1 to 3, 1:1 to 2, 1:4 to 5, 1:4 to 5, 1:1 to 2, 1:1 to 2, and 1:4 to 5, respectively.

[0040] In some embodiments, the cell number concentration in the single cell suspension is 1×10 5 / ml~1×10 7 pcs / ml.

[0041] A fourth aspect of the present invention provides a use of the above-described kit for detecting early precursor T cell acute lymphocytes for non-diagnostic purposes.

[0042] A fifth aspect of the present invention provides a method for detecting early precursor T cells acute lymphocytes for non-diagnostic purposes, the method comprising the following steps:

[0043] Performing flow cytometry detection on the cells to be tested using the antibody composition or the kit as described above;

[0044] According to the results of flow cytometry, CD45-SSC was used to set the gate, and the CD45dim area was obtained as the target cell population based on the expression of CD45-SSC;

[0045] Analyzing the expression of each fluorescent antibody pair in the target cell population, and obtaining an immune marker score based on the expression of the fluorescent antibody pair;

[0046] Among them, the fluorescent antibody pairs include: CD7-CD45, CD7-CD34, CD7-CD99, CD7-CD2, CD7-CD3, CD7-CD4, CD7-CD5, CD7-CD8, CD7-CD56, CD7-HLA-DR, CD7-CD33, CD7-CD117, CD7-CD13, CD7-CD11b, CD7-CD64, CD7-CD38, CD7-cCD3, CD7-TdT, CD7-MPO, CD7-CD1a, CD7-CD10, and CD7-cCD22.

[0047] In the present invention, after extensive research and experiments, the inventors divided a variety of antibodies that recognize early precursor T cell acute lymphocytes, such as CD99, CD8, HLA-DR, CD117, CD13, CD11b, TdT, and cCD22, into three specific groups of antibodies, and combined them to obtain an antibody composition. When using this antibody composition to detect early precursor T cell acute lymphocytes, it is possible to enhance the targeting of the detection to lock on cells with abnormal T-lineage phenotypes, enhance the identification of abnormal myeloid differentiation, and accurately confirm the origin of the lymphocyte lineage, thereby enabling rapid, accurate, and comprehensive detection of early precursor T cell acute lymphocytes at one time, greatly improving the accuracy of detecting ETP-ALL. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a scatter plot of CD45-SSC expression, an immunophenotype of cells in an ETP-ALL sample to be tested, detected using the third round of antibody composition in Example 3 of the present invention.

[0049] Figure 2 Figure 5 is a scatter plot of the CD7-CD45 immunophenotype of all nucleated cells in ETP-ALL samples.

[0050] Figure 3 This is a scatter plot of CD7-CD34 expression in all nucleated cells in ETP-ALL samples.

[0051] Figure 4 This is a scatter plot of CD7-CD99 expression in all nucleated cells in ETP-ALL samples.

[0052] Figure 5 This is a scatter plot of CD7-CD2 expression in all nucleated cells in ETP-ALL samples.

[0053] Figure 6 This is a scatter plot of CD7-CD3 expression in all nucleated cells in ETP-ALL samples.

[0054] Figure 7 It is a scatter plot of CD7-CD4 expression in all nucleated cells in ETP-ALL samples.

[0055] Figure 8 It is a scatter plot of CD7-CD5 expression in all nucleated cells in ETP-ALL samples.

[0056] Figure 9 This is a scatter plot of CD7-CD8 expression in all nucleated cells in ETP-ALL samples.

[0057] Figure 10 This is a scatter plot of CD7-CD56 expression in all nucleated cells in ETP-ALL samples.

[0058] Figure 11 It is a scatter plot of CD7-HLA-DR expression in all nucleated cells in ETP-ALL samples.

[0059] Figure 12 This is a scatter plot of CD7-CD33 expression in all nucleated cells in ETP-ALL samples.

[0060] Figure 13 This is a scatter plot of CD7-CD117 expression in all nucleated cells in ETP-ALL samples.

[0061] Figure 14 This is a scatter plot of CD7-CD13 expression in all nucleated cells in ETP-ALL samples.

[0062] Figure 15 This is a scatter plot of CD7-CD11b expression in all nucleated cells in ETP-ALL samples.

[0063] Figure 16 This is a scatter plot of CD7-CD64 expression in all nucleated cells in ETP-ALL samples.

[0064] Figure 17 This is a scatter plot of CD7-CD38 expression in all nucleated cells in ETP-ALL samples.

[0065] Figure 18 It is a scatter plot of CD7-cCD3 expression in all nucleated cells in ETP-ALL samples.

[0066] Figure 19 It is a scatter plot of CD7-TdT expression in all nucleated cells in ETP-ALL samples.

[0067] Figure 20 It is a scatter plot of CD7-MPO expression in all nucleated cells in ETP-ALL samples.

[0068] Figure 21 Figure 2 is a scatter plot of the CD7-CD1a immunophenotype of all nucleated cells in ETP-ALL samples.

[0069] Figure 22 Figure 3 is a scatter plot of the CD7-CD10 immunophenotype of all nucleated cells in ETP-ALL samples.

[0070] Figure 23 Figure 2 is a scatter plot of the CD7-cCD22 immunophenotype of all nucleated cells in ETP-ALL samples.

[0071] Figure 24 This is a scatter plot of CD45-SSC expression, an immunophenotype of cells in a T-ALL sample to be tested, detected using the third round of antibody composition in Example 3 of the present invention.

[0072] Figure 25 It is a scatter plot of the CD7-CD45 immunophenotype of all nucleated cells in T-ALL samples.

[0073] Figure 26 It is a scatter plot of CD7-CD34 expression in all nucleated cells in T-ALL samples.

[0074] Figure 27 It is a scatter plot of CD7-CD99 expression in all nucleated cells in T-ALL samples.

[0075] Figure 28 It is a scatter plot of CD7-CD2 expression in all nucleated cells in T-ALL samples.

[0076] Figure 29It is a scatter plot of CD7-CD3 expression in all nucleated cells in T-ALL samples.

[0077] Figure 30 It is a scatter plot of CD7-CD4 expression in all nucleated cells in T-ALL samples.

[0078] Figure 31 It is a scatter plot of CD7-CD5 expression in all nucleated cells in T-ALL samples.

[0079] Figure 32 It is a scatter plot of CD7-CD8 expression in all nucleated cells in T-ALL samples.

[0080] Figure 33 It is a scatter plot of CD7-CD56 expression in all nucleated cells in T-ALL samples.

[0081] Figure 34 It is a scatter plot of CD7-HLA-DR expression in all nucleated cells in T-ALL samples.

[0082] Figure 35 It is a scatter plot of CD7-CD33 expression in all nucleated cells in T-ALL samples.

[0083] Figure 36 It is a scatter plot of CD7-CD117 expression in all nucleated cells in T-ALL samples.

[0084] Figure 37 It is a scatter plot of CD7-CD13 expression in all nucleated cells in T-ALL samples.

[0085] Figure 38 This is a scatter plot of CD7-CD11b expression in all nucleated cells in T-ALL samples.

[0086] Figure 39 It is a scatter plot of CD7-CD64 expression in all nucleated cells in T-ALL samples.

[0087] Figure 40 It is a scatter plot of CD7-CD38 expression in all nucleated cells in T-ALL samples.

[0088] Figure 41 It is a scatter plot of CD7-cCD3 expression in all nucleated cells in T-ALL samples.

[0089] Figure 42 It is a scatter plot of CD7-TdT expression in all nucleated cells in T-ALL samples.

[0090] Figure 43It is a scatter plot of CD7-MPO expression in all nucleated cells in T-ALL samples.

[0091] Figure 44 It is a scatter plot of the CD7-CD1a immunophenotype of all nucleated cells in T-ALL samples.

[0092] Figure 45 It is a scatter plot of the CD7-CD10 immunophenotype of all nucleated cells in T-ALL samples.

[0093] Figure 46 Figure 3 is a scatter plot of the CD7-cCD22 immunophenotype of all nucleated cells in a T-ALL sample. DETAILED DESCRIPTION

[0094] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0095] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0097] Example 1 Screening of Antibody Compositions for Detecting Early Precursor T Cell Acute Lymphocytes

[0098] In this embodiment, in the detection of early precursor T cell acute lymphocytes, due to the high homology between the surface antigen epitopes of early precursor T cells and normal T cell precursors, myeloid cells or hematopoietic stem cells, traditional antibodies are prone to cross-reactions. The inventors, through long-term research and analysis, designed antigen immunogens based on the conformational epitopes of ETP-ALL specific mutant proteins and their spatial folding characteristics, breaking through the interference of epitope similarity. After a large amount of research and screening experiments, the following antibodies were obtained for the detection of early precursor T cell acute lymphocytes. The fluorescent labels and dosages of the relevant monoclonal antibodies in the antibodies are shown in Table 1.

[0099] Table 1

[0100]

[0101]

[0102]

[0103]

[0104] Based on the antibodies provided in Table 1, three antibody panels were designed in this example to conduct three rounds of detection and screening. The antibodies used in the three antibody panels are shown in Table 2.

[0105] Table 2

[0106]

[0107] Note: Intracellular antibodies: TdT, MPO, cCD3, cCD22, the rest are cell membrane antibodies

[0108] Twenty clinically confirmed ETP-ALL and non-ETP-ALL cases were used for testing using the three antibody panels described above. The specific testing process is as follows:

[0109] 1. Prepare the membrane antibodies (excluding intracellular antibodies) in the antibody combination in Table 2. The specific liquid addition amounts are shown in Table 1.

[0110] 2. Sample processing.

[0111] The concentration of the sample to be tested was adjusted to 1×10 6 / ml to prepare a single cell suspension.

[0112] 3. Sample testing.

[0113] (1) Take the flow cytometry tubes, mark the corresponding test round numbers and corresponding tube numbers 1, 2, 3, etc., add the corresponding prepared membrane antibodies, and then add 100 μl of the suspension in step 2, vortex and shake to mix, and incubate at room temperature in the dark for 15 minutes.

[0114] (2) Add 400 μl of Bc hemolysin to each of the incubated flow tubes, vortex and shake, and let it stand until the hemolysis is clear. After the hemolysis is clear, centrifuge flow tubes 1 and 2 at 1500 r / min for 5 minutes, discard the supernatant, and perform intracellular antibody detection on the flow tubes that need to add intracellular antibodies according to the following steps; add 2 ml of calf serum to the remaining flow tubes, vortex and shake, centrifuge at 1500 r / min for 5 minutes, discard the supernatant, and add 400 μl of 1% paraformaldehyde to resuspend.

[0115] (3) Intracellular antibody experimental operation: After the cell surface marker staining is completed according to the above (1, 2), 450uL of 1XFACS membrane permeabilization reagent is added, mixed and incubated in the dark for 5 minutes, and then washed with PBS and the supernatant is discarded. Anti-intracellular fluorescent antibodies (TdT, MPO, cCD3, cCD22) are added according to the above antibody amount and incubated in the dark at room temperature for 30 minutes. 2ml of calf serum is added, vortexed, centrifuged at 1500r / min for 5 minutes, the supernatant is discarded, and 400μl of 1% paraformaldehyde is added for resuspending.

[0116] (4) Beckman Coulter Navios ten-color flow cytometer was used to detect the flow tubes and analyze their immunophenotype.

[0117] 4. Data analysis.

[0118] (1) Establish a template for the antibody expression pattern of healthy people

[0119] First, based on the flow cytometry test results of cell populations from 20 normal control groups, the cells were grouped by setting a gate, preferably using CD45-SSC (side scattered light) to set the gate, circle the target cell population, and then analyze the expression of each fluorescent antibody in this cell population. The following antibody pairs were selected: CD7-CD45, CD7-CD34, CD7-CD99, CD7-CD2, CD7-CD3, CD7-CD4, CD7-CD5, CD7-CD8, CD7-CD56, CD7-HLA-DR, CD7-CD33, CD7-CD117, CD7-CD13, CD7-CD11b, CD7-CD64, CD7-CD38, CD7-cCD3, CD7-TdT, CD7-MPO, CD7-CD1a, CD7-CD10, and CD7-cCD22.

[0120] (2) Establish the antibody expression pattern of the cells to be tested

[0121] Obtain flow cytometry test result data of the cells to be tested, set gates to group the cells according to the above-mentioned antibody expression pattern of the normal control population, circle the target cell population, and then analyze the expression of each fluorescent antibody in the target cell population according to the method of the antibody expression pattern of the normal control population to obtain the antibody expression pattern of the cells to be tested.

[0122] (3) Analyze the antibody expression pattern of the cells to be tested

[0123] If the antibody expression pattern of the cells to be tested falls within the antibody expression pattern template of the normal control population, the cells to be tested are determined to be a cell population with normal immunophenotype;

[0124] If the antibody expression pattern of the cells to be tested does not fall within the antibody expression pattern template of the normal control population, the cells to be tested are determined to be ETP-ALL cells.

[0125] 5. The test results are shown in Table 3 below:

[0126] Table 3

[0127]

[0128] Comparative analysis of the screening experimental data in Table 3 shows that the antibody group in the third round has better targeting than other antibody groups and can accurately distinguish ETP-ALL from non-ETP-ALL cases. Based on the comparison of various antibody groups, the antibody group in the third round has the following advantages compared with other antibody groups:

[0129] Combination tube 1 adds CD99 (a key marker for early T cell development) and CD8-APC700, allowing it to accurately locate the weak CD5 expression and CD8-negative characteristics of ETP-ALL. Combination tube 2, through the combined use of HLA-DR and CD117 / CD13 / CD11b, can enhance the identification of abnormal myeloid differentiation. Combination tube 3 introduces TdT and cCD22, combined with cCD3 to achieve ETP-ALL specific targeting. Therefore, each tube contains CD45-KO, which accurately delineates the primitive cell population by CD45 expression intensity. Specific grouping of each antibody enables the test to be designed in a progressive manner. Combination tube 1 targets abnormal T-lineage phenotypes, combination tube 2 excludes myeloid differentiation, and combination tube 3 confirms lymphoid origin, thereby quickly, accurately, and comprehensively detecting early precursor T-cell acute lymphocytes in one go. In addition, intracellular / cell membrane markers are layered for detection (such as MPO and cCD3) to avoid cross-interference. Furthermore, the choice of fluorophore and its combination with antibodies can also impact the accuracy of results: Using high-resolution fluorophores such as APC700 / APC750 optimizes signal separation for weakly expressed antigens (such as CD7). Adjusting CD38-PB to a dedicated channel for strongly expressed markers improves the signal-to-noise ratio. Using a multi-tube cross-validation strategy for key markers (such as CD34 / CD45) ensures data reliability.

[0130] Based on the above results, it can be concluded that the third round of antibody group has the highest accuracy in detecting early precursor T cell acute lymphocytes, and this antibody group will be further studied in the future.

[0131] The antibody group is an antibody composition obtained by combining the first group of antibodies, the second group of antibodies and the third group of antibodies;

[0132] The first panel of antibodies includes CD99, CD4, CD34, CD56, CD5, CD3, CD8, CD2, CD7, and CD45 antibodies;

[0133] The second group of antibodies includes: HLA-DR, CD33, CD34, CD117, CD13, CD11b, CD7, CD64, CD38 and CD45 antibodies;

[0134] The third group of antibodies includes: TdT, MPO, CD10, CD1a, cCD3, CD7, CD19, cCD22 and CD45 antibodies.

[0135] Example 2 System and method for detecting early precursor T cells and acute lymphocytes

[0136] This embodiment provides a system for detecting early precursor T-cell acute lymphoblastic leukemia (ETP-ALL), comprising:

[0137] A detection module, wherein the detection module performs flow cytometry detection on the cells to be detected;

[0138] a data acquisition module, wherein the data acquisition module acquires flow cytometry detection result data of cells to be tested stained with the antibody composition in Table 3 of Example 1;

[0139] The data analysis module analyzes the acquired data and determines whether the acquired cells are tumorous abnormal promyelocytic cells based on predetermined analysis logic and judgment criteria.

[0140] The specific workflow for detecting early precursor T-cell acute lymphoblastic leukemia using the above system of this embodiment is as follows:

[0141] 1. Prepare the membrane antibodies (excluding intracellular antibodies) in the third round of antibody combination in Table 2. The specific liquid addition amounts are shown in Table 1.

[0142] 2. Sample processing.

[0143] The concentration of the sample to be tested was adjusted to 1×10 6 / ml to prepare a single cell suspension.

[0144] 3. Sample testing.

[0145] (1) Take the flow cytometry tubes, mark the corresponding test round numbers and the corresponding tube numbers 1, 2, and 3, add the corresponding prepared membrane antibodies, and then add 100 μl of the suspension in step 2, vortex and shake to mix, and incubate at room temperature in the dark for 15 minutes.

[0146] (2) Add 400 μl of Bc hemolysin to each of the incubated flow tubes, vortex and shake, and let it stand until the hemolysis is clear. After the hemolysis is clear, centrifuge flow tubes 1 and 2 at 1500 r / min for 5 minutes, discard the supernatant, and perform intracellular antibody detection on the flow tubes that need to add intracellular antibodies according to the following steps; add 2 ml of calf serum to the remaining flow tubes, vortex and shake, centrifuge at 1500 r / min for 5 minutes, discard the supernatant, and add 400 μl of 1% paraformaldehyde to resuspend.

[0147] (3) Intracellular antibody experimental operation: After the cell surface marker staining is completed according to the above (1, 2), 450uL of 1XFACS membrane permeabilization reagent is added, mixed and incubated in the dark for 5 minutes, and then washed with PBS and the supernatant is discarded. Anti-intracellular fluorescent antibodies (TdT, MPO, cCD3, cCD22) are added according to the above antibody amount and incubated in the dark at room temperature for 30 minutes. 2ml of calf serum is added, vortexed, centrifuged at 1500r / min for 5 minutes, the supernatant is discarded, and 400μl of 1% paraformaldehyde is added for resuspending.

[0148] (4) Beckman Coulter Navios ten-color flow cytometer was used to detect the flow tubes and analyze their immunophenotype.

[0149] Example 3 Verification of the Accuracy of Antibody Composition Detection

[0150] The detection method of Example 2 was used to detect tumor cells in two test cells (one ETP-ALL sample and one non-ETP-ALL T-ALL control sample), and antibody expression pattern analysis was performed to verify the accuracy of the detection method of the present invention.

[0151] 1. ETP-ALL Sample

[0152] This sample is a bone marrow fluid sample from a clinically confirmed ETP-ALL patient. The antibody expression pattern was analyzed according to the following steps:

[0153] Obtain the flow cytometry test result data of the sample to be tested. Use CD45-SSC (side scattered light) to set the gate, and divide the cell population into 5 areas according to the expression of CD45-SSC, namely granulocyte area (middle upper area in the figure), monocyte area (right upper area in the figure), lymphocyte area (right lower area in the figure), CD45neg area (left lower area in the figure) and CD45dim area (middle lower area in the figure) 5 areas (as shown in the figure). Figure 1 The CD45dim region is where the target cell population is located, and this group of cells will be subjected to immunophenotypic analysis below.

[0154] Figure 2The CD7-CD45 immunophenotype scatter plot of all nucleated cells shows that the percentage of CD7+CD45dim+ abnormal cells is 92.25% (red in the figure), which is a significantly increased proportion, suggesting a high possibility of AL.

[0155] Figure 3 This is a scatter plot of the CD7-CD34 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD34 positive (partially expressed).

[0156] Figure 4 This is a scatter plot of the CD7-CD99 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD99 negative (not expressed).

[0157] Figure 5 This is a scatter plot of CD7-CD2 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD2 negative (not expressed), and CD7+CD2+ normal T / NK lymphocytes can be seen (the blue group in the upper right corner of the figure).

[0158] Figure 6 This is a scatter plot of CD7-CD3 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD3 negative (not expressed), and CD7+CD3+ normal T lymphocytes can be seen (the blue group in the upper right corner of the figure).

[0159] Figure 7 This is a scatter plot of CD7-CD4 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD4 negative (not expressed), and CD7+CD4+ normal T lymphocytes can be seen (the blue group in the upper right corner of the figure).

[0160] Figure 8 This is a scatter plot of CD7-CD5 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD5 negative (not expressed), and CD7+CD5+ normal T lymphocytes can be seen (the blue group in the upper right corner of the figure).

[0161] Figure 9 This is a scatter plot of CD7-CD8 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD8 negative (no expression), and CD7+CD8+ normal T / NK lymphocytes can be seen (the blue group in the upper right corner of the figure).

[0162] Figure 10 This is a scatter plot of CD7-CD56 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD56 negative (not expressed), and CD7+CD56+ normal NK lymphocytes can be seen (the blue group in the upper right corner of the figure).

[0163] Figure 11The figure is a scatter plot of the CD7-HLA-DR expression of all nucleated cells. It can be seen from the figure that the CD7+ abnormal cells are HLA-DR negative (not expressed).

[0164] Figure 12 This is a scatter plot of the CD7-CD33 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD33 positive (expression).

[0165] Figure 13 This is a scatter plot of the CD7-CD117 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD117 negative (not expressed).

[0166] Figure 14 This is a scatter plot of CD38-CD13 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD13 positive (partially expressed).

[0167] Figure 15 This is a scatter plot of the CD7-CD11b expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD11b positive (partially expressed).

[0168] Figure 16 This is a scatter plot of CD7-CD64 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD64 negative (not expressed).

[0169] Figure 17 This is a scatter plot of the CD7-CD38 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD38 positive (expression).

[0170] Figure 18 The left figure shows the expression of CD7-cCD3 in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are cCD3 positive (expression). The right figure shows the normal expression of CD7-cCD3 in normal T lymphocytes.

[0171] Figure 19 The figure shows the scatter plot of CD7-TdT expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are TdT negative (no expression), indicating that the abnormal cells are T-ALL cells.

[0172] Figure 20 The figure is a scatter plot of the CD7-MPO expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are MPO negative (not expressed).

[0173] Figure 21 This is a scatter plot of the CD7-CD1a immunophenotype of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD1a negative (not expressed).

[0174] Figure 22 This is a scatter plot of the CD7-CD10 immunophenotype of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD10 negative (not expressed).

[0175] Figure 23 The figure is a scatter plot of the CD7-cCD22 immunophenotype of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are cCD22 negative (not expressed).

[0176] In summary, the abnormal cells in the sample to be tested expressed CD7, cCD3, and CD38, partially expressed CD34, and did not express MPO and cCD22, which was consistent with the immune phenotype of acute T-lymphocytic leukemia (T-ALL). In addition, they did not express CD99, CD1a, CD3, CD5, CD8, CD10, CD4, CD2, CD117, CD64, and cCD22, but expressed CD13, CD33, and CD11b. According to the 11-item immune marker immune scoring system for the diagnosis of ETP-ALL proposed in the Chinese Expert Consensus on the Diagnosis and Treatment of Early Precursor T-cell Acute Lymphoblastic Leukemia in Adults (2023 Edition), the score of this case was 11 points (≥8 points), and it was diagnosed as early precursor T-cell acute lymphoblastic leukemia (ETP-ALL), which was consistent with the clinical diagnosis.

[0177] 2. Control samples of T-ALL other than ETP-ALL

[0178] This sample is a control bone marrow fluid specimen from a clinically confirmed non-ETP-ALL T-ALL (cortical T-ALL stage). Antibody expression pattern analysis was performed as follows:

[0179] Obtain the flow cytometry test result data of the sample to be tested. Use CD45-SSC (side scattered light) to set the gate, and divide the cell population into 5 areas according to the expression of CD45-SSC, namely granulocyte area (middle upper area in the figure), monocyte area (right upper area in the figure), lymphocyte area (right lower area in the figure), CD45neg area (left lower area in the figure) and CD45dim area (middle lower area in the figure) 5 areas (as shown in the figure). Figure 24 The CD45dim region is where the target cell population is located, and this group of cells will be subjected to immunophenotypic analysis below.

[0180] Figure 25 The CD7-CD45 immunophenotype scatter plot of all nucleated cells shows that the percentage of CD7+CD45dim+ abnormal cells is 91.79% (red in the figure), which is a significantly increased proportion, suggesting a high possibility of AL.

[0181] Figure 26This is a scatter plot of the CD7-CD34 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD34 positive (partially expressed).

[0182] Figure 27 The figure is a scatter plot of the CD7-CD99 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD99 positive (partially expressed).

[0183] Figure 28 This is a scatter plot of CD7-CD2 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD2 positive (partially expressed).

[0184] Figure 29 This is a scatter plot of CD7-CD3 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD3 positive (expression).

[0185] Figure 30 This is a scatter plot of CD7-CD4 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD4 positive (partially expressed).

[0186] Figure 31 This is a scatter plot of CD7-CD5 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD5 positive (partially expressed).

[0187] Figure 32 This is a scatter plot of the CD7-CD8 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are partially CD8 positive (partially expressed).

[0188] Figure 33 This is a scatter plot of the CD7-CD56 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD56 negative (not expressed).

[0189] Figure 34 The figure is a scatter plot of the CD7-HLA-DR expression of all nucleated cells. It can be seen from the figure that CD117+ abnormal cells are HLA-DR negative (not expressed).

[0190] Figure 35 This is a scatter plot of the CD7-CD33 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD33 negative (not expressed).

[0191] Figure 36 This is a scatter plot of the CD7-CD117 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD117 negative (not expressed).

[0192] Figure 37This is a scatter plot of the CD7-CD13 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD13 negative (not expressed).

[0193] Figure 38 This is a scatter plot of the CD7-CD11b expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD11b negative (not expressed).

[0194] Figure 39 This is a scatter plot of CD7-CD64 expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD64 negative (not expressed).

[0195] Figure 40 This is a scatter plot of the CD7-CD38 expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD38 positive (expression).

[0196] Figure 41 The left figure shows the expression of CD7-cCD3 in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are cCD3 positive (expression). The right figure shows the normal expression of CD7-cCD3 in normal T lymphocytes.

[0197] Figure 42 The figure shows the scatter plot of CD7-TdT expression in all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are TdT positive (expression), indicating that the abnormal cells are T-ALL cells.

[0198] Figure 43 The figure is a scatter plot of the CD7-MPO expression of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are MPO negative (not expressed).

[0199] Figure 44 The CD7-CD1a immunophenotype scatter plot of all nucleated cells shows that the CD7+ abnormal cells are CD1a positive (expressing), suggesting that the tumor cells originate from the cortical T-ALL stage.

[0200] Figure 45 This is a scatter plot of the CD7-CD10 immunophenotype of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are CD10 negative (not expressed).

[0201] Figure 46 The figure is a scatter plot of the CD7-cCD22 immunophenotype of all nucleated cells. It can be seen from the figure that CD7+ abnormal cells are cCD22 negative (not expressed).

[0202] The abnormal cells in the sample to be tested expressed CD7, cCD3, CD38, CD1a, and CD3, partially expressed CD99, CD34, CD5, CD8, CD4, and CD2, but did not express CD10, MPO, CD19, and cCD22, which was consistent with the immunophenotype of acute T-lymphocytic leukemia (T-ALL). It was considered to be derived from cortical T-ALL stage tumor cells. In addition, it did not express myeloid-related antigens CD117, CD64, CD13, CD33, and CD11b. According to the 11-item immune marker immune scoring system for the diagnosis of ETP-ALL proposed in the Chinese Expert Consensus on the Diagnosis and Treatment of Early Precursor T-cell Acute Lymphoblastic Leukemia in Adults (2023 Edition), the score of this case was -4 points (<8 points), which did not meet the diagnosis of early precursor T-cell acute lymphoblastic leukemia (ETP-ALL). It was diagnosed as non-ETP-ALL T-ALL (cortical T-ALL stage), which was consistent with the clinical diagnosis.

[0203] The results of this example show that the antibody composition designed by the present invention uses the CD7+CD45dim region as the main target cell population, covering multiple antibodies that recognize early precursor T cell acute lymphocytes, such as CD99, CD8, HLA-DR, CD117, CD13, CD11b, TdT, and cCD22, and the antibodies are divided into three specific groups. The combined detection of these three groups of antibodies can enhance the targeting of the detection to lock cells with abnormal T-lineage phenotypes, enhance the identification of abnormal myeloid differentiation, and accurately confirm the origin of the lymphocyte lineage, thereby enabling rapid, accurate, and one-time comprehensive detection of early precursor T cell acute lymphocytes.

[0204] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0205] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An antibody composition for detecting early precursor T cells and acute lymphocytes, characterized in that: The antibody composition includes a first group of antibodies, a second group of antibodies, and a third group of antibodies; The first panel of antibodies includes CD99, CD4, CD34, CD56, CD5, CD3, CD8, CD2, CD7, and CD45 antibodies; The second group of antibodies includes: HLA-DR, CD33, CD34, CD117, CD13, CD11b, CD7, CD64, CD38 and CD45 antibodies; The third group of antibodies includes: TdT, MPO, CD10, CD1a, cCD3, CD7, CD19, cCD22 and CD45 antibodies; Each of the antibodies is a monoclonal antibody labeled with a detection marker.

2. The antibody composition according to claim 1, wherein The CD99, HLA-DR and TdT antibodies are labeled with the same fluorescein; The CD4, CD33 and MPO antibodies are labeled with the same fluorescein; The CD34 and CD10 antibodies are labeled with the same fluorescein; The CD56, CD117 and CD1a antibodies are labeled with the same fluorescein; The CD5 and CD13 antibodies are labeled with the same fluorescein; The CD3, CD11b and cCD3 antibodies are labeled with the same fluorescein; The CD8, the CD7 antibody in the second group of antibodies, and the CD7 antibody in the third group of antibodies are labeled with the same fluorescent dye; The CD2, CD64 and CD19 antibodies are labeled with the same fluorescein; The CD38, cCD22 and CD7 antibodies in the first group of antibodies are labeled with the same fluorescent dye; The CD45 antibody is labeled with a fluorescent dye; The fluorescein is selected from the group consisting of FITC, PE, ECD, PECY5.5, PECY7, APC, APC-700, APC-750, PB, and KO.

3. The antibody composition according to claim 2, wherein The CD99, HLA-DR and TdT antibodies are labeled with fluorescein FITC; The CD4, CD33 and MPO antibodies are labeled with fluorescein PE; The CD34 and CD10 antibodies are labeled with fluorescein ECD; The CD56, CD117 and CD1a antibodies are labeled with fluorescein PECY5.5; The CD5 and CD13 antibodies are labeled with fluorescein PECY7; The CD3, CD11b and cCD3 antibodies are labeled with fluorescein APC; The CD8, the CD7 antibody in the second group of antibodies, and the CD7 antibody in the third group of antibodies are labeled with fluorescein APC-700; The CD2, CD64 and CD19 antibodies are labeled with fluorescein APC-750; The CD38, cCD22 and CD7 antibodies in the first group of antibodies are labeled with fluorescein PB; The CD45 antibody was labeled with fluorescein KO.

4. Use of the antibody composition according to any one of claims 1 to 3 for detecting early precursor T cell acute lymphocytes for non-diagnostic purposes.

5. A kit for detecting early precursor T cells and acute lymphocytes, characterized in that: The kit comprises the antibody composition for detecting early precursor T cell acute lymphocytes according to any one of claims 1 to 3.

6. The kit according to claim 5, wherein The kit also includes reagents for flow cytometry detection.

7. The kit according to claim 6, wherein In the flow cytometry detection, the single cell suspension of the sample to be tested is divided into three parts, and the first group of antibodies, the second group of antibodies and the third group of antibodies are added respectively for incubation, and then the flow cytometry detection is performed.

8. The kit according to claim 7, wherein The amount of the first group of antibodies added per 100 μl of the sample to be tested is 40 μl to 50 μl, wherein the amount of the CD99 antibody added is 15 to 25 μl, and the amount of each of the other antibodies added is 1 μl to 5 μl, and the titer of each antibody in the first group of antibodies is 1:1 to 7; and / or, The amount of the second group of antibodies added per 100 μl of the sample to be tested is 15 μl to 20 μl, wherein the amount of each antibody in the second group of antibodies added is 1 μl to 5 μl, and the titer of each antibody in the second group of antibodies is 1:1 to 5; and / or, The amount of the third group of antibodies added per 100 μl of the sample to be tested is 40 μl to 50 μl, wherein the amount of the cCD22 antibody added is 15 to 25 μl, and the amount of each of the other antibodies added is 1 μl to 5 μl, and the titer of each antibody in the third group of antibodies is 1:1 to 5; and / or, The cell number concentration in the single cell suspension is 1×10 5 / ml~1×10 7 pcs / ml.

9. Use of the kit according to any one of claims 5 to 8 for detecting early precursor T cell acute lymphocytes for non-diagnostic purposes.

10. A method for detecting early precursor T cells and acute lymphocytes for non-diagnostic purposes, characterized in that: The method comprises the following steps: Performing flow cytometry detection on the cells to be tested using the antibody composition according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 8; According to the results of flow cytometry, CD45-SSC was used to set the gate, and the CD45dim area was obtained as the target cell population based on the expression of CD45-SSC; Analyzing the expression of each fluorescent antibody pair in the target cell population, and obtaining an immune marker score based on the expression of the fluorescent antibody pair; Among them, the fluorescent antibody pairs include: CD7-CD45, CD7-CD34, CD7-CD99, CD7-CD2, CD7-CD3, CD7-CD4, CD7-CD5, CD7-CD8, CD7-CD56, CD7-HLA-DR, CD7-CD33, CD7-CD117, CD7-CD13, CD7-CD11b, CD7-CD64, CD7-CD38, CD7-cCD3, CD7-TdT, CD7-MPO, CD7-CD1a, CD7-CD10, and CD7-cCD22.

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