Antibody composition and kit for detecting mother cell plasma cell-like dendritic cells and application of antibody composition and kit

Abstract

The invention discloses an antibody composition for detecting mother cell plasma cell-like dendritic cells. The antibody composition comprises a first group of antibodies, a second group of antibodies, a third group of antibodies and a fourth group of antibodies, the first group of antibodies comprises a CD36 antibody, a CD4 antibody, a CD14 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 CD56 antibody, a CD117 antibody, a CD123 antibody, a CD19 antibody, a CD38 antibody and a CD45 antibody; the third group of antibodies comprises a CD303 antibody, a CD304 antibody, a CD41 antibody, a CD56 antibody, a CD13 antibody, a CD85j antibody, a CD64 antibody, a CD15 antibody and a CD45 antibody; and the fourth group of antibodies comprises TdT, MPO, CD56, cCD3, CD10, cCD22 and CD45 antibodies. The antibody composition can be used for accurately detecting tumor abnormal BPDCN cells.

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 blastic plasmacytoid dendritic cells, a kit and applications thereof. Background Art

[0002] Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a hematologic malignancy composed of immature cells that have differentiated into plasmacytoid dendritic cells (pDCs). The median survival is less than two years, and its clinical manifestations are widely heterogeneous and unique. Clinically, this disease is often poorly understood and, due to its morphological similarities to acute myeloid leukemia and acute lymphoblastic leukemia, it is difficult to differentiate. Consequently, it is often misdiagnosed as other diseases, such as acute myeloid leukemia (AML) and T-lymphoblastic lymphoma.

[0003] The diagnosis of BPDCN was previously primarily based on histopathology and histochemistry, but histochemistry is slow to produce results and cannot simultaneously localize multiple markers, resulting in a certain misdiagnosis rate. The WHO 2022 Classification of Lymphoid and Hematopoietic Tissue Neoplasms newly proposed a diagnostic basis based on immunophenotypic markers as follows: expected positive markers include CD123, CD4, CD56, CD303, CD304, TCF4, and TCL1; expected negative markers include CD3, CD14, CD19, CD34, lysozyme, and MPO. The diagnosis method is based on the expression of CD123 and pDCs or the expression of any three pDC markers. Differential diagnosis from AML is particularly crucial. Some cases of AML can show overlap with the clinical, morphological, and immunophenotypic features of BPDCN. In this case, a comprehensive differential diagnosis based on the absence of pDC markers and lineage-specific markers is necessary.

[0004] However, due to the uneven technical capabilities of flow cytometry platforms in different hospitals in China, the antibody combinations, experimental operations and data analysis processes for screening blastic plasmacytoid dendritic cell neoplasm (BPDCN) diseases are different, which is not conducive to the accuracy of BPDCN detection results. Summary of the Invention

[0005] The object of the present invention is to provide an antibody composition capable of accurately detecting blastic plasmacytoid dendritic cells.

[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 blastic plasmacytoid dendritic cells, the antibody composition comprising a first group of antibodies, a second group of antibodies, a third group of antibodies, and a fourth group of antibodies;

[0008] The first panel of antibodies includes CD36, CD4, CD14, CD56, CD5, CD3, CD8, CD2, CD7, and CD45 antibodies;

[0009] The second group of antibodies includes: HLA-DR, CD33, CD34, CD56, CD117, CD123, CD19, CD38 and CD45 antibodies;

[0010] The third group of antibodies includes: CD303, CD304, CD41, CD56, CD13, CD85j, CD64, CD15 and CD45 antibodies;

[0011] The fourth group of antibodies includes: TdT, MPO, CD56, cCD3, CD10, cCD22 and CD45 antibodies;

[0012] Each of the antibodies is a monoclonal antibody labeled with a detection marker.

[0013] In some embodiments, the CD36, HLA-DR, CD303 and TdT antibodies are labeled with the same fluorescein;

[0014] The CD4, CD33, CD304 and MPO antibodies are labeled with the same fluorescein;

[0015] The CD14, CD34 and CD41 antibodies are labeled with the same fluorescein;

[0016] The CD56 antibody is labeled with a fluorescent dye;

[0017] The CD5, CD117 and CD13 antibodies are labeled with the same fluorescein;

[0018] The CD3, CD123, CD85j and cCD3 antibodies are labeled with the same fluorescein;

[0019] The CD8 antibody is labeled with a fluorescent dye;

[0020] The CD2, CD19, CD64 and CD10 antibodies are labeled with the same fluorescein;

[0021] The CD7, CD38, CD15 and cCD22 antibodies are labeled with the same fluorescein;

[0022] The CD45 antibody is labeled with a fluorescent dye;

[0023] The fluorescein is selected from the group consisting of FITC, PE, ECD, PECY5.5, PECY7, APC, APC-700, APC-750, PB, and KO.

[0024] In some embodiments, the CD36, HLA-DR, CD303 and TdT antibodies are labeled with fluorescein FITC;

[0025] The CD4, CD33, CD304 and MPO antibodies are labeled with fluorescein PE;

[0026] The CD14, CD34 and CD41 antibodies are labeled with fluorescein ECD;

[0027] The CD56 antibody is labeled with fluorescein PECY5.5;

[0028] The CD5, CD117 and CD13 antibodies are labeled with fluorescein PECY7;

[0029] The CD3, CD123, CD85j and cCD3 antibodies are labeled with fluorescein APC;

[0030] The CD8 antibody is labeled with fluorescein APC-700;

[0031] The CD2, CD19, CD64 and CD10 antibodies are labeled with fluorescein APC-750;

[0032] The CD7, CD38, CD15 and cCD22 antibodies are labeled with fluorescein PB;

[0033] The CD45 antibody was labeled with fluorescein KO.

[0034] A second aspect of the present invention provides a use of the above-mentioned antibody composition for detecting blastic plasmacytoid dendritic cells in detecting blastic plasmacytoid dendritic cells for non-diagnostic purposes.

[0035] A third aspect of the present invention provides a kit for detecting blastic plasmacytoid dendritic cells, wherein the kit comprises the antibody composition for detecting blastic plasmacytoid dendritic cells as described above.

[0036] In some embodiments, the kit further comprises reagents for flow cytometry detection.

[0037] In some embodiments, in the flow cytometry detection, the single cell suspension of the sample to be tested is divided into four parts, and the first group of antibodies, the second group of antibodies, the third group of antibodies and the fourth group of antibodies are added respectively for incubation, and then the flow cytometry detection is performed.

[0038] In some embodiments, the amount of the first group of antibodies added per 100 μl of the sample to be tested is 15 μl to 25 μl, wherein the amount of each antibody in the first group of antibodies added is 1 μl to 5 μl, and the titer of each antibody in the first group of antibodies is 1:3 to 7; specifically, the titers of CD36, CD4, CD14, CD56, CD5, CD3, CD8, CD2, CD7 and CD45 antibodies in the first group of antibodies are 1:4 to 6, 1:3 to 5, 1:4 to 6, 1:6 to 7, 1:4 to 6, 1:4 to 6, 1:4 to 6, 1:4 to 6, 1:4 to 6, and 1:4 to 6, respectively.

[0039] In some embodiments, the amount of the second group of antibodies added per 100 μl of the sample to be tested is 20 μl to 30 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 1:2 to 7; specifically, the titers of HLA-DR, CD33, CD34, CD56, CD117, CD123, CD19, CD38 and CD45 antibodies in the second group of antibodies are 1:4 to 6, 1:4 to 6, 1:2 to 3, 1:6 to 7, 1:4 to 6, 1:2 to 3, 1:2 to 3, 1:4 to 6, and 1:4 to 6, respectively.

[0040] In some embodiments, the amount of the third group of antibodies added per 100 μl of the sample to be tested is 20 μl to 30 μl, wherein the amount of each antibody in the third group of antibodies added is 1 μl to 5 μl, and the titer of each antibody in the third group of antibodies is 1:1 to 7; specifically, the titers of CD303, CD304, CD41, CD56, CD13, CD85j, CD64, CD15 and CD45 antibodies in the third group of antibodies are 1:1 to 2, 1:1 to 2, 1:3 to 5, 1:6 to 7, 1:4 to 6, 1:2 to 3, 1:4 to 6, 1:4 to 6, and 1:4 to 6, respectively.

[0041] In some embodiments, the amount of the fourth group of antibodies added per 100 μl of the sample to be tested is 30 μl to 40 μl, wherein the amount of the cCD22 antibody added is 18 to 22 μl, and the amount of the remaining antibodies added is 1 μl to 5 μl, and the titer of each antibody in the fourth group of antibodies is 1:2 to 7; specifically, the titers of TdT, MPO, CD56, cCD3, CD10, cCD22 and CD45 antibodies in the fourth group of antibodies are 1:3 to 5, 1:3 to 5, 1:6 to 7, 1:4 to 6, 1:4 to 6, 1:4 to 6, and 1:4 to 6, respectively.

[0042] In some embodiments, the cell number concentration in the single cell suspension is 1×10 5 / ml~1×10 7pcs / ml.

[0043] A fourth aspect of the present invention provides a use of the above-described kit for detecting blastic plasmacytoid dendritic cells for non-diagnostic purposes.

[0044] A fifth aspect of the present invention provides a method for detecting blastic plasmacytoid dendritic cells for non-diagnostic purposes, the method comprising the following steps:

[0045] Performing flow cytometry detection on the cells to be tested using the antibody composition or the kit as described above;

[0046] 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;

[0047] 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;

[0048] Wherein, the fluorescent antibody pairs include: CD56-CD45, CD56-CD36, CD56-CD4, CD56-CD7, CD56-CD5, CD56-CD3, CD56-CD2, CD14-CD36, CD3-CD5, CD3-CD2, CD3-CD7, CD3-CD4, CD3-CD8, CD8-CD4, CD3-CD56, CD56-HLA-DR, CD56-CD33, CD56-CD38, CD56-CD117, CD56-CD123, CD56-CD19, CD34-HLA-DR, CD34-CD4 5. CD34-CD117, CD34-CD33, CD34-CD38, CD34-CD123, CD34-CD56, CD34-CD19, HLA-DR-CD33, CD56-CD303, CD56-CD304, CD56-CD15, CD56-C D13, CD56-CD85j, CD56-CD64, CD56-CD41, CD15-CD13, CD64-CD85j, CD41-CD45, CD56-TdT, CD56-MPO, CD56-cCD22, CD56-cCD3, CD56-CD10.

[0049] In the present invention, after a large number of experiments, the inventors screened out an antibody composition for identifying blastic plasmacytoid dendritic cells with the CD56+CD45dim+ region as the main target cell population, covering CD4, CD123, CD36, CD303, CD304, CD85j, CD64, CD19, CD7, CD13, CD33, CD34, CD117, cCD22, cCD3, TdT, MPO, etc. The antibody composition can be used to quickly, simply and effectively exclude monocytes that are most easily confused with blastic plasmacytoid dendritic cells, and accurately detect tumor-related abnormal blastic plasmacytoid dendritic cells, thereby enabling rapid, accurate and comprehensive detection of tumor-related abnormal blastic plasmacytoid dendritic cells at one time. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a scatter plot of CD45-SSC expression, an immunophenotype of cells in a BPDCN sample to be tested, detected using an antibody composition in Example 3 of the present invention.

[0051] Figure 2 The figure is a scatter plot of the CD56-CD45 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0052] Figure 3 This is a scatter plot of the CD56-CD36 expression of all nucleated cells in the BPDCN sample to be tested.

[0053] Figure 4 The figure is a scatter plot of the CD56-CD4 expression of all nucleated cells in the BPDCN sample to be tested.

[0054] Figure 5 This is a scatter plot of the CD56-CD7 expression in all nucleated cells in the BPDCN sample to be tested.

[0055] Figure 6 The figure is a scatter plot of the CD56-CD5 expression in all nucleated cells in the BPDCN sample to be tested.

[0056] Figure 7 This is a scatter plot of the CD56-CD3 expression of all nucleated cells in the BPDCN sample to be tested.

[0057] Figure 8 This is a scatter plot of the CD56-CD2 expression of all nucleated cells in the BPDCN sample to be tested.

[0058] Figure 9 The figure is a scatter plot of the CD14-CD36 expression of all nucleated cells in the BPDCN sample to be tested.

[0059] Figure 10It is a scatter plot of CD3-CD5 expression of lymphocytes in the BPDCN samples to be tested.

[0060] Figure 11 This is a scatter plot of CD3-CD2 expression in lymphocytes in the BPDCN sample to be tested.

[0061] Figure 12 This is a scatter plot of CD3-CD7 expression in lymphocytes in the BPDCN samples to be tested.

[0062] Figure 13 This is a scatter plot of CD3-CD4 expression in lymphocytes in the BPDCN sample to be tested.

[0063] Figure 14 This is a scatter plot of CD3-CD8 expression in lymphocytes in the BPDCN sample to be tested.

[0064] Figure 15 This is a scatter plot of CD8-CD4 expression in lymphocytes in the BPDCN sample to be tested.

[0065] Figure 16 The figure is a scatter plot of CD3-CD56 expression in lymphocytes in the BPDCN samples to be tested.

[0066] Figure 17 The figure is a scatter plot of the CD56-HLA-DR expression of all nucleated cells in the BPDCN sample to be tested.

[0067] Figure 18 The figure is a scatter plot of the CD56-CD33 expression of all nucleated cells in the BPDCN sample to be tested.

[0068] Figure 19 This is a scatter plot of the CD56-CD38 expression of all nucleated cells in the BPDCN sample to be tested.

[0069] Figure 20 This is a scatter plot of the CD56-CD117 expression in all nucleated cells in the BPDCN sample to be tested.

[0070] Figure 21 The figure is a scatter plot of the CD56-CD123 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0071] Figure 22 The figure is a scatter plot of the CD56-CD19 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0072] Figure 23 The figure is a scatter plot of the CD34-HLA-DR immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0073] Figure 24 The figure is a scatter plot of the CD34-CD45 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0074] Figure 25 The figure is a scatter plot of the CD34-CD117 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0075] Figure 26 The figure is a scatter plot of the CD34-CD33 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0076] Figure 27 The figure is a scatter plot of the CD34-CD38 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0077] Figure 28 The figure is a scatter plot of the CD34-CD123 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0078] Figure 29 The figure is a scatter plot of the CD34-CD56 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0079] Figure 30 The figure is a scatter plot of the CD34-CD19 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0080] Figure 31 The figure is a scatter plot of the HLA-DR-CD33 immunophenotype of all nucleated cells in the BPDCN sample to be tested.

[0081] Figure 32 This is a scatter plot of the CD56-CD303 expression in all nucleated cells in the BPDCN sample to be tested.

[0082] Figure 33 This is a scatter plot of the CD56-CD304 expression in all nucleated cells in the BPDCN sample to be tested.

[0083] Figure 34 This is a scatter plot of the CD56-CD15 expression of all nucleated cells in the BPDCN sample to be tested.

[0084] Figure 35 This is a scatter plot of the CD56-CD13 expression of all nucleated cells in the BPDCN sample to be tested.

[0085] Figure 36 This is a scatter plot of the CD56-CD85j expression in all nucleated cells in the BPDCN sample to be tested.

[0086] Figure 37This is a scatter plot of the CD56-CD64 expression in all nucleated cells in the BPDCN sample to be tested.

[0087] Figure 38 The figure is a scatter plot of the CD56-CD41 expression of all nucleated cells in the BPDCN sample to be tested.

[0088] Figure 39 This is a scatter plot of the CD15-CD13 expression of all nucleated cells in the BPDCN sample to be tested.

[0089] Figure 40 The figure is a scatter plot of the CD64-CD85j expression of all nucleated cells in the BPDCN sample to be tested.

[0090] Figure 41 The figure is a scatter plot of the CD41-CD45 expression of all nucleated cells in the BPDCN sample to be tested.

[0091] Figure 42 The figure is a scatter plot of the CD56-TdT expression in all nucleated cells in the BPDCN samples to be tested.

[0092] Figure 43 The figure is a scatter plot of the CD56-MPO expression in all nucleated cells in the BPDCN samples to be tested.

[0093] Figure 44 The figure is a scatter plot of the CD56-cCD22 expression of all nucleated cells in the BPDCN samples to be tested.

[0094] Figure 45 The figure is a scatter plot of the CD56-cCD3 expression of all nucleated cells in the BPDCN samples to be tested.

[0095] Figure 46 This is a scatter plot of the CD56-CD10 expression of all nucleated cells in the BPDCN sample to be tested.

[0096] Figure 47 This is a scatter plot of CD45-SSC expression, an immunophenotype of cells in a test AML-M5 sample, detected using an antibody composition in Example 3 of the present invention.

[0097] Figure 48 The figure is a scatter plot of the CD56-CD45 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0098] Figure 49 The figure is a scatter plot of the CD56-CD36 expression in all nucleated cells of the AML-M5 sample to be tested.

[0099] Figure 50The figure is a scatter plot of the CD56-CD4 expression of all nucleated cells in the AML-M5 sample to be tested.

[0100] Figure 51 This is a scatter plot of the CD56-CD7 expression in all nucleated cells of the AML-M5 sample to be tested.

[0101] Figure 52 The figure is a scatter plot of the CD56-CD5 expression in all nucleated cells of the AML-M5 sample to be tested.

[0102] Figure 53 This is a scatter plot of the CD56-CD3 expression in all nucleated cells of the AML-M5 sample to be tested.

[0103] Figure 54 The figure is a scatter plot of the CD56-CD2 expression in all nucleated cells of the AML-M5 sample to be tested.

[0104] Figure 55 This is a scatter plot of the CD14-CD36 expression of all nucleated cells in the AML-M5 sample to be tested.

[0105] Figure 56 This is a scatter plot of CD3-CD5 expression in lymphocytes of the AML-M5 sample to be tested.

[0106] Figure 57 This is a scatter plot of CD3-CD2 expression in lymphocytes of the AML-M5 sample to be tested.

[0107] Figure 58 This is a scatter plot of CD3-CD7 expression in lymphocytes of the AML-M5 sample to be tested.

[0108] Figure 59 The figure is a scatter plot of CD3-CD4 expression in lymphocytes of the AML-M5 sample to be tested.

[0109] Figure 60 This is a scatter plot of CD3-CD8 expression in lymphocytes of the AML-M5 sample to be tested.

[0110] Figure 61 The figure is a scatter plot of the CD8-CD4 expression of lymphocytes in the AML-M5 sample to be tested.

[0111] Figure 62 This is a scatter plot of CD3-CD56 expression in lymphocytes of the AML-M5 sample to be tested.

[0112] Figure 63 The figure is a scatter plot of the CD56-HLA-DR expression of all nucleated cells in the AML-M5 sample to be tested.

[0113] Figure 64 This is a scatter plot of the CD56-CD33 expression in all nucleated cells of the AML-M5 sample to be tested.

[0114] Figure 65 The figure is a scatter plot of the CD56-CD38 expression in all nucleated cells of the AML-M5 sample to be tested.

[0115] Figure 66 This is a scatter plot of the CD56-CD117 expression in all nucleated cells of the AML-M5 sample to be tested.

[0116] Figure 67 The figure is a scatter plot of the CD56-CD123 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0117] Figure 68 The figure is a scatter plot of the CD56-CD19 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0118] Figure 69 The figure is a scatter plot of the CD34-HLA-DR immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0119] Figure 70 The figure is a scatter plot of the CD34-CD45 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0120] Figure 71 The figure is a scatter plot of the CD34-CD117 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0121] Figure 72 The figure is a scatter plot of the CD34-CD33 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0122] Figure 73 The figure is a scatter plot of the CD34-CD38 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0123] Figure 74 The figure is a scatter plot of the CD34-CD123 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0124] Figure 75 The figure is a scatter plot of the CD34-CD56 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0125] Figure 76 The figure is a scatter plot of the CD34-CD19 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0126] Figure 77The figure is a scatter plot of the HLA-DR-CD33 immunophenotype of all nucleated cells in the AML-M5 sample to be tested.

[0127] Figure 78 This is a scatter plot of the CD56-CD303 expression in all nucleated cells of the AML-M5 sample to be tested.

[0128] Figure 79 The figure is a scatter plot of the CD56-CD304 expression in all nucleated cells of the AML-M5 sample to be tested.

[0129] Figure 80 This is a scatter plot of the CD56-CD15 expression of all nucleated cells in the AML-M5 sample to be tested.

[0130] Figure 81 This is a scatter plot of the CD56-CD13 expression of all nucleated cells in the AML-M5 sample to be tested.

[0131] Figure 82 This is a scatter plot of the CD56-CD85j expression in all nucleated cells of the AML-M5 sample to be tested.

[0132] Figure 83 The figure is a scatter plot of the CD56-CD64 expression in all nucleated cells of the AML-M5 sample to be tested.

[0133] Figure 84 The figure is a scatter plot of the CD56-CD41 expression in all nucleated cells of the AML-M5 sample to be tested.

[0134] Figure 85 This is a scatter plot of the CD15-CD13 expression of all nucleated cells in the AML-M5 sample to be tested.

[0135] Figure 86 The figure is a scatter plot of the CD64-CD85j expression in all nucleated cells of the AML-M5 sample to be tested.

[0136] Figure 87 The figure is a scatter plot of the CD41-CD45 expression of all nucleated cells in the AML-M5 sample to be tested.

[0137] Figure 88 The figure is a scatter plot of the CD56-TdT expression in all nucleated cells of the AML-M5 sample to be tested.

[0138] Figure 89 The figure is a scatter plot of the CD56-MPO expression in all nucleated cells of the AML-M5 sample to be tested.

[0139] Figure 90The figure is a scatter plot of the CD56-cCD22 expression in all nucleated cells of the AML-M5 sample to be tested.

[0140] Figure 91 The figure is a scatter plot of the CD56-cCD3 expression in all nucleated cells of the AML-M5 sample to be tested.

[0141] Figure 92 This is a scatter plot of the CD56-CD10 expression in all nucleated cells of the AML-M5 sample to be tested. DETAILED DESCRIPTION

[0142] 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.

[0143] 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.

[0144] 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.

[0145] In the present invention, in response to the deficiencies of the existing technology, an antibody composition for rapid detection of blastic plasmacytoid dendritic cells is provided, which includes four groups of antibodies; the first group of antibodies includes CD36, CD4, CD14, CD56, CD5, CD3, CD8, CD2, CD7 and CD45 antibodies; the second group of antibodies includes: HLA-DR, CD33, CD34, CD56, CD117, CD123, CD19, CD38 and CD45 antibodies; the third group of antibodies includes: CD303, CD304, CD41, CD56, CD13, CD85j, CD64, CD15 and CD45 antibodies; the fourth group of antibodies includes: TdT, MPO, CD56, cCD3, CD10, cCD22 and CD45 antibodies; each of the antibodies is a monoclonal antibody labeled with a detection marker.

[0146] Among them, the antibody composition has the following advantages:

[0147] 1. Breakthrough integration of specific markers:

[0148] In the second group of antibodies: CD123+CD33- double labeling locking: CD123 (IL-3Rα) is the core marker of BPDCN (sensitivity > 95%). Co-labeling with CD33 (myeloid marker) can exclude CD123+AML (such as M0 / M5 subtypes). The typical phenotype of BPDCN is CD123++ / CD33- / CD34±, while AML-M5 is CD123+ / CD33++.

[0149] In the third group of antibodies: CD303 / CD304 / CD85j triple verification; CD303 (BDCA-2) and CD304 (BDCA-4) are pDC-specific markers, and combined with CD85j (LILRB1) can distinguish: BPDCN: CD303+ / CD304+ / CD85j+ (strong expression), reactive pDC proliferation: CD303+ / CD304+ / CD85j-, other pDC tumors: CD303± / CD304± / CD85j-.

[0150] 2. Cross-tube collaborative optimization of fluorescence channels:

[0151] Spectral interference control strategy: separate highly expressed antigens (such as CD56-PECy5.5) and low expressed antigens (such as CD8-APC700) into different tubes to avoid signal leakage; use long-wavelength dyes (APC750, PB) to label weakly expressed antigens (such as CD10, CD7) to reduce background interference.

[0152] Cross-tube compensation correction design: CD8-APC700 in the first set of antibodies and CD19-APC750 in the second set of antibodies are excited by different lasers (635nm vs. 488nm) to eliminate compensation dependency. CD56-PECy5.5 is used as an "anchor marker" throughout all tubes to achieve multi-tube data alignment.

[0153] 3. Upgrade of interference signal elimination system:

[0154] Lineage cross-expression exclusion module: The first group of antibodies excludes monocytic leukemia by co-labeling with CD36 (monocyte / erythroid marker) and CD14 (monocyte marker) (CD36+ / CD14+ vs BPDCN CD36- / CD14-); the fourth group of antibodies excludes B-ALL by combining CD10 and cCD22 (B lineage markers) (CD10+ / cCD22+ vs BPDCN CD10- / cCD22-).

[0155] Activation state interference suppression: remove nonspecific activation markers (such as CD71, CD64), introduce CD38 (plasma cell differentiation marker) and CD117 (stem cell factor receptor) for dynamic grouping, BPDCN: CD38- / CD117- (90% of cases), plasma cell neoplasms: CD38++ / CD117-, myeloid neoplasms: CD38± / CD117+.

[0156] Therefore, the antibody composition can quickly, simply and effectively exclude monocytes that are most easily confused with blastic plasmacytoid dendritic cells, and accurately detect tumor-related abnormal blastic plasmacytoid dendritic cells, thereby enabling rapid, accurate and comprehensive detection of tumor-related abnormal blastic plasmacytoid dendritic cells at one time, greatly improving the accuracy of BPDCN detection.

[0157] The present invention is described in detail below with reference to specific embodiments.

[0158] Example 1 Screening of Antibody Compositions for Detecting Blastic Plasmacytoid Dendritic Cells

[0159] In this embodiment, in the detection of blastic plasmacytoid dendritic cells, due to the epitope homology between the surface markers of blastic plasmacytoid dendritic cells (such as CD123, CD303, etc.) and other immune cells (such as monocytes and lymphocytes), traditional antibodies are prone to cross-reaction. The inventors have obtained the following antibodies for the detection of blastic plasmacytoid dendritic cells through long-term research and analysis, and after a large number of research and screening experiments. The fluorescent labeling and dosage of the relevant monoclonal antibodies in the antibodies are shown in Table 1.

[0160] Table 1

[0161]

[0162]

[0163]

[0164]

[0165] 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.

[0166] Table 2

[0167]

[0168]

[0169]

[0170] Twenty clinically confirmed BPDCN and 20 non-BPDCN cases were used for testing using the above screening protocol. The specific testing process is as follows:

[0171] 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.

[0172] 2. Sample processing.

[0173] The concentration of the sample to be tested was adjusted to 1×10 6 / ml to prepare a single-cell suspension.

[0174] 3. Sample testing.

[0175] (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.

[0176] (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.

[0177] (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.

[0178] (4) Beckman Coulter Navios ten-color flow cytometer was used to detect the flow tubes and analyze their immunophenotype.

[0179] 4. Data analysis.

[0180] (1) Establish a template for the antibody expression pattern of healthy people

[0181] First, based on the flow cytometry test results of 20 normal control cell populations, 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: CD56-CD45, CD56-CD36, CD56-CD4, CD56-CD7, CD56-CD5, CD56-CD3, CD56-CD2, CD14-CD36, CD3-CD5, CD3-CD2, CD3-CD7, CD3-CD4, CD3-CD8, CD8-CD4, CD3-CD56, CD56-HLA-DR, CD56-CD33, CD56-CD38, CD56-CD117 , CD56-CD123, CD56-CD19, CD34-HLA-DR, CD34-CD45, CD34-CD117, CD34-CD33, CD34-CD38, CD34-CD123, CD34-CD56, CD34-CD19, HLA-DR-CD33, CD56-CD303, CD56-C D304, CD56-CD15, CD56-CD13, CD56-CD85j, CD56-CD64, CD56-CD41, CD15-CD13, CD64-CD85j, CD41-CD45, CD56-TdT, CD56-MPO, CD56-cCD22, CD56-cCD3, CD56-CD10.

[0182] (2) Establish the antibody expression pattern of the cells to be tested

[0183] 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.

[0184] (3) Analyze the antibody expression pattern of the cells to be tested

[0185] 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;

[0186] 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 BPDCN cells.

[0187] The test results are shown in Table 3 below:

[0188] Table 3

[0189]

[0190] Comparative analysis of the screening data in Table 3 shows that the antibody panel in round 3 has better targeting than other antibody panels and can accurately distinguish BPDCN from non-BPDCN. Further research on this antibody panel will be conducted in the future.

[0191] The antibody group includes a first group of antibodies, a second group of antibodies, a third group of antibodies and a fourth group of antibodies;

[0192] The first panel of antibodies includes CD36, CD4, CD14, CD56, CD5, CD3, CD8, CD2, CD7, and CD45 antibodies;

[0193] The second group of antibodies includes: HLA-DR, CD33, CD34, CD56, CD117, CD123, CD19, CD38 and CD45 antibodies;

[0194] The third group of antibodies includes: CD303, CD304, CD41, CD56, CD13, CD85j, CD64, CD15 and CD45 antibodies;

[0195] The fourth group of antibodies includes: TdT, MPO, CD56, cCD3, CD10, cCD22 and CD45 antibodies.

[0196] Example 2 System and Method for Detecting Blastic Plasmacytoid Dendritic Cells

[0197] This embodiment provides a system for detecting blastic plasmacytoid dendritic cells, comprising:

[0198] A detection module, wherein the detection module performs flow cytometry detection on the cells to be detected;

[0199] a data acquisition module, which acquires flow cytometry detection result data of cells to be tested stained with the third round of antibody composition of Example 1;

[0200] The data analysis module analyzes the acquired data and determines whether the acquired cells are blastic plasmacytoid dendritic cells according to predetermined analysis logic and judgment criteria.

[0201] The specific workflow for detecting blastic plasmacytoid dendritic cells using the above system of this embodiment is as follows:

[0202] 1. Prepare the membrane antibodies (excluding intracellular antibodies) in the third round of antibody combination in Table 2 (i.e., the present invention). The specific liquid addition amounts are shown in Table 1.

[0203] 2. Sample processing.

[0204] The concentration of the sample to be tested was adjusted to 1×10 6 / ml to prepare a single-cell suspension.

[0205] 3. Sample testing.

[0206] (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.

[0207] (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.

[0208] (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.

[0209] (4) Beckman Coulter Navios ten-color flow cytometer was used to detect the flow tubes and analyze their immunophenotype.

[0210] Example 3 Verification of the Accuracy of Antibody Composition Detection

[0211] The detection method of Example 2 was used to detect tumor cells in two test cells (one BPDCN sample and one non-BPDCN AML-M5 control sample), and antibody expression pattern analysis was performed to verify the accuracy of the detection results of the detection method of the present invention.

[0212] 1. BPDCN Sample

[0213] This sample is a bone marrow fluid sample from a clinically confirmed case of BPDCN. The antibody expression pattern was analyzed according to the following steps:

[0214] 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.

[0215] Figure 2 The CD56-CD45 immunophenotype scatter plot of all nucleated cells shows that 87.97% of the abnormal cells are CD56+CD45dim+ (red in the figure), a significantly increased proportion, suggesting a high possibility of AL / NHL.

[0216] Figure 3 This is a scatter plot of the CD56-CD36 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD36 positive (expression).

[0217] Figure 4 This is a scatter plot of CD56-CD4 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are partially CD4 positive (partially expressed).

[0218] Figure 5 This is a scatter plot of the CD56-CD7 expression of all nucleated cells. It can be seen from the figure that a small number of CD56+ abnormal cells are CD7 positive (a small amount of expression).

[0219] Figure 6 This is a scatter plot of the CD56-CD5 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD5 negative (no expression).

[0220] Figure 7 This is a scatter plot of the CD56-CD3 expression of all nucleated cells. It can be seen from the figure that the CD56+ abnormal cells are CD3 negative (not expressed), which can rule out that this group of tumor cells is not T-ALL cells.

[0221] Figure 8 This is a scatter plot of the CD56-CD2 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD2 negative (not expressed).

[0222] Figure 9This is a scatter plot of the CD14-CD36 expression of all nucleated cells. It can be seen from the figure that the red abnormal cells are CD36 positive (expression) and CD14 negative (no expression), which can rule out that they are not mature monocytes.

[0223] Figure 10 The figure is a scatter plot of CD3-CD5 expression in lymphocytes. It can be seen from the figure that the proportion of CD3+CD5+ normal T lymphocytes in lymphocytes is 73.69%.

[0224] Figure 11 This is a scatter plot of CD3-CD2 expression in lymphocytes. It can be seen from the figure that all CD3+T lymphocytes are CD2 positive.

[0225] Figure 12 The figure is a scatter plot of CD3-CD7 expression on lymphocytes. It can be seen from the figure that the expression rate of CD7 on CD3+T lymphocytes is 63.10%. The loss of a small amount of CD7 expression on such T cells is common in disease states and is a concomitant manifestation.

[0226] Figure 13 This is a scatter plot of CD3-CD4 expression on lymphocytes. The figure shows that the CD4 expression rate of CD3+ T lymphocytes is 36.18%, indicating that the CD3+CD4+ T lymphocyte subset accounts for 36.18%. This ratio is basically normal.

[0227] Figure 14 This is a scatter plot of CD3-CD8 expression on lymphocytes. The figure shows that the expression rate of CD8 on CD3+ T lymphocytes is 35.00%, indicating that the CD3+CD8+ T lymphocyte subset accounts for 35.00%. This ratio is basically normal.

[0228] Figure 15 This is a scatter plot of CD8-CD4 expression in lymphocytes, and the proportion is basically normal.

[0229] Figure 16 The figure shows the scatter plot of CD3-CD56 expression on lymphocytes. It can be seen that the proportion of CD3-CD56+ NK lymphocytes in lymphocytes is 6.10%, which is basically normal.

[0230] Figure 17 The figure is a scatter plot of the CD56-HLA-DR expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are HLA-DR positive (expression).

[0231] Figure 18 This is a scatter plot of the CD56-CD33 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD33 negative (not expressed).

[0232] Figure 19 The scatter plot shows the expression of CD56 and CD38 in all nucleated cells. The CD56+ abnormal cells are CD38 positive (expression). The expression of CD38 is not strong, suggesting that the tumor cells are not plasma cell tumors.

[0233] Figure 20 The figure is a scatter plot of the CD56-CD117 expression of all nucleated cells. It can be seen from the figure that a small number of CD56+ abnormal cells are CD117 positive (a small amount of expression).

[0234] Figure 21 This is a scatter plot of the CD56-CD123 immunophenotype of all nucleated cells. The figure shows that CD56+ abnormal cells are strongly positive for CD123 (strong expression). The simultaneous expression of CD123 and CD56 should be considered in the differential diagnosis of AML / BPDCN / precursor NK tumors.

[0235] Figure 22 This is a scatter plot of the CD56-CD19 immunophenotype of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD19 negative (not expressed).

[0236] Figure 23 This is a scatter plot of the CD34-HLA-DR immunophenotype of all nucleated cells. It can be seen from the figure that the abnormal cells are CD34 negative (no expression), suggesting that CD34+ AML-M0, M1, and M2 types of AL can be excluded.

[0237] Figure 24 The figure shows the CD34-CD45 immunophenotype scatter plot of all nucleated cells. It can be seen from the figure that in addition to the red abnormal population, there are also CD34+ primitive cells that occupy 0.02% of the total number of nucleated cells (shown as the brown population).

[0238] Figure 25 The scatter plot shows the CD34-CD117 immunophenotype of all nucleated cells. The figure shows that CD34+ primitive cells are CD117 positive (brown group shown). No obvious abnormalities were found in the phenotype.

[0239] Figure 26 The CD34-CD33 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD33 is positive for CD34+ primitive cells (the brown group shown). No obvious abnormalities were observed in the phenotype.

[0240] Figure 27 The CD34-CD38 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD38 is positive for CD34+ primitive cells (brown group shown). No obvious abnormalities were observed in the phenotype.

[0241] Figure 28 The CD34-CD123 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD34+ primitive cells are CD123 positive (brown group shown). No obvious abnormalities were observed in the phenotype.

[0242] Figure 29 The CD34-CD56 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD34+ primitive cells are CD56 negative (brown group shown). No obvious abnormalities were observed in the phenotype.

[0243] Figure 30 The CD34-CD19 immunophenotype of all nucleated cells is shown in a scatter plot. It can be seen that CD34+ primitive cells are CD19 negative (brown group shown). No obvious abnormalities were observed in the phenotype.

[0244] Figure 31 This is a scatter plot of the HLA-DR-CD33 immunophenotype of all nucleated cells. It can be seen from the figure that in addition to CD34+ primitive cells and red abnormal cells, there are also normal CD33+HLA-DR+ monocytes (Figure V++ area).

[0245] Figure 32 The figure shows the scatter plot of CD56-CD303 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD303 negative (no expression), indicating that they are not normal mature pDC cells.

[0246] Figure 33 The figure shows the scatter plot of CD56-CD304 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD304 positive (expression), suggesting that they are cells derived from pDC.

[0247] Figure 34 The figure shows the scatter plot of CD56-CD15 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD15 negative (no expression), indicating that tumor cells do not express markers of myeloid differentiation.

[0248] Figure 35 The scatter plot shows the expression of CD56-CD13 in all nucleated cells. It can be seen from the figure that the CD56+ abnormal cells are CD13 negative (no expression), which basically excludes AML-M5.

[0249] Figure 36 The figure shows the scatter plot of CD56-CD85j expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD85j positive (expression), suggesting that they are pDCs or cells of myeloid origin.

[0250] Figure 37The scatter plot shows the expression of CD56 and CD64 in all nucleated cells. The abnormal CD56+ cells are negative for CD64 (no expression), which means that AML-M5 can be ruled out.

[0251] Figure 38 The scatter plot shows the expression of CD56 and CD41 in all nucleated cells. The abnormal CD56+ cells are negative for CD41 (no expression), which means that AML-M7 can be ruled out.

[0252] Figure 39 The figure shows the scatter plot of CD15-CD13 expression in all nucleated cells. It can be seen from the figure that abnormal cells are negative for both CD13 and CD15 (no expression). Normal granulocyte and monocytic cells expressing CD15 and CD13 can be seen.

[0253] Figure 40 The figure shows the scatter plot of CD64-CD85j expression in all nucleated cells. It can be seen from the figure that abnormal cells are CD85j positive and CD64 negative (no expression). Normal granulocyte and monocytic cells expressing CD85j and CD64 can be seen.

[0254] Figure 41 The figure is a scatter plot of CD41-CD45 expression in all nucleated cells, from which CD41-expressing platelets can be seen (S-+ area in the figure).

[0255] Figure 42 The scatter plot shows the expression of CD56-TdT in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are TdT positive (expression), indicating that the abnormal cells are derived from early primitive cells.

[0256] Figure 43 This is a scatter plot of CD56-MPO expression in all nucleated cells. The figure shows that CD56+ abnormal cells are MPO-negative (no expression). This indicates that the abnormal cells are MPO-negative, and combined with the absence of CD13 / CD64 / CD15 / CD41 expression, AML-M1 / M2 / M3 / M4 / M5 / M7 can be excluded.

[0257] Figure 44 The scatter plot shows the expression of CD56-cCD22 in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are cCD22 negative (no expression). Combined with the lack of CD19 expression, B-ALL is excluded.

[0258] Figure 45 The scatter plot shows the expression of CD56-cCD3 in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are cCD3 negative (no expression), which excludes T-ALL.

[0259] Figure 46 This is a scatter plot of the CD56-CD10 expression of all nucleated cells. It can be seen from the figure that a small number of CD56+ abnormal cells are CD10 positive (a small amount of expression).

[0260] The abnormal cells in this sample expressed CD4, CD123, CD56, CD304, CD36, CD38, CD85j, and HLA-DR, with limited expression of CD7 and CD10. MPO, cCD22, cCD3, CD2, CD3, CD5, CD8, CD13, CD14, CD15, CD41, CD64, and CD19 were absent, excluding AML / B-ALL / T-ALL and precursor NK cell tumors. According to the WHO 2022 Classification of Lymphoid and Hematopoietic Tumors, the diagnostic basis for immunophenotyping is as follows: expected positive markers include CD123, CD4, CD56, CD303, CD304, TCF4, and TCL1, and expected negative markers include CD3, CD14, CD19, CD34, lysozyme, and MPO. The diagnosis is based on the expression of CD123 and pDC, or any three pDC markers. This specimen expressed four positive indicators: CD4, CD123, CD56, and CD304. The expected negative markers were CD3, CD14, CD19, CD34, lysozyme (monocyte marker: this test uses CD64 / CD13 / CD15 as a substitute), and MPO, all of which were negative. This is consistent with the diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN), which is consistent with the clinical diagnosis.

[0261] 2. Non-BPDCN AML-M5 control samples

[0262] This sample is a control bone marrow fluid specimen from a clinically confirmed non-BPDCN AML-M5. Antibody expression pattern analysis was performed using the following steps:

[0263] 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 47 The CD45dim region is where the target cell population is located, and this group of cells will be subjected to immunophenotypic analysis below.

[0264] Figure 48The CD56-CD45 immunophenotype scatter plot of all nucleated cells shows that the percentage of CD56+CD45dim+ abnormal cells is 83.31% (red in the figure), which is a significantly increased proportion, suggesting a high possibility of AL / NHL.

[0265] Figure 49 This is a scatter plot of the CD56-CD36 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD36 positive (expression).

[0266] Figure 50 This is a scatter plot of CD56-CD4 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are partially CD4 positive (partially expressed).

[0267] Figure 51 This is a scatter plot of the CD56-CD7 expression of all nucleated cells. It can be seen from the figure that a small number of CD56+ abnormal cells are CD7 positive (a small amount of expression).

[0268] Figure 52 This is a scatter plot of the CD56-CD5 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD5 negative (no expression).

[0269] Figure 53 This is a scatter plot of the CD56-CD3 expression of all nucleated cells. It can be seen from the figure that the CD56+ abnormal cells are CD3 negative (not expressed), which can rule out that this group of tumor cells is not T-ALL cells.

[0270] Figure 54 This is a scatter plot of the CD56-CD2 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD2 negative (not expressed).

[0271] Figure 55 This is a scatter plot of the CD14-CD36 expression of all nucleated cells. It can be seen from the figure that the red abnormal cells are CD36 positive (expression) and CD14 positive (expression), and are likely to be abnormal monocytes.

[0272] Figure 56 The figure is a scatter plot of CD3-CD5 expression in lymphocytes. It can be seen from the figure that the proportion of CD3+CD5+ normal T lymphocytes in lymphocytes is 66.17%.

[0273] Figure 57 This is a scatter plot of CD3-CD2 expression in lymphocytes. It can be seen from the figure that all CD3+T lymphocytes are CD2 positive.

[0274] Figure 58The figure is a scatter plot of CD3-CD7 expression on lymphocytes. It can be seen from the figure that the expression rate of CD7 on CD3+T lymphocytes is 55.11%. The loss of a small amount of CD7 expression on such T cells is common in disease states and is a concomitant manifestation.

[0275] Figure 59 This is a scatter plot of CD3-CD4 expression on lymphocytes. The figure shows that the CD4 expression rate of CD3+ T lymphocytes is 40.64%, indicating that the CD3+CD4+ T lymphocyte subset accounts for 40.64%. This ratio is basically normal.

[0276] Figure 60 The following is a scatter plot of CD3-CD8 expression on lymphocytes. The figure shows that the expression rate of CD8 on CD3+ T lymphocytes is 25.06%, indicating that the CD3+CD8+ T lymphocyte subset accounts for 25.06%, which is slightly low.

[0277] Figure 61 This is a scatter plot of CD8-CD4 expression in lymphocytes, and the proportion is basically normal.

[0278] Figure 62 The following is a scatter plot of CD3-CD56 expression on lymphocytes. The figure shows that CD3-CD56+ NK lymphocytes account for 6.28% of lymphocytes, which is basically normal.

[0279] Figure 63 The figure is a scatter plot of the CD56-HLA-DR expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are HLA-DR positive (expression).

[0280] Figure 64 The figure shows the scatter plot of CD56-CD33 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD33 positive (expression), indicating that tumor cells express the mononuclear marker CD33.

[0281] Figure 65 The scatter plot shows the expression of CD56 and CD38 in all nucleated cells. The CD56+ abnormal cells are CD38 positive (expression). The expression of CD38 is not strong, suggesting that the tumor cells are not plasma cell tumors.

[0282] Figure 66 The figure is a scatter plot of the CD56-CD117 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD117 negative (no expression).

[0283] Figure 67This is a scatter plot of the CD56-CD123 immunophenotype of all nucleated cells. It can be seen that CD56+ abnormal cells are partially positive for CD123 (partial expression). When both CD123 and CD56 are expressed, the diagnosis of AML / BPDCN / precursor NK tumors should be considered.

[0284] Figure 68 This is a scatter plot of the CD56-CD19 immunophenotype of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD19 negative (not expressed).

[0285] Figure 69 This is a scatter plot of the CD34-HLA-DR immunophenotype of all nucleated cells. It can be seen from the figure that the abnormal cells are CD34 negative (no expression), suggesting that CD34+ AML-M0, M1, and M2 types of AL can be excluded.

[0286] Figure 70 This is a scatter plot of the CD34-CD45 immunophenotype of all nucleated cells. It can be seen from the figure that in addition to the red abnormal population, there are also CD34+ primitive cells that occupy 0.05% of the total number of nucleated cells (shown as the brown population).

[0287] Figure 71 The scatter plot shows the CD34-CD117 immunophenotype of all nucleated cells. The figure shows that CD34+ primitive cells are CD117 positive (brown group shown). No obvious abnormalities were found in the phenotype.

[0288] Figure 72 The CD34-CD33 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD33 is positive for CD34+ primitive cells (the brown group shown). No obvious abnormalities were observed in the phenotype.

[0289] Figure 73 The CD34-CD38 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD38 is positive for CD34+ primitive cells (brown group shown). No obvious abnormalities were observed in the phenotype.

[0290] Figure 74 The CD34-CD123 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that some CD34+ primitive cells are CD123 positive (brown population shown). No obvious abnormalities were observed in the phenotype.

[0291] Figure 75 The CD34-CD56 immunophenotype of all nucleated cells is shown in the scatter plot. It can be seen that CD34+ primitive cells are CD56 negative (brown group shown). No obvious abnormalities were observed in the phenotype.

[0292] Figure 76The CD34-CD19 immunophenotype of all nucleated cells is shown in a scatter plot. It can be seen that CD34+ primitive cells are CD19 negative (brown group shown). No obvious abnormalities were observed in the phenotype.

[0293] Figure 77 This is a scatter plot of the HLA-DR-CD33 immunophenotype of all nucleated cells. It can be seen from the figure that abnormal cells appear in the position of normal monocytes (V++ area in the figure).

[0294] Figure 78 The figure shows the scatter plot of CD56-CD303 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD303 negative (no expression), indicating that they are not normal mature pDC cells.

[0295] Figure 79 The figure shows the scatter plot of CD56-CD304 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD304 positive (expression), suggesting that they are cells of non-pDC origin.

[0296] Figure 80 The figure shows the scatter plot of CD56-CD15 expression in all nucleated cells. It can be seen from the figure that some CD56+ abnormal cells are CD15 positive (partial expression), indicating that tumor cells express markers of myeloid differentiation.

[0297] Figure 81 The figure shows the scatter plot of CD56-CD13 expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are partially CD13 positive (partially expressed), indicating that tumor cells express markers of myeloid differentiation.

[0298] Figure 82 The figure shows the scatter plot of CD56-CD85j expression in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD85j positive (expression), indicating that they are cells of monocytic origin.

[0299] Figure 83 The scatter plot shows the expression of CD56 and CD64 in all nucleated cells. The abnormal CD56+ cells are strongly CD64 positive (strong expression), indicating that AML-M5 may be a possibility.

[0300] Figure 84 The scatter plot shows the expression of CD56 and CD41 in all nucleated cells. The abnormal CD56+ cells are negative for CD41 (no expression), which means that AML-M7 can be ruled out.

[0301] Figure 85The scatter plot shows the expression of CD15 and CD13 in all nucleated cells. The abnormal cells are partially positive for both CD13 and CD15 (partial expression), indicating that AML-M5 is the case.

[0302] Figure 86 This is a scatter plot of the CD64-CD85j expression of all nucleated cells. It can be seen from the figure that abnormal cells are CD85j positive and CD64 strongly positive (strong expression).

[0303] Figure 87 The figure is a scatter plot of CD41-CD45 expression in all nucleated cells, from which CD41-expressing platelets can be seen (S-+ area in the figure).

[0304] Figure 88 The scatter plot shows the expression of CD56-TdT in all nucleated cells. It can be seen from the figure that some CD56+ abnormal cells are TdT positive (partially expressed), indicating that the abnormal cells are derived from early primitive cells.

[0305] Figure 89 The scatter plot shows the expression of CD56-MPO in all nucleated cells. The figure shows that the CD56+ abnormal cells are partially MPO positive (partially expressed), indicating that the abnormal cells are partially MPO (myeloperoxidase) positive, supporting AML-M5.

[0306] Figure 90 The scatter plot shows the expression of CD56-cCD22 in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are cCD22 negative (no expression). Combined with the lack of CD19 expression, B-ALL is excluded.

[0307] Figure 91 The scatter plot shows the expression of CD56-cCD3 in all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are cCD3 negative (no expression), which excludes T-ALL.

[0308] Figure 92 This is a scatter plot of the CD56-CD10 expression of all nucleated cells. It can be seen from the figure that CD56+ abnormal cells are CD10 negative (no expression).

[0309] The abnormal cells in this sample expressed CD4, CD123, CD56, CD64, CD33, CD36, CD38, CD85j, and HLA-DR, and partially expressed MPO, CD13, CD14, and CD15. They did not express CD7, CD10, cCD22, cCD3, CD2, CD3, CD5, CD8, CD41, CD19, CD303, and CD304, excluding the diagnosis of BPDCN / B-ALL / T-ALL / precursor NK tumors. According to the WHO 2022 Classification of Lymphoid and Hematopoietic Tumors, the diagnostic basis for immunophenotyping is as follows: expected positive markers include CD123, CD4, CD56, CD303, CD304, TCF4, and TCL1, and expected negative markers include CD3, CD14, CD19, CD34, lysozyme, and MPO. The diagnosis is based on the expression of CD123 and pDC, or any three pDC markers. This specimen expressed three positive indicators: CD4, CD123, and CD56, which can be easily confused with BPDCN. However, the expected negative markers included CD3, CD14, CD19, CD34, lysozyme (monocyte marker: this test uses CD64 / CD13 / CD15 instead), and MPO was expressed in the monocyte marker and MPO. This does not meet the diagnostic requirements of BPDCN immune markers and is not consistent with the diagnosis of BPDCN. Taking into account the expression of all antibody markers in this protocol, the sample results in this case are consistent with AML-M5, which is consistent with the clinical diagnosis.

[0310] The results of this example show that the antibody composition designed by the present invention uses the CD56+CD45dim+ region as the main target cell population, and covers CD4, CD123, CD36, CD303, CD304, CD85j, CD64, CD19, CD7, CD13, CD33, CD34, CD117, cCD22, CCD3, TdT, MPO and other antibodies to identify BPDCN cells. The antibody composition can exclude monocytes that are easily confused with BPDCN cells, accurately distinguish BPDCN cells from AML cells, and thus accurately detect tumorous abnormal BPDCN cells.

[0311] 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.

[0312] 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 blastic plasmacytoid dendritic cells, characterized in that: The antibody composition includes a first group of antibodies, a second group of antibodies, a third group of antibodies, and a fourth group of antibodies; The first panel of antibodies includes CD36, CD4, CD14, CD56, CD5, CD3, CD8, CD2, CD7, and CD45 antibodies; The second group of antibodies includes: HLA-DR, CD33, CD34, CD56, CD117, CD123, CD19, CD38 and CD45 antibodies; The third group of antibodies includes: CD303, CD304, CD41, CD56, CD13, CD85j, CD64, CD15 and CD45 antibodies; The fourth group of antibodies includes: TdT, MPO, CD56, cCD3, CD10, 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 CD36, HLA-DR, CD303 and TdT antibodies are labeled with the same fluorescein; The CD4, CD33, CD304 and MPO antibodies are labeled with the same fluorescein; The CD14, CD34 and CD41 antibodies are labeled with the same fluorescein; The CD56 antibody is labeled with a fluorescent dye; The CD5, CD117 and CD13 antibodies are labeled with the same fluorescein; The CD3, CD123, CD85j and cCD3 antibodies are labeled with the same fluorescein; The CD8 antibody is labeled with a fluorescent dye; The CD2, CD19, CD64 and CD10 antibodies are labeled with the same fluorescein; The CD7, CD38, CD15 and cCD22 antibodies are labeled with the same fluorescein; 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 CD36, HLA-DR, CD303 and TdT antibodies are labeled with fluorescein FITC; The CD4, CD33, CD304 and MPO antibodies are labeled with fluorescein PE; The CD14, CD34 and CD41 antibodies are labeled with fluorescein ECD; The CD56 antibody is labeled with fluorescein PECY5.5; The CD5, CD117 and CD13 antibodies are labeled with fluorescein PECY7; The CD3, CD123, CD85j and cCD3 antibodies are labeled with fluorescein APC; The CD8 antibody is labeled with fluorescein APC-700; The CD2, CD19, CD64 and CD10 antibodies are labeled with fluorescein APC-750; The CD7, CD38, CD15 and cCD22 antibodies are labeled with fluorescein PB; The CD45 antibody was labeled with fluorescein KO. 4 . Use of the antibody composition for detecting blastic plasmacytoid dendritic cells according to claim 1 in detecting blastic plasmacytoid dendritic cells for non-diagnostic purposes.

5. A kit for detecting blastic plasmacytoid dendritic cells, characterized in that: The kit comprises the antibody composition for detecting blastic plasmacytoid dendritic cells 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 four parts, and the first group of antibodies, the second group of antibodies, the third group of antibodies and the fourth 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 15 μl to 25 μl, wherein the amount of each antibody in the first group of antibodies added is 1 μl to 5 μl, and the titer of each antibody in the first group of antibodies is 1:3 to 7; and / or, The amount of the second group of antibodies added per 100 μl of the sample to be tested is 20 μl to 30 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 1:2 to 7; and / or, The amount of the third group of antibodies added per 100 μl of the sample to be tested is 20 μl to 30 μl, wherein the amount of each antibody in the third group of antibodies added is 1 μl to 5 μl, and the titer of each antibody in the third group of antibodies is 1:1 to 7; and / or, The amount of the fourth group of antibodies added per 100 μl of the sample to be tested is 30 μl to 40 μl, wherein the amount of the cCD22 antibody added is 18 to 22 μl, and the amount of the remaining antibodies added is 1 μl to 5 μl, and the titer of each antibody in the fourth group of antibodies is 1:2 to 7; 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 blastic plasmacytoid dendritic cells for non-diagnostic purposes.

10. A method for detecting blastic plasmacytoid dendritic cells 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: CD56-CD45, CD56-CD36, CD56-CD4, CD56-CD7, CD56-CD5, CD56-CD3, CD56-CD2, CD14-CD36, CD3-CD5, CD3-CD2, CD3-CD7, CD3-CD4, CD3-CD8, CD8-CD4, CD3-CD56, CD56-HLA-DR, CD56-CD33, CD56-CD38, CD56-CD117, CD56-CD123, CD56-CD19, CD34-HLA-DR, CD34-CD45, CD34-CD117, CD34-CD33, CD34-CD38, CD34-CD123, CD34-CD56, CD34-CD19, HLA-DR-CD33, CD56-CD303, CD56-CD304, CD56-CD15, CD56-CD13, CD56-CD85j, CD56-CD64, CD56-CD41, CD15-CD13, CD64-CD85j, CD41-CD45, CD56-TdT, CD56-MPO, CD56-cCD22, CD56-cCD3, CD56-CD10.

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