Antibody combination, kit and application thereof for detecting NK large granular lymphocytes

Abstract

The invention discloses an antibody combination for detecting NK large granular lymphocytes, comprising a first group of antibodies, a second group of antibodies and a third group of antibodies; the first group of antibodies comprises CD158a, CD159c (NKG2C), CD3, CD56, CD159a (NKG2A), CD158b, CD7, CD5, CD158e and CD45 antibodies; the second group of antibodies comprises: CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45 antibodies; the third group of antibodies comprises: Granzyme B, Perforin, CD3, CD56, Ki67, CD2 and CD45 antibodies; each antibody is a monoclonal antibody marked with a detection marker. The antibody combination can detect NK large granular lymphocytes quickly and easily, with high accuracy, high sensitivity and high specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of immunological detection, and specifically relates to an antibody combination for detecting NK large granular lymphocytes, a kit and applications thereof. Background Art

[0002] NK large granular lymphocytic leukemia (NK-LGLL) is a clonal NK lymphocyte proliferative disease. Most patients are middle-aged and elderly (median age 60 years old). Its important diagnostic indicator is the unexplained continuous increase of peripheral blood NK cells (usually >2*10 9 / L) for more than 6 months, and the proportion of mature granular lymphocytes in peripheral blood morphology is often increased. This disease often accumulates in peripheral blood, bone marrow, liver, spleen, etc., and can cause symptoms related to hemocyte reduction. In some elderly people, especially those with granulocytopenia, NK large granular lymphocytic leukemia (NK-LGLL) screening can identify clonal NK large granular lymphocytes earlier, which is convenient for clinical better early identification and diagnosis and treatment of this disease.

[0003] NK large granular lymphocytic leukemia (NK-LGLL) needs to be distinguished from reactive NK increase. Reactive NK cell increase is often secondary to viral infection, autoimmune disease, malignant tumor, etc. After the inducement is removed, NK cells can decrease on their own. However, both of these conditions can show an increase in the absolute value of lymphocytes in blood routine examinations, and the proportion of granular lymphocytes in peripheral blood cell morphology increases, which makes it difficult to identify. In the WHO hematopoietic and lymphoid tissue tumors, it is mentioned that large granular lymphocytic leukemia (NK-LGLL) shows unique characteristics through flow cytometry immunophenotyping. Surface CD3 is negative, CD16 is positive, weak CD56 expression, there may be reduced or lost expression of CD2, CD7 and CD57, and abnormal uniform expression of CD8. In NK-LGLL, the expression of the killer cell immunoglobulin-like receptor (KIR) family of NK cell receptors is abnormal; limited KIR isotype expression or a complete lack of detectable KIR can be seen. Therefore, accurate identification can be performed by detecting the expression of KIR by flow cytometry.

[0004] Currently, KIR indicators for detecting NK cells include CD158a / CD158b / CD158e, etc., which are combined with NK surface markers such as CD56 and CD16 for detection. However, because there is no systematic detection solution on the market, different testing institutions may have different antibody combinations, or the results may be misjudged due to incomplete and unsystematic antibody combinations.

[0005] Therefore, there is a need for a systematic antibody combination and detection kit for detecting NK large granular lymphocytes on the market, with standard detection schemes, operating procedures and analysis ideas to ensure the accuracy and comparability of the results, and to assist in the accurate diagnosis of NK large granular lymphocytic leukemia (NK-LGLL) in clinical practice. Summary of the invention

[0006] The object of the present invention is to provide an antibody combination capable of rapidly and accurately detecting NK large granular lymphocytes.

[0007] The above-mentioned purpose is achieved by the following technical solutions.

[0008] The first aspect of the present invention provides an antibody combination for detecting NK large granular lymphocytes, comprising a first group of antibodies, a second group of antibodies and a third group of antibodies;

[0009] The first group of antibodies includes CD158a, CD159c (NKG2C), CD3, CD56, CD159a (NKG2A), CD158b, CD7, CD5, CD158e and CD45 antibodies;

[0010] The second group of antibodies includes: CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45 antibodies;

[0011] The third group of antibodies includes: Granzyme B, Perforin, CD3, CD56, Ki67, CD2 and CD45 antibodies; each of the antibodies is a monoclonal antibody marked with a detection marker.

[0012] In some of these embodiments, the CD158a, CD94 and Granzyme B antibodies are labeled with the same fluorescein;

[0013] The CD159c (NKG2C), CD161 and Perforin antibodies are labeled with the same fluorescein;

[0014] The CD3 and CD16 antibodies are labeled with the same fluorescein;

[0015] The CD56 antibody is labeled with a fluorescent substance;

[0016] The CD159a (NKG2A), CD45RO and Ki67 antibodies are labeled with the same fluorescein;

[0017] The CD158b and CD3 antibodies are labeled with the same fluorescein;

[0018] The CD7 and CD8 antibodies are labeled with the same fluorescein;

[0019] The CD5, CD45RA and CD2 antibodies are labeled with the same fluorescein;

[0020] The CD158e and CD57 antibodies are labeled with the same fluorescein;

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

[0022] The fluorescein is selected from: FITC, PE, ECD, PE-Cy TM 5.5, PE-Cy7, APC, APC-700, APC-750, PB, KO.

[0023] In some embodiments, the CD158a, CD94 and Granzyme B antibodies are labeled with fluorescein FITC; the CD159c (NKG2C), CD161 and Perforin antibodies are labeled with fluorescein PE; the CD3 and CD16 antibodies are labeled with fluorescein ECD; the CD56 antibody is labeled with fluorescein PE-Cy TM 5.5; the CD159a (NKG2A), CD45RO and Ki67 antibodies are labeled with fluorescein PE-Cy7; the CD158b and CD3 antibodies are labeled with fluorescein APC; the CD7 and CD8 antibodies are labeled with fluorescein APC-700; the CD5, CD45RA and CD2 antibodies are labeled with fluorescein APC-750; the CD158e and CD57 antibodies are labeled with fluorescein PB; the CD45 antibody is labeled with fluorescein KO;

[0024] Among them, the product number of CD3 antibody labeled with fluorescein ECD is A07748, and the product number of CD56 antibody labeled with fluorescein PE-Cy TM The catalog number of 5.5 is B49189, the catalog number of CD7 antibody labeled with fluorescein APC700 is B36290, the catalog number of CD5 antibody labeled with fluorescein APC750 is B92413, the catalog number of CD45 antibody labeled with fluorescein KO is B36294, the catalog number of CD16 antibody labeled with fluorescein ECD is B49216, the catalog number of CD45 RO antibody labeled with fluorescein PECY7 is B13648, the catalog number of CD3 antibody labeled with fluorescein APC is IM2467, the catalog number of CD45 RA antibody labeled with fluorescein APC750 is B49194, the catalog number of GranzymeB antibody labeled with fluorescein FITC is B46038, and the catalog number of CD2 antibody labeled with fluorescein APC750 is B01681. The manufacturer is BeckmanCoulter;

[0025] The catalog number of CD158a antibody labeled with fluorescein FITC is 339504, the catalog number of CD159c (NKG2C) antibody labeled with fluorescein PE is 375004, the catalog number of CD159a (NKG2C) antibody labeled with fluorescein PECY7 is 375114, the catalog number of CD158b antibody labeled with fluorescein APC is 312612, the catalog number of CD158e antibody labeled with fluorescein PB is 312714, the catalog number of CD94 antibody labeled with fluorescein FITC is 305504, the catalog number of CD161 antibody labeled with fluorescein PE is 339904, and the catalog number of CD57 antibody labeled with fluorescein PB is 393326. The manufacturer is biolegend;

[0026] The product number of Ki67 antibody labeled with fluorescein APC is 561283, manufacturer BD;

[0027] The product number of Perforin antibody labeled with fluorescein PE is 1P-765-T100, and the manufacturer is Exbio.

[0028] A second aspect of the present invention provides a kit for detecting NK large granular lymphocytes, wherein the kit comprises the antibody combination for detecting NK large granular lymphocytes as described above.

[0029] In some embodiments, the kit further comprises reagents for flow cytometry detection.

[0030] In some of the embodiments, in the flow cytometry detection, the single cell suspension of the sample to be detected 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.

[0031] In some embodiments, the volume ratio of the single cell suspension to the first group of antibodies is 100 μl: 35 μl-36 μl, wherein the amount of each antibody in the first group of antibodies is 1.5 μl-5 μl, and the cell number concentration in the single cell suspension is 1×10 5 / ml~1×10 6 / ml; and / or,

[0032] The volume ratio of the single cell suspension to the second group of antibodies is 100 μl: 31 μl-33 μl, wherein the amount of each antibody in the second group of antibodies is 1 μl-5 μl; and / or,

[0033] The volume ratio of the single cell suspension to the third group of antibodies is 100 μl:30 μl-32 μl, wherein the dosage of each antibody in the third group of antibodies is 1 μl-10 μl;

[0034] The cell number concentration in the single cell suspension is 1×10 5 / ml~1×106 Pieces / ml.

[0035] The third aspect of the present invention provides a use of the above-mentioned antibody combination or the above-mentioned kit in assisting the detection of NK large granular lymphocytes.

[0036] A fourth aspect of the present invention provides a method for detecting NK large granular lymphocytes, the method comprising the following steps:

[0037] Prepare single cell suspension of samples;

[0038] 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 as described above are added respectively for incubation;

[0039] After the incubation, the sample cells to be tested are subjected to flow cytometry detection.

[0040] In some embodiments, after the sample cells to be tested are subjected to flow cytometry detection, the method further comprises:

[0041] The data obtained after flow cytometry detection is analyzed to determine whether the antibody expression pattern of the sample cells to be tested falls into the expression pattern template of the antibodies of the normal control population; wherein the antibody expression pattern of the sample cells to be tested and the expression pattern of the antibodies of the normal control population are established by the following steps:

[0042] The data of the flow cytometry detection results are used to group the nucleated cells by setting a gate, circle the target cell population, and then analyze the expression of each fluorescent antibody pair in the target cell population; preferably, the fluorescent antibody pair includes: CD45-SSC, CD3-CD56, CD3-CD7, CD3-CD5, CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, CD56-CD159c; CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, CD56-CD57; CD3-CD2, CD56-Granzyme B, CD56-Perforin, CD56-Ki67.

[0043] The technical solution provided by the present invention has the following advantages and effects:

[0044] In the present invention, the inventors have screened out three groups of antibody combinations for identifying NK large granular lymphocytes, including CD3-CD56+ region as the main target cell population, covering KIR series (CD158a, CD158b, CD158e), CD159a (NKG2A), CD159c (NKG2C), CD94, CD161, Granzyme B, Perforin, CD45RA, CD45RO, Ki67, etc., after a lot of research and experiments. The antibody combination can be used to detect NK large granular lymphocytes quickly, easily, with high accuracy, high sensitivity and high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a scatter plot of CD45-SSC expression of the immunophenotype of normal NK lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

[0046] Figure 2 This is a scatter plot of the CD3-CD56 expression of the immunophenotype of normal NK lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

[0047] Figure 3 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD3-CD7 of the normal NK lymphocytes to be tested.

[0048] Figure 4 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD3-CD5 of the normal NK lymphocytes to be tested.

[0049] Figure 5 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-CD7 of the normal NK lymphocytes to be tested.

[0050] Figure 6 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-FS of the normal NK lymphocytes to be tested.

[0051] Figure 7 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-CD158a of the normal NK lymphocytes to be tested.

[0052] Figure 8 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-CD158b of the normal NK lymphocytes to be tested.

[0053] Fig. 9This is a scatter plot of the expression of the immunophenotype CD56-CD158e of normal NK lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

[0054] Fig.10 This is a scatter plot of the expression of the immunophenotype CD56-CD159a of normal NK lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

[0055] Fig.11 This is a scatter plot of the expression of the immunophenotype CD56-CD159c of normal NK lymphocytes to be tested using the first group of antibodies in Example 3 of the present invention.

[0056] Fig.12 This is a scatter plot of KIR-negative NK cells circled by the CD158a-CD158e and CD158b-CD158e logic gates for detecting the immunophenotype of normal NK lymphocytes to be tested in Example 3 of the present invention using the first group of antibodies.

[0057] Fig.13 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD3-CD56 of the normal NK lymphocytes to be tested.

[0058] Fig.14 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD3-CD8 of the normal NK lymphocytes to be tested.

[0059] Fig.15 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD16 of the normal NK lymphocytes to be tested.

[0060] Fig.16 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD94 of the normal NK lymphocytes to be tested.

[0061] Fig.17 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD161 of the normal NK lymphocytes to be tested.

[0062] Fig.18 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD45RO of the normal NK lymphocytes to be tested.

[0063] Fig.19 This is a scatter plot of the expression of CD56-CD45RA in the immune phenotype of normal NK lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

[0064] Fig. 20This is a scatter plot of the expression of CD56-CD57 of the immunophenotype of normal NK lymphocytes to be tested using the second group of antibodies in Example 3 of the present invention.

[0065] Fig.21 This is a scatter plot of the expression of CD3-CD56 of the immunophenotype of normal NK lymphocytes to be tested using the third group of antibodies in Example 3 of the present invention.

[0066] Fig. 22 This is a scatter plot of the expression of the immunophenotype CD3-CD2 of normal NK lymphocytes to be tested using the third group of antibodies in Example 3 of the present invention.

[0067] Fig.23 This is a scatter plot of the expression of CD56-Granzyme B in detecting the immunophenotype of normal NK lymphocytes to be tested using the third group of antibodies in Example 3 of the present invention.

[0068] Fig.24 This is a scatter plot of CD56-Perforin expression of the immunophenotype of normal NK lymphocytes to be tested using the third group of antibodies in Example 3 of the present invention.

[0069] Fig.25 This is a scatter plot of the expression of CD56-Ki67 of the immunophenotype of normal NK lymphocytes to be tested using the third group of antibodies in Example 3 of the present invention.

[0070] Fig.26 This is a scatter plot of CD45-SSC expression of the immunophenotype of abnormal NK lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

[0071] Fig. 27 This is a scatter plot of the CD3-CD56 expression of the immunophenotype of abnormal NK lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

[0072] Fig.28 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD3-CD7 of the abnormal NK lymphocytes to be tested.

[0073] Fig.29 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD3-CD5 of the abnormal NK lymphocytes to be tested.

[0074] Fig.30 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-CD7 of the abnormal NK lymphocytes to be tested.

[0075] Fig.31 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-FS of the abnormal NK lymphocytes to be tested.

[0076] Fig.32 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-CD158a of the abnormal NK lymphocytes to be tested.

[0077] Fig.33 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD56-CD158b of the abnormal NK lymphocytes to be tested.

[0078] Fig.34 This is a scatter plot of the expression of the immunophenotype CD56-CD158e of abnormal NK lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

[0079] Fig.35 This is a scatter plot of the expression of the immunophenotype CD56-CD159a of abnormal NK lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

[0080] Fig.36 This is a scatter plot of the expression of the immunophenotype CD56-CD159c of abnormal NK lymphocytes to be detected using the first group of antibodies in Example 3 of the present invention.

[0081] Fig.37 This is a scatter plot of KIR-negative NK cells circled by the CD158a-CD158e and CD158b-CD158e logic gates for detecting the immunophenotype of abnormal NK lymphocytes to be detected in Example 3 of the present invention using the first group of antibodies.

[0082] Fig.38 This is a scatter plot of the expression of CD3-CD56, the immunophenotype of abnormal NK lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

[0083] Fig.39 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD3-CD8 of the abnormal NK lymphocytes to be tested.

[0084] Fig.40 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD16 of the abnormal NK lymphocytes to be tested.

[0085] Fig.41 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD94 of the abnormal NK lymphocytes to be tested.

[0086] Fig.42 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD161 of the abnormal NK lymphocytes to be tested.

[0087] Fig.43 The second group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD56-CD45RO of the abnormal NK lymphocytes to be tested.

[0088] Fig.44 This is a scatter plot of the expression of CD56-CD45RA in detecting the immunophenotype of abnormal NK lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

[0089] Fig.45 This is a scatter plot of the expression of CD56-CD57 of the immunophenotype of abnormal NK lymphocytes to be detected using the second group of antibodies in Example 3 of the present invention.

[0090] Fig.46 This is a scatter plot of the expression of CD3-CD56, the immunophenotype of abnormal NK lymphocytes to be detected using the third group of antibodies in Example 3 of the present invention.

[0091] Fig.47 This is a scatter plot of the expression of the immunophenotype CD3-CD2 of abnormal NK lymphocytes to be detected using the third group of antibodies in Example 3 of the present invention.

[0092] Fig.48 This is a scatter plot of the expression of CD56-Granzyme B in detecting the immunophenotype of abnormal NK lymphocytes to be detected using the third group of antibodies in Example 3 of the present invention.

[0093] Fig.49 This is a scatter plot of CD56-Perforin expression of the immunophenotype of abnormal NK lymphocytes to be detected using the third group of antibodies in Example 3 of the present invention.

[0094] Fig.50 This is a scatter plot of the expression of CD56-Ki67 of the immunophenotype of abnormal NK lymphocytes to be detected using the third group of antibodies in Example 3 of the present invention.

[0095] Fig.51 This is a scatter plot of CD45-SSC expression of the immunophenotype of NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0096] Fig.52 This is a scatter plot of CD3-CD56 expression of the immunophenotype of NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0097] Fig.53 This is a scatter plot of the CD3-CD7 expression of the immunophenotype of the NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0098] Fig.54 This is a scatter plot of the CD56-CD7 expression of the immunophenotype of the NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0099] Fig.55 This is a scatter plot of the expression of CD56-FS, the immunophenotype of NK lymphocytes to be tested, detected using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0100] Fig.56 This is a scatter plot of the expression of CD56-CD158a of the immunophenotype of NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0101] Fig.57 This is a scatter plot of the expression of CD56-CD158b of the immunophenotype of NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0102] Fig.58 This is a scatter plot of the expression of CD56-CD158e, the immunophenotype of NK lymphocytes to be tested, detected using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0103] Fig.59 This is a scatter plot of KIR-negative NK cells circled by the CD158a-CD158e and CD158b-CD158e logic gates for detecting the immunophenotype of NK lymphocytes to be tested using the antibody combination recommended in NCCN in Example 4 of the present invention.

[0104] Fig.60 This is a scatter plot of the CD56-CD16 expression of the immunophenotype of the NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0105] Fig.61 This is a scatter plot of the expression of CD56-CD94, the immunophenotype of NK lymphocytes to be tested, detected using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0106] Fig.62 This is a scatter plot of the expression of CD56-CD161, the immunophenotype of NK lymphocytes to be tested, detected using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0107] Fig.63 This is a scatter plot of the CD56-CD57 expression of the immunophenotype of the NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0108] Fig.64This is a scatter plot of the CD3-CD2 expression of the immunophenotype of the NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0109] Fig.65 This is a scatter plot of CD56-Granzyme B expression in Example 4 of the present invention using the antibody combination recommended by NCCN to detect the immunophenotype of NK lymphocytes to be tested.

[0110] Fig.66 This is a scatter plot of CD56-Perforin expression in detecting the immunophenotype of NK lymphocytes to be tested using the antibody combination recommended by NCCN in Example 4 of the present invention.

[0111] Fig.67 This is a scatter plot of the expression of the immunophenotype CD45-SS of the NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention.

[0112] Fig.68 This is a scatter plot of the expression of the immunophenotype CD3-CD56 of the NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention.

[0113] Fig.69 This is a scatter plot of the expression of the immunophenotype CD3-CD7 of the NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention.

[0114] Fig.70 This is a scatter plot of the expression of the immunophenotype CD56-CD7 of the NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention.

[0115] Fig.71 This is a scatter plot of the expression of CD56-FS of the immunophenotype of NK lymphocytes to be detected using the first group of antibodies of the present invention in Example 4 of the present invention.

[0116] Fig.72 This is a scatter plot of the expression of the immunophenotype CD5-CD7 of the NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention.

[0117] Fig.73 This is a scatter plot of the immunophenotype CD56-CD158a expression of NK lymphocytes to be detected using the first group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD5+NK).

[0118] Fig.74 This is a scatter plot of the immunophenotype CD56-CD158b expression of NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD5+NK).

[0119] Fig.75 This is a scatter plot of the immunophenotype CD56-CD158e expression of NK lymphocytes to be detected using the first group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD5+NK).

[0120] Fig.76 This is a scatter plot of the immunophenotype CD56-CD159a expression of NK lymphocytes to be tested using the first group of antibodies in the present invention in Example 4 of the present invention (as CD56+CD5+NK).

[0121] Fig.77 This is a scatter plot of the immunophenotype CD56-CD159c expression of NK lymphocytes to be tested using the first group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD5+NK).

[0122] Fig.78 This is a scatter plot of KIR-negative NK cells circled by the second group of antibodies of the present invention in Example 4 of the present invention to detect the immunophenotype CD158a-CD158e and CD158b-CD158e logic gates of the NK lymphocytes to be tested.

[0123] Fig.79 This is a scatter plot of the expression of the immunophenotype CD3-CD8 of the NK lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention.

[0124] Fig.80 This is a scatter plot of the expression of CD56-CD16 of the immunophenotype of NK lymphocytes to be detected using the second group of antibodies of the present invention in Example 4 of the present invention.

[0125] Fig.81 This is a scatter plot of the expression of the immunophenotype CD56-CD8 of the NK lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention.

[0126] Fig.82 This is a scatter plot of the immunophenotype CD56-CD94 expression of NK lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD8+NK).

[0127] Fig.83 This is a scatter plot of the immunophenotype CD56-CD161 expression of NK lymphocytes to be detected using the second group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD8+NK).

[0128] Fig.84 This is a scatter plot of the immunophenotype CD56-CD45RO expression of NK lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD8+NK).

[0129] Fig.85 This is a scatter plot of the immunophenotype CD56-CD45RA expression of NK lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD8+NK).

[0130] Fig.86 This is a scatter plot of the immunophenotype CD56-CD57 expression of NK lymphocytes to be tested using the second group of antibodies of the present invention in Example 4 of the present invention (as CD56+CD8+NK).

[0131] Fig.87 This is a scatter plot of the expression of the immunophenotype CD3-CD2 of the NK lymphocytes to be tested using the third group of antibodies of the present invention in Example 4 of the present invention.

[0132] Fig.88 This is a scatter plot of the expression of CD56-Granzyme B in detecting the immunophenotype of NK lymphocytes to be tested using the third group of antibodies in the present invention in Example 4 of the present invention.

[0133] Fig.89 This is a scatter plot of the expression of CD56-Perforin, the immunophenotype of NK lymphocytes to be tested, detected using the third group of antibodies of the present invention in Example 4 of the present invention.

[0134] Fig.90 This is a scatter plot of the expression of CD56-Ki67 of the immunophenotype of NK lymphocytes to be tested using the third group of antibodies of the present invention in Example 4 of the present invention. DETAILED DESCRIPTION

[0135] In order to facilitate the 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 disclosure of the present invention more thorough and comprehensive.

[0136] The experimental methods in the following examples without specifying specific conditions are usually 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.

[0137] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0138] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or under conditions recommended by the manufacturer. The various commonly used reagents used in the examples are all commercially available products.

[0139] In the present invention, in view of the deficiencies of the prior art, an antibody combination, a kit and a system for rapid detection of NK large granular lymphocytes are provided. The antibody combination includes three groups of antibodies, with the CD3-CD56+ region as the main target cell population, covering the KIR series (CD158a, CD158b, CD158e), CD159a (NKG2A), CD159c (NKG2C), CD94, CD161, Granzyme B, Perforin, CD45RA, CD45RO, Ki67 and other antibodies that recognize NK large granular lymphocytes, and a comprehensive logical analysis strategy can better identify tumorous NK large granular lymphocytes. The present invention reasonably applies various antibody indicators and covers a wide range. The antibody combination can be used through multi-parameter flow detection technology to quickly and easily (it only takes 2 to 3 hours from sample receipt to test results) and highly sensitively detect NK large granular lymphocytes. The present invention also optimizes the template for result analysis. Before optimization, only a single gate (CD3-CD56+) was used to circle out NK lymphocytes to see whether the KIR series (CD158a, CD158b, CD158e) was expressed. However, because CD56 of tumor NK cells may sometimes be lost, the (CD3-CD56+) gate is inaccurate, resulting in deviations in the analysis results. After optimization, CD3-CD56+ and CD3-CD5+ can be used in combination to circle out suspicious NK cells, and then CD56-CD158b, CD56-CD158e, CD56-CD159a, CD56-CD159c, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, CD56-CD57, CD3-CD2, and CD56-Granzyme B, CD56-Perforin and other methods are used to judge whether NK lymphocytes are clonally active, and CD56-Ki67 is used to judge the proliferation index of tumorous NK cells. If Ki67 expression is high, it is necessary to exclude the possibility of invasive NK lymphocyte leukemia, because it has a high mortality rate and needs to be promptly reported to the clinic. In addition, the present invention has a fixed analysis template, and the data can be directly analyzed by dragging it in, which is simple to operate, reduces the requirements for the professional knowledge level of the analyst, and makes up for the limitations of existing detection technologies.

[0140] In some embodiments, an antibody combination for detecting NK large granular lymphocytes is provided, comprising:

[0141] 1. The first group of antibodies includes CD158a, CD159c (NKG2C), CD3, CD56, CD159a (NKG2A), CD158b, CD7, CD5, CD158e and CD45 antibodies. In addition to CD45 as the leukocyte gating antibody, the first group of antibodies can identify suspicious NK cells (CD3-CD56+) through the backbone antibodies CD3 and CD56. NK lymphocytes expressing CD3-CD56+ are circled, and CD3-CD5 and CD56-CD7 are observed to see whether there are abnormal CD3-CD5+CD7+ cells. Abnormal NK lymphocytes can be initially suspected, and then the clonality of this group of NK lymphocytes can be confirmed by combining CD158a, CD159c (NKG2C), CD159a (NKG2A), CD158b, CD158e and other indicators. The first group of antibodies added CD159c (NKG2C) and CD159a (NKG2A), two markers that can complement the determination of the clonality of KIR-negative NK cells.

[0142] 2. The second group of antibodies includes CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45 antibodies. Use CD3-CD56+ to circle NK lymphocytes, and then check whether CD94, CD161, CD16 and CD57 are expressed to determine whether they are cytotoxic cells; then combine the uniform expression of CD45RA, the lack of CD45RO, and the expression of the first tube of combined antibodies to determine whether it is NK large granular lymphocyte leukemia.

[0143] 3. The third group of antibodies includes Granzyme B, Perforin, CD3, CD56, Ki67, CD2 and CD45 antibodies. Use CD3-CD56+ to circle NK lymphocytes, and then check whether Granzyme B and Perforin are expressed to determine whether they are cytotoxic cells; in addition, CD56-Ki67 can be combined to judge the proliferation index of tumor NK cells. If Ki67 expression is high, it is necessary to exclude the possibility of invasive NK lymphocyte leukemia. If Ki67 expression is low, it indicates NK large granular lymphocyte leukemia.

[0144] In some embodiments, each of the antibodies is a monoclonal antibody labeled with a detectable marker.

[0145] In some embodiments, the monoclonal antibody is a fluorescein-labeled antibody, so that the above indicators can be detected simultaneously in a ten-color or more flow cytometer, which has a certain convenience; the fluorescein is selected from: FITC, PE, ECD, PE-Cy TM 5.5, PE-Cy7, APC, APC-700, APC-750, PB, KO.

[0146] In some embodiments, the CD158a, CD94 and Granzyme B antibodies are labeled with fluorescein FITC; the CD159c (NKG2C), CD161 and Perforin antibodies are labeled with fluorescein PE; the CD3 and CD16 antibodies are labeled with fluorescein ECD; the CD56 antibody is labeled with fluorescein PE-Cy TM 5.5; the CD159a (NKG2A), CD45RO and Ki67 antibodies are labeled with fluorescein PE-Cy7; the CD158b and CD3 antibodies are labeled with fluorescein APC; the CD7 and CD8 antibodies are labeled with fluorescein APC-700; the CD5, CD45RA and CD2 antibodies are labeled with fluorescein APC-750; the CD158e and CD57 antibodies are labeled with fluorescein PB; the CD45 antibody is labeled with fluorescein KO.

[0147] The embodiment of the present invention also provides a use of the above-mentioned antibody combination in assisting the detection of NK large granular lymphocytes.

[0148] The embodiment of the present invention further provides a kit for detecting NK large granular lymphocytes, wherein the kit comprises the above-mentioned antibody combination for detecting NK large granular lymphocytes.

[0149] The embodiment of the present invention also provides a use of the above-mentioned kit for detecting NK cell large granular lymphocytes in assisting the detection of NK cell large granular lymphocytes.

[0150] Specifically, in the application of assisting the detection of NK cells and large granular lymphocytes, the detection is performed by using flow cytometry.

[0151] The present invention also provides a method for detecting NK large granular lymphocytes, the method comprising the following steps:

[0152] Prepare single cell suspension of samples;

[0153] 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 as described above are added respectively for incubation;

[0154] After the incubation, the sample cells to be tested are subjected to flow cytometry detection.

[0155] The embodiment of the present invention further provides a system for detecting NK large granular lymphocytes, comprising:

[0156] A detection module, wherein the detection module performs flow cytometry detection on the cells to be detected, and comprises: a single cell suspension module, for preparing a single cell suspension; an incubation module, for incubating the single cell suspension with the reagent composition in a dark environment and performing an antigen-antibody reaction; a resuspension module, for adding hemolysin to the incubated cells, centrifuging, washing, and then resuspending the cells to prepare a suspension; and a determination module, for performing flow cytometry determination on the resuspended suspension;

[0157] A data acquisition module, which acquires data of flow cytometry detection results of cells to be tested stained with the antibody combination;

[0158] The data analysis module analyzes the acquired data and determines whether the acquired NK lymphocytes are tumorous large granular NK lymphocytes according to a predetermined analysis logic and judgment criteria; it includes: a normal control population antibody expression pattern template and a test cell antibody expression pattern, and the predetermined judgment criteria are: if the test cell antibody expression pattern falls into the normal control population antibody expression pattern template, the test cell is judged as a NK lymphocyte population with a normal immune phenotype; if the test cell antibody expression pattern does not fall into the normal control population antibody expression pattern template, the test cell is judged as a suspected tumor cell population.

[0159] The technical process of the system for detecting NK large granular lymphocytes in the present invention can refer to the conventional flow cytometry technology, and the equipment and consumables used are selected from flow cytometry analysis equipment and consumables, such as flow-specific tubes, oscillators, pipettes and other consumables.

[0160] In some embodiments, the normal control population antibody expression pattern template is established by the following method: obtaining flow cytometry detection result data of the normal control population cell population, grouping the nucleated cells by setting a gate, circling the target cell population (i.e., NK lymphocytes), and then analyzing the expression of each fluorescent antibody in the target cell population to obtain the normal control population antibody expression pattern template.

[0161] In some embodiments, the antibody expression pattern of the cells to be tested is established by the following method: obtaining flow cytometry test result data of the cells to be tested, setting gates to group the nucleated cells according to the antibody expression pattern of the normal control population, circling the target cell population (i.e., NK lymphocytes), and then analyzing the expression of each fluorescent antibody in the target cell population according to the antibody expression pattern of the normal control population to obtain the antibody expression pattern of the cells to be tested.

[0162] In some embodiments, the gating uses CD45-SSC (side scattered light) gating, and divides the cell population into a granulocyte population, a monocyte population, and a lymphocyte population according to the expression of CD45 (CD45 is a common leukocyte antigen, so CD45 is used as a gating antibody, and the expression intensity of CD45 of normal blood cells is: lymphocyte>monocyte>granulocyte, SSC is side scattered light, which is related to the granularity of the cell, and the normal expression intensity of side scattered light is: granulocyte>monocyte>lymphocyte; combined with the expression characteristics of CD45 and SSC, lymphocytes, monocytes and granulocytes can be divided into zones, among which lymphocytes are at the position with the strongest CD45 and the lowest SSC; the CD45 of monocytes is slightly weaker than that of lymphocytes and the SSC is slightly higher than that of lymphocytes; the CD45 of granulocytes is weaker than that of monocytes and the SSC signal is the highest. According to this expression pattern, the cell population is divided into granulocytes, monocytes, and lymphocytes), so as to further analyze the target cells in the lymphocyte population; and then analyze the fluorescence expression intensity corresponding to the antibodies in the target cells; the antibody pairs include: CD45-SSC, CD3-CD56, CD3-CD7, CD3-CD5, CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, CD56-CD159c; CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, CD56-CD57; CD3-CD2, CD56-Granzyme B, CD56-Perforin, CD56-Ki67.

[0163] The specific sources of some antibodies used in the following examples are: CD3ECD (Cat. No. A07748), CD56PE-Cy TM 5.5 (Cat. No. B49189), CD7 APC700 (Cat. No. B36290), CD5 APC750 (Cat. No. B92413), CD45KO (Cat. No. B36294), CD16ECD (Cat. No. B49216), CD45 RO PECY7 (Cat. No. B13648), CD3 APC (Cat. No. IM2467), CD45 RA APC750 (Cat. No. B49194), GranzymeB FITC (Cat. No. B46038), CD2APC750 (Cat. No. B01681), manufacturer BeckmanCoulter;

[0164] CD158a FITC (Cat. No. 339504), CD159c (NKG2C) PE (Cat. No. 375004), CD159a (NKG2C) PECY7 (Cat. No. 375114), CD158b APC (Cat. No. 312612), CD158e PB (Cat. No. 312714), CD94 FITC (Cat. No. 305504), CD161 PE (Cat. No. 339904), CD57 PB (Cat. No. 393326), manufacturer: biolegend;

[0165] Ki67APC (Cat. No. 561283), manufacturer BD;

[0166] Perforin PE (Cat. No. 1P-765-T100), manufacturer: Exbio.

[0167] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0168] Example 1 Antibody combination for detecting NK large granular lymphocytes

[0169] The antibody combination for detecting NK large granular lymphocytes in this embodiment includes a first group of antibodies, a second group of antibodies and a third group of antibodies, wherein:

[0170] The first group of antibodies includes: CD158a, CD159c (NKG2C), CD3, CD56, CD159a (NKG2A), CD158b, CD7, CD5, CD158e and CD45 antibodies;

[0171] The second group of antibodies includes: CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45 antibodies;

[0172] The third group of antibodies includes: Granzyme B, Perforin, CD3, CD56, Ki67, CD2 and CD45 antibodies.

[0173] The fluorescent labels and dosages of the relevant monoclonal antibodies in the above antibodies are shown in Table 1.

[0174] Table 1

[0175]

[0176]

[0177] Note: The commercially available antibodies are tested in a concentration gradient to determine the optimal dosage, and the above-mentioned amounts of monoclonal antibodies are respectively placed in flow cytometry tubes numbered 1, 2, and 3.

[0178] Example 2 System and method for detecting NK large granular lymphocytes

[0179] This embodiment provides a system for rapid detection of NK large granular lymphocytes, comprising: a detection module, a data acquisition module and a data analysis module. The detection module performs flow cytometry detection on the cells to be detected; the data acquisition module obtains flow cytometry detection result data of the cells to be detected stained with the detection reagent composition described in Example 1; the data analysis module analyzes the above-obtained data and determines whether the cells to be detected are tumorous NK lymphocytes according to a predetermined judgment standard.

[0180] The specific workflow of detecting NK large granular lymphocytes using the above system of this embodiment is as follows:

[0181] 1. Prepare the antibodies in the antibody combination of Example 1 as shown in Table 1.

[0182] 2. Sample processing.

[0183] The concentration of the sample to be tested was adjusted to 1×10 6 / ml to make a single cell suspension.

[0184] 3. Sample testing.

[0185] (1) Take flow tubes, mark them as 1, 2, and 3, and add the first group of antibodies, the second group of antibodies, and the third group of antibodies in Example 1 (add membrane antibodies CD3 / CD56 / CD2 / CD45 first) in an amount of 35.5 μl, 31 μl, and 11 μl, respectively. Then, add 100 μl of the suspension in step 2, vortex and mix, and incubate at room temperature in the dark for 15 min.

[0186] (2) After incubation, add 400 μl of Bc hemolysin to each of the flow tubes 1, 2, and 3, vortex and shake, and let stand until the hemolysis is clear. After the hemolysis is clear, centrifuge the flow tubes 1 and 2 at 1500 r / min for 5 min, discard the supernatant, and perform intracellular antibody detection in the third tube according to the following steps; add 2 ml of calf serum to tubes 1 and 2, vortex and shake, centrifuge at 1500 r / min for 5 min, discard the supernatant, and add 400 μl of 1% paraformaldehyde to resuspend.

[0187] (3) Intracellular antibody experiment operation: After the cell surface marker staining is completed in the above-mentioned tube 3 according to the above-mentioned (1, 2), 450uL of 1X FACS permeabilization agent is added, mixed and incubated in the dark for 5 minutes, and then PBS is added to wash once and the supernatant is discarded. Anti-intracellular fluorescent antibodies (Granzyme B, Perforin, Ki67) are added according to the above-mentioned 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 5min, the supernatant is discarded, and 400μl of 1% paraformaldehyde is added for resuspending.

[0188] (4) Use BeckmanCoulter Navios ten-color flow cytometer to detect flow tubes 1, 2, and 3 and analyze their immunophenotypes.

[0189] 4. Data analysis.

[0190] (1) Establish a template for the antibody expression pattern of healthy people

[0191] According to the flow cytometry test results of 40 normal control cell populations, the cells were grouped by setting a gate, preferably using CD45-SSC (side scattered light) to set a gate, circle the target cell population, and then analyze the expression of each fluorescent antibody for this cell population, and select the following antibody pairs: CD45-SSC, CD3-CD56, CD3-CD7, CD3-CD5, CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, CD56-CD159c; CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, CD56-CD57; CD3-CD2, CD56-Granzyme B, CD56-Perforin, CD56-Ki67.

[0192] (2) Establishing the antibody expression pattern of the cells to be tested

[0193] Obtain flow cytometry test result data of the cells to be tested, set gates to group the cells according to the 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 antibody expression pattern of the normal control population to obtain the antibody expression pattern of the cells to be tested.

[0194] (3) Analyze the antibody expression pattern of the cells to be tested

[0195] If the antibody expression pattern of the cells to be tested falls into the antibody expression pattern template of the normal control population, the cells to be tested are determined to be a NK lymphocyte population with a normal immunophenotype;

[0196] If the antibody expression pattern of the cells to be tested does not fall into the antibody expression pattern template of the normal control population, the cells to be tested are determined to be NK large granular lymphocytes.

[0197] Example 3 Verification of the accuracy of the detection method of the present invention

[0198] The detection method of Example 2 was used to detect NK large granular lymphocytes of two cells to be tested (one normal sample + one abnormal sample), and the antibody expression pattern was analyzed to verify the accuracy of the detection method of the present invention.

[0199] 1. Normal Sample

[0200] This sample is a clinical sample with normal physical examination and no obvious large granular lymphocytes in peripheral blood smear morphology analysis. The antibody expression pattern analysis was performed as follows:

[0201] 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 three areas according to the expression of CD45-SSC, namely granulocytes (the middle upper area in the figure), monocytes (the upper right area in the figure), and lymphocytes (the lower right area in the figure) (as shown in the figure). Figure 1 As shown in the figure, the lymphocyte area is the target cell population. It can be seen that lymphocytes account for 22.03% of the total number of nuclear cells, which is a normal proportion. The immunophenotype analysis of this group of cells will be performed below.

[0202] Figure 2 This is a scatter plot of the CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3-CD56+ NK lymphocytes account for 14.80% of the lymphocytes, which is a normal proportion.

[0203] Figure 3 The scatter plot of CD3-CD7 expression of lymphocyte population shows that CD3-CD7+ NK lymphocytes account for 14.58% of lymphocytes, which is basically the same as Figure 2 The NK cells shown were in equal proportions, with no obvious abnormal CD7- NK cells.

[0204] Figure 4 This is a scatter plot of CD3-CD5 expression in the lymphocyte population. You can focus on the presence or absence of CD3-CD5+ NK lymphocytes. There are no CD3-CD5+ NK lymphocytes in this figure.

[0205] Figure 5 to Figure 12The scatter plots are respectively the expression of CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, and CD56-CD159c of the NK lymphocyte population. It can be seen that the CD7 expression of the CD3-CD56+ NK lymphocytes is normal, and the focus is on analyzing the clonal indicators of the CD3-CD56+ NK lymphocytes (CD158a, CD158b, CD158, CD159a, CD159c) (normal NK lymphocytes express the above markers in small or partial amounts, and the judgment criteria for clonal NK lymphocytes are: CD159a>88% or CD158a>53% or CD158b>72% or CD158e>54% or KIR negative (CD158a, CD158b, CD158e)>72%). As shown in the figure, the CD158a expression rate of NK cells is 17.11% (<53%), which is normal; the CD158b expression rate is 39.72% (<72%); the CD158e expression rate is 29.11% (<54%); the CD159a expression rate is 10.15% (<88%); and the KIR negative (CD158a, CD158b, CD158e) cells are 40.16% (<72%). From the expression of the above clonal indicators, it can be judged that the NK cells of this specimen are normal NK lymphocytes.

[0206] Fig.13 Circle the NK lymphocytes which are CD3-CD56+ in the second group of antibodies.

[0207] Figure 14-Figure 20 This is a scatter plot of the expression of CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, and CD56-CD57 of CD3-CD56+NK lymphocytes. It can be seen that CD8 of NK cells is partially positive, and the expression of CD94 / CD161 / CD57 is normal. In addition, the partial expression of CD45RA and CD45RO indicates that the NK cells in this specimen are normal NK lymphocytes.

[0208] Fig.21 For the CD3-CD56+ in the third group of antibodies, circle the NK lymphocytes.

[0209] Figure 22-Figure 25 This is a scatter plot of the expression of CD2, Granzyme B, Perforin, and Ki67 of CD3-CD56+ NK lymphocytes. It can be seen that CD2, Granzyme B, and Perforin of NK cells are all expressed, while Ki67 is negative, and there is no abnormality in the expression, indicating that the NK cells in this specimen are normal NK lymphocytes.

[0210] The cell population of the sample to be tested was determined to be a cell population with normal immunophenotype in the antibody expression pattern template of the normal control population, which was consistent with the morphological analysis results and clinical symptoms.

[0211] 2. Abnormal Samples

[0212] This sample was clinically diagnosed as NK large granular lymphocytic leukemia by morphological analysis of peripheral blood smears, which showed obvious large granular lymphocytes.

[0213] Follow these steps to analyze antibody expression patterns:

[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 3 areas according to the expression of CD45-SSC, such as Fig.26 As shown, there are three areas, namely, granulocytes (the middle upper area in the figure), monocytes (the upper right area in the figure), and lymphocytes (the lower right area in the figure). The lymphocyte area is the target cell population of this implementation case. It can be seen that lymphocytes account for 15.12% of the total number of nuclear cells, which is a normal proportion. The immunophenotype analysis of this group of cells will be performed below.

[0215] Fig. 27 This is a scatter plot of CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3-CD56+ NK lymphocytes account for 32.93% of lymphocytes, and the proportion has increased significantly.

[0216] Fig.28 The scatter plot of CD3-CD7 expression of lymphocyte population shows that CD3-CD7+ NK lymphocytes account for 32.10% of lymphocytes, which is basically the same as Figure 2 The NK cells shown were in equal proportions, with no obvious abnormal CD7- NK cells.

[0217] Fig.29 This is a scatter plot of CD3-CD5 expression in the lymphocyte population. From the figure, we can see that there are CD3-CD5+ NK lymphocytes, indicating that the NK cell phenotype is abnormal.

[0218] Figure 30-Figure 37 The scatter plots of the expression of CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, and CD56-CD159c of the NK lymphocyte population were used to analyze the clonal indexes of NK lymphocytes (CD158a, CD158b, CD158, CD159a, and CD159c). Figure 32-Figure 37As shown, the CD158a expression rate of NK cells was 2.56% (<53%), the CD158b expression rate was 86.46% (>72%), indicating that they were clonal NK lymphocytes, the CD158e expression rate was 1.76% (<54%), the CD159a expression rate was 12.26% (<88%), and the KIR negative (CD158a, CD158b, CD158e) cells were 5.58% (<72%). From the expression of the above clonal indicators, it can be judged that the NK cells of this specimen clonally express CD158b and are clonal NK lymphocytes.

[0219] Fig.38 Circle the NK lymphocytes which are CD3-CD56+ in the second group of antibodies.

[0220] Figure 39-Figure 45 This is a scatter plot of the expression of CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, and CD56-CD57 of CD3-CD56+NK lymphocytes. It can be seen that CD8 of NK cells is uniformly positive, which is an abnormal expression (normal NK lymphocytes are partially positive and not aggregated), and there is no abnormality in the expression of CD16 and CD161. However, the expression of CD94 is weakened. In addition, the uniform expression of CD45RA, the lack of CD45RO, and the enhanced expression of CD57 all indicate that the NK cells in this specimen are abnormal NK lymphocytes.

[0221] Fig.46 For the CD3-CD56+ in the third group of antibodies, circle the NK lymphocytes.

[0222] Figure 47-Figure 50 The scatter plot is of the expression of CD2, Granzyme B, Perforin, and Ki67 of CD3-CD56+ NK lymphocytes. It can be seen that a small part of the CD2 of the NK cells is lost, there is no abnormality in the expression of Granzyme B, the expression of Perforin is enhanced, and Ki67 is negative, indicating that the NK cells in this specimen are inert abnormal NK lymphocytes.

[0223] The above immunophenotypes all indicate that the cell population of the sample to be tested is not in the antibody expression pattern template of the normal control population, and is a tumorous, inert NK large granular lymphocyte, which is consistent with the morphological analysis results and clinical diagnosis.

[0224] Example 4 Comparison of the detection accuracy of the antibody combination of the present invention and the antibody combination of the existing method

[0225] Compare the antibody combination of Example 1 of the present invention and the detection antibody combination recommended in NCCN (CD158a, CD3, CD56, CD158b, CD7, CD158e, CD94, CD161, CD16, CD57, Granzyme B, Perforin, CD2) The fluorescein labeling of each antibody refers to Table 1, and the dosage also refers to Table 1), and the accuracy when detecting the same cells to be tested.

[0226] 1. Use the detection antibodies recommended by NCCN (CD158a, CD3, CD56, CD158b, CD7, CD158e, CD94, CD161, CD16, CD57, Granzyme B, Perforin, CD2) to obtain the flow cytometry test result data of the sample to be tested.

[0227] CD45-SSC (side scattered light) was used to set the gate. According to the expression of CD45-SSC, the cell population was divided into three regions: granulocytes (the middle upper region in the figure), monocytes (the upper right region in the figure), and lymphocytes (the lower right region in the figure). Fig.51 As shown in the figure, the lymphocyte area is the target cell population. It can be seen that lymphocytes account for 13.68% of the total number of nuclear cells, which is a normal proportion. The immunophenotype analysis of this group of cells will be performed below.

[0228] Fig.52 This is a scatter plot of the CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3-CD56+ NK lymphocytes account for 13.69% of the lymphocytes, which is a normal proportion.

[0229] Figure 53-Figure 66 The scatter plot is of the expression of CD7, CD56-FS, CD158a, CD158b, CD158e, CD16, CD94, CD161, CD57, CD2, Granzyme B and Perforin of the NK lymphocyte population. It can be seen that there is no obvious abnormality in the expression of CD7, CD16, CD94, CD161, CD57, CD2, Granzyme B and Perforin of NK lymphocytes. Analysis of the NK lymphocyte clonality index CD158a expression rate is 8.54% (<53%), CD158b expression rate is 36.25% (<72%), CD158e expression rate is 11.17% (<54%), KIR negative (CD158a, CD158b, CD158e) cells are 46.39% (<72%), which are considered to be polyclonal normal NK lymphocytes.

[0230] 2. Use the antibody combination of Example 1 of the present invention to detect the sample to be tested, and obtain flow cytometry test result data of the sample to be tested.

[0231] CD45-SSC (side scattered light) was also used to set the gate. According to the expression of CD45-SSC, the cell population was divided into three regions, namely granulocytes (the middle upper region in the figure), monocytes (the upper right region in the figure), and lymphocytes (the lower right region in the figure). Fig.67 As shown), the lymphocyte area is the target cell population. It can be seen that lymphocytes account for 13.80% of the total number of nuclear cells, which is a normal proportion. The immunophenotype analysis of this group of cells will be performed below.

[0232] Fig.68 This is a scatter plot of the CD3-CD56 immunophenotype of lymphocytes. It can be seen from the figure that CD3-CD56+ NK lymphocytes account for 14.21% of lymphocytes, which is a normal proportion.

[0233] Figure 69-Figure 90 This is a scatter plot of the expression of CD7, CD56-FS, CD5-CD7, CD158a, CD158b, CD158e, CD16, CD161, CD57, CD2, Granzyme B, and Perforin in the NK lymphocyte population. It can be seen that 48.21% of NK lymphocytes are CD5 positive. Circle this group of CD5+NK lymphocytes and analyze their CD16, CD94, CD161, CD57, CD2, Granzyme B, and Perforin. B. There was no obvious abnormality in the expression of Perforin (no significant difference from the above-mentioned NCCN recommended detection scheme), but the loss of CD94, uniform expression of CD8, negativity of CD45RO, and uniform expression of CD45RA all indicated that this group of NK cells had abnormal phenotype. Further analysis of the clonal indicators of this group of CD5+NK lymphocytes showed that the expression rate of CD158a was 0.19% (<53%), the expression rate of CD158b was 0.19% (<72%), the expression rate of CD158e was 0.38% (<54%), and the KIR negative (CD158a, CD158b, CD158e) cells were 99.50% (>72%), which were clonal NK lymphocytes. Because Ki67 was negative, this group of 6.85% CD3-CD56+CD5+NK lymphocytes were neoplastic large granular NK lymphocytes.

[0234] The results of this embodiment show that the antibody combination designed by the present invention takes the CD3-CD56+ region as the main target cell population, covering the KIR series (CD158a, CD158b, CD158e), CD159a (NKG2A), CD159c (NKG2C), CD5, CD8, CD94, CD161, Granzyme B, Perforin, CD45RA, CD45RO, Ki67 and other antibody combinations for identifying NK large granular lymphocytes. The comprehensive logical analysis strategy can better identify tumorous NK large granular lymphocytes, and has better accuracy than the detection antibodies recommended in NCCN.

[0235] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0236] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An antibody combination for detecting NK large granular lymphocytes, characterized in that: The antibody combination consists of a first group of antibodies, a second group of antibodies and a third group of antibodies; The first group of antibodies is CD158a, CD159c (NKG2C), CD3, CD56, CD159a (NKG2A), CD158b, CD7, CD5, CD158e and CD45 antibodies; The second group of antibodies is CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45 antibodies; The third group of antibodies includes Granzyme B, Perforin, CD3, CD56, Ki67, CD2 and CD45 antibodies; each of the antibodies is a monoclonal antibody marked with a detection marker.

2. The antibody combination according to claim 1, characterized in that The CD158a, CD94 and Granzyme B antibodies are labeled with the same fluorescein; The CD159c (NKG2C), CD161 and Perforin antibodies are labeled with the same fluorescein; The CD3 antibodies in the first group of antibodies and the third group of antibodies and the CD16 antibodies in the second group of antibodies are labeled with the same fluorescent substance; The CD56 antibody is labeled with a fluorescent substance; The CD159a (NKG2A), CD45RO and Ki67 antibodies are labeled with the same fluorescein; The CD158b antibody and the CD3 antibody in the second group of antibodies are labeled with the same fluorescein; The CD7 and CD8 antibodies are labeled with the same fluorescein; The CD5, CD45RA and CD2 antibodies are labeled with the same fluorescein; The CD158e and CD57 antibodies are labeled with the same fluorescein; The CD45 antibody is labeled with a fluorescent substance; The fluorescein is selected from: FITC, PE, ECD, PE-Cy TM 5.5, PE-Cy7, APC, APC-700, APC-750, PB, KO.

3. The antibody combination according to claim 2, characterized in that The CD158a, CD94 and Granzyme B antibodies are labeled with fluorescein FITC; the CD159c (NKG2C), CD161 and Perforin antibodies are labeled with fluorescein PE; the CD3 antibodies in the first group of antibodies and the third group of antibodies and the CD16 antibodies in the second group of antibodies are labeled with fluorescein ECD; the CD56 antibody is labeled with fluorescein PE-Cy TM 5.5; the CD159a (NKG2A), CD45RO and Ki67 antibodies are labeled with fluorescein PE-Cy7; the CD158b antibody and the CD3 antibody in the second group of antibodies are labeled with fluorescein APC; the CD7 and CD8 antibodies are labeled with fluorescein APC-700; the CD5, CD45RA and CD2 antibodies are labeled with fluorescein APC-750; the CD158e and CD57 antibodies are labeled with fluorescein PB; the CD45 antibody is labeled with fluorescein KO.

4. A kit for detecting NK large granular lymphocytes, characterized in that: The kit comprises the antibody combination for detecting NK large granular lymphocytes according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes reagents for flow cytometry detection.

6. The kit according to claim 5, characterized in that In the flow cytometry detection, the single cell suspension of the sample to be detected 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.

7. The kit according to claim 6, characterized in that The volume ratio of the single cell suspension to the first group of antibodies is 100 μl: 35 μl-36 μl, wherein the dosage of each antibody in the first group of antibodies is 1.5 μl-5 μl, and the cell number concentration in the single cell suspension is 1×10 5 / ml~1×10 6 pcs / ml; and / or, The volume ratio of the single cell suspension to the second group of antibodies is 100 μl: 31 μl-33 μl, wherein the amount of each antibody in the second group of antibodies is 1 μl-5 μl; and / or, The volume ratio of the single cell suspension to the third group of antibodies is 100 μl:30 μl-32 μl, wherein the dosage of each antibody in the third group of antibodies is 1 μl-10 μl; The cell number concentration in the single cell suspension is 1×10 5 / ml~1×10 6 Pieces / ml.

8. Use of the antibody combination according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 7 for assisting the detection of NK large granular lymphocytes for non-diagnostic purposes.

9. A method for detecting NK large granular lymphocytes for non-diagnostic purposes, characterized in that: The method comprises the following steps: Prepare single cell suspension of samples; Dividing the single cell suspension of the sample to be tested into three portions, and adding the first group of antibodies, the second group of antibodies, and the third group of antibodies described in any one of claims 1 to 3 respectively for incubation; After the incubation, the sample cells to be tested are subjected to flow cytometry detection.

10. The method according to claim 9, characterized in that After the sample cells are tested by flow cytometry, the following steps are also included: The data obtained after flow cytometry detection is analyzed to determine whether the antibody expression pattern of the sample cells to be tested falls into the expression pattern template of the antibodies of the normal control population; wherein the antibody expression pattern of the sample cells to be tested and the expression pattern of the antibodies of the normal control population are established by the following steps: The data of flow cytometry detection results are used to group the nucleated cells by setting gates, circle the target cell population, and then analyze the expression of each fluorescent antibody pair in the target cell population.

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