CN118671340B - Antibody combination, kit and application thereof for detecting NK large granular lymphocytes - Google Patents
Antibody combination, kit and application thereof for detecting NK large granular lymphocytes Download PDFInfo
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Abstract
The invention discloses an antibody combination for detecting NK large granule lymphocytes, which comprises a first group of antibodies, a second group of antibodies and a third group of antibodies, wherein the first group of antibodies comprises antibodies CD158a, CD159C (NKG 2C), CD3, CD56, CD159a (NKG 2A), CD158B, CD7, CD5, CD158e and CD45, the second group of antibodies comprises antibodies CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45, the third group of antibodies comprises antibodies Granzyme B, performin, CD3, CD56, ki67, CD2 and CD45, and each antibody is a monoclonal antibody marked with a detection marker. The antibody combination can rapidly, simply and conveniently detect NK large particle lymphocytes with high accuracy, high sensitivity and high specificity.
Description
Technical Field
The invention belongs to the technical field of immunological detection, and particularly relates to an antibody combination for detecting NK large-particle lymphocytes, a kit and application thereof.
Background
NK large granule lymphocytic leukemia (NK-LGLL) is a clonal NK lymphoproliferative disease, most of patients are middle-aged and elderly (middle-aged 60 years), and an important diagnostic index is continuous increase of peripheral blood NK cell unknown reasons (usually >2 x 10 9/L) for more than 6 months, and the proportion of common mature granule lymphomas in peripheral blood forms is increased. This disease often accumulates peripheral blood, bone marrow, liver, spleen, etc., and can lead to symptoms associated with cytopenia. In some elderly, especially in patients with granulocytopenia, NK large granule lymphocytic leukemia (NK-LGLL) screening can be carried out, so that cloned NK large granule lymphocytic cells can be recognized early, and the early recognition and diagnosis of the disease can be carried out better in clinic.
NK large granule lymphocytic leukemia (NK-LGLL) needs to be distinguished from reactive NK cell increase, which is often secondary to viral infection, autoimmune disease, malignant tumor, etc., and NK cells can be reduced by themselves after the predisposition is released. However, in both cases, the absolute value of lymphocytes can be increased in blood routine, and the proportion of granular lymphocytes in peripheral blood cell morphology is increased, which is difficult to distinguish. The large granular lymphocytic leukemia (NK-LGLL) mentioned in WHO hematopoietic and lymphoid tissue tumors shows unique features by flow cytometry immunophenotyping. Surface CD3 negative, CD16 positive, weak CD56 expression, the possible presence of reduced or lost expression of CD2, CD7 and CD57, and abnormal uniform expression of CD 8. In NK-LGLL, the expression of the killer cell immunoglobulin-like receptor (KIR) family of NK cell receptors is aberrant, limited KIR isoform expression or complete lack of detectable KIR can be seen. Thus, the expression condition of KIR can be detected by flow cytometry to carry out precise identification.
Currently, the KIR indexes for detecting NK cells comprise indexes such as CD158a/CD158b/CD158e and the like, and the indexes are detected by combining with NK surface marks such as CD56 and CD16, but because a detection scheme of a system does not exist in the market, different detection mechanisms can have different antibody combinations, or the situation that the result is misjudged because the antibody combination is incomplete and the system is not caused.
Therefore, a systematic antibody combination and detection kit for detecting NK large-particle lymphocytes are required in the market, and standard detection schemes, operation flows and analysis ideas are provided for ensuring the accuracy and comparability of results, and facilitating the accurate diagnosis of clinical NK large-particle lymphoblastic leukemia (NK-LGLL).
Disclosure of Invention
The invention aims to provide an antibody combination capable of rapidly detecting and accurately detecting NK large-particle lymphocytes.
The technical scheme for achieving the aim comprises the following steps.
The first aspect of the invention provides an antibody combination for detecting NK large particle lymphocytes, comprising a first group of antibodies, a second group of antibodies and a third group of antibodies;
The first set of antibodies comprises CD158a, CD159C (NKG 2C), CD3, CD56, CD159a (NKG 2A), CD158b, CD7, CD5, CD158e, and CD45 antibodies;
the second set of antibodies includes 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, and each of the antibodies is a monoclonal antibody marked with a detection marker.
In some of these embodiments, the CD158a, CD94 and Granzyme B antibodies label the same fluorescein;
The CD159C (NKG 2C), CD161 and Perforin antibodies label the same fluorescein;
the CD3 and CD16 antibodies label the same fluorescein;
The CD56 antibody labels a fluorescein;
The CD159a (NKG 2A), CD45RO and Ki67 antibodies label the same fluorescein;
The CD158b and CD3 antibodies label the same fluorescein;
The CD7 and CD8 antibodies label the same fluorescein;
The CD5, CD45RA and CD2 antibodies label the same fluorescein;
The CD158e and CD57 antibodies label the same fluorescein;
The CD45 antibody labels a fluorescein;
the fluorescein is selected from FITC, PE, ECD, PE-Cy TM 5.5.5, PE-Cy7, APC-700, APC-750, PB, KO.
In some of these embodiments, the CD158a, CD94 and Granzyme B antibodies label fluorescein FITC, the CD159C (NKG 2C), CD161 and performin antibodies label fluorescein PE, the CD3 and CD16 antibodies label fluorescein ECD, the CD56 antibodies label fluorescein PE-Cy TM 5.5.5, the CD159a (NKG 2A), CD45RO and Ki67 antibodies label fluorescein PE-Cy7, the CD158B and CD3 antibodies label fluorescein APC, the CD7 and CD8 antibodies label fluorescein APC-700, the CD5, CD45RA and CD2 antibodies label fluorescein APC-750, the CD158e and CD57 antibodies label fluorescein PB, the CD45 antibodies label fluorescein KO;
Wherein, the CD3 antibody-labeled fluorescein ECD has the product number of A07748, the CD56 antibody-labeled fluorescein PE-Cy TM 5.5.5 has the product number of B49189, the CD7 antibody-labeled fluorescein APC700 has the product number of B36290, the CD5 antibody-labeled fluorescein APC750 has the product number of B92413, the CD45 antibody-labeled fluorescein KO has the product number of B36294, the CD16 antibody-labeled fluorescein ECD has the product number of B49216, the CD45 RO antibody-labeled fluorescein PECY7 has the product number of B13648, the CD3 antibody-labeled fluorescein APC has the product number of IM2467, the CD45 RA antibody-labeled fluorescein APC750 has the product number of B49194, the product number of GranzymeB antibody-labeled fluorescein FITC has the product number of B46038, the CD2 antibody-labeled fluorescein 750 has the product number of B01681, and the manufacturer BeckmanCoulter;
The CD158a antibody labeled fluorescein FITC accession number 339504, the CD159C (NKG 2C) antibody labeled fluorescein PE accession number 375004, the CD159a (NKG 2C) antibody labeled fluorescein PECY accession number 375114, the CD158b antibody labeled fluorescein APC accession number 312612, the CD158e antibody labeled fluorescein PB accession number 312714, the CD94 antibody labeled fluorescein FITC accession number 305504, the CD161 antibody labeled fluorescein PE accession number 339904, the CD57 antibody labeled fluorescein PB accession number 393326, manufacturer biolegend;
the product number of the Ki67 antibody labeled fluorescein APC is 561283, and the manufacturer BD;
the Perforin antibody labeled fluorescein PE is provided with a product number of 1P-765-T100, manufacturer Exbio.
In a second aspect, the invention provides a kit for detecting NK large particle lymphocytes, comprising the combination of antibodies for detecting NK large particle lymphocytes as described above.
In some of these embodiments, the kit further comprises reagents for flow cytometry detection.
In some embodiments, the flow cytometry is performed by dividing a single cell suspension of a sample to be tested into three parts, and incubating the three parts with a first group of antibodies, a second group of antibodies, and a third group of antibodies.
In some embodiments, the volume ratio of the single cell suspension to the first set of antibodies is 100. Mu.l to 36. Mu.l, wherein each antibody in the first set of antibodies is used in an amount of 1.5. Mu.l to 5. Mu.l, the concentration of cells in the single cell suspension is 1X 10 5 cells/ml to 1X 10 6 cells/ml, and/or,
The volume ratio of the single cell suspension to the second group of antibodies is 100 mu l to 33 mu l, wherein the dosage of each antibody in the second group of antibodies is 1 mu l to 5 mu l, and/or,
The volume ratio of the single cell suspension to the third group of antibodies is 100 mu l to 32 mu l, wherein the dosage of each antibody in the third group of antibodies is 1 mu l to 10 mu l;
The cell number concentration in the single cell suspension is 1X 10 5/ml to 1X 10 6/ml.
In a third aspect, the invention provides the use of an antibody combination as described above or a kit as described above for the assisted detection of NK large particle lymphocytes.
In a fourth aspect, the present invention provides a method for detecting NK large particle lymphocytes, said method comprising the steps of:
Preparing a sample single cell suspension;
dividing single cell suspension of a sample to be detected into three parts, and respectively adding the first group of antibodies, the second group of antibodies and the third group of antibodies for incubation;
And performing flow cytometry detection on the cells of the sample to be detected after incubation.
In some embodiments, after performing flow cytometry on the sample cells to be tested, the method further comprises:
analyzing the data obtained after the flow cytometry detection, and judging whether the antibody expression pattern of the cells of the sample to be detected falls into an expression pattern template of the antibodies of the normal control population, wherein the antibody expression pattern of the cells of the sample to be detected and the expression pattern of the antibodies of the normal control population are established through the following steps:
Grouping nucleated cells by gating, circling a target cell group, and analyzing the expression of each fluorescent antibody pair in the target cell group, wherein the fluorescent antibody pair preferably comprises ：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.
The technical scheme provided by the invention has the following advantages and effects:
In the invention, through a great deal of research and experiments, the inventor screens out three groups of cell groups which take a CD3-CD56+ region as a main purpose, covers a plurality of NK large-particle lymphocyte-recognizing antibody combinations such as KIR series (CD158a、CD158b、CD158e)、CD159a(NKG2A)、CD159c(NKG2C)、CD94、CD161、Granzyme B、Perforin、CD45RA、CD45RO、Ki67 and the like, and can rapidly, simply, conveniently, high-accuracy, high-sensitivity and high-specificity detect NK large-particle lymphocytes by adopting the antibody combinations.
Drawings
FIG. 1 is a plot of immunophenotype CD45-SSC expression using a first set of antibodies for detecting normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 2 is a plot of immunophenotype CD3-CD56 expression using a first set of antibodies to detect normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 3 shows the immunophenotype CD3-CD7 expression of normal NK lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 4 shows the immunophenotype CD3-CD5 expression of normal NK lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 5 shows the detection of immunophenotype CD56-CD7 expression of normal NK lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 6 shows the immunophenotype CD56-FS expression of normal NK lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 7 shows the immunophenotype CD56-CD158a expression of normal NK lymphocytes to be tested using the first set of antibodies in example 3 of the present invention.
FIG. 8 shows the use of a first set of antibodies for detecting immunophenotype CD56-CD158b expression of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 9 is a plot of immunophenotype CD56-CD158e expression using a first set of antibodies to detect normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 10 is a plot of immunophenotype CD56-CD159a expression using a first set of antibodies to test normal NK lymphocytes in example 3 of the present invention.
FIG. 11 is a plot of immunophenotype CD56-CD159c expression using a first set of antibodies to detect normal NK lymphocytes tested in example 3 of the present invention.
FIG. 12 is a scatter plot of KIR negative NK cells logically gated by CD158a-CD158e, CD158b-CD158e using a first panel of antibodies for detecting the immunophenotype of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 13 shows the use of a second set of antibodies for detecting immunophenotype CD3-CD56 expression of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 14 shows the use of a second set of antibodies for detecting immunophenotype CD3-CD8 expression of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 15 shows the use of a second set of antibodies for detecting immunophenotype CD56-CD16 expression of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 16 shows the use of a second set of antibodies for detecting immunophenotype CD56-CD94 expression of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 17 shows the use of a second set of antibodies for detecting immunophenotype CD56-CD161 expression of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 18 shows the immunophenotype CD56-CD45RO expression of normal NK lymphocytes to be tested using the second set of antibodies in example 3 of the present invention.
FIG. 19 is a plot of immunophenotype CD56-CD45RA expression using a second set of antibodies to detect normal NK lymphocytes tested in example 3 of the present invention.
FIG. 20 is a scatter plot of immunophenotype CD56-CD57 expression of normal NK lymphocytes tested for the detection of the second set of antibodies in example 3 of the present invention.
FIG. 21 is a scatter plot of immunophenotype CD3-CD56 expression of normal NK lymphocytes to be tested using the third set of antibodies in example 3 of the present invention.
FIG. 22 is a plot of immunophenotype CD3-CD2 expression using a third set of antibodies to detect normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 23 is a plot of immunophenotype CD56-Granzyme B expression using a third set of antibodies to detect normal NK lymphocytes tested in example 3 of the present invention.
FIG. 24 is a plot of immunophenotype CD56-Perforin expression using a third set of antibodies for detection of normal NK lymphocytes to be tested in example 3 of the present invention.
FIG. 25 is a plot of immunophenotype CD56-Ki67 expression using a third set of antibodies to test normal NK lymphocytes in example 3 of the present invention.
FIG. 26 is a plot of immunophenotype CD45-SSC expression using a first set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 27 is a plot of immunophenotype CD3-CD56 expression using a first set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 28 shows the immunophenotype CD3-CD7 expression of NK lymphocytes for the test of the abnormality using the first group of antibodies in example 3 of the present invention.
FIG. 29 shows the immunophenotype CD3-CD5 expression of NK lymphocytes to be tested for the first group of antibodies in example 3 of the present invention.
FIG. 30 shows the immunophenotype CD56-CD7 expression of NK lymphocytes to be tested for the first group of antibodies in example 3 of the present invention.
FIG. 31 shows the immunophenotype CD56-FS expression of NK lymphocytes to be tested for using the first set of antibodies in example 3 of the present invention.
FIG. 32 shows the immunophenotype CD56-CD158a expression of NK lymphocytes to be tested for the first group of antibodies in example 3 of the present invention.
FIG. 33 shows the immunophenotype CD56-CD158b expression of NK lymphocytes to be tested for the first group of antibodies in example 3 of the present invention.
FIG. 34 is a plot of immunophenotype CD56-CD158e expression using a first set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 35 is a plot of immunophenotype CD56-CD159a expression using a first set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 36 is a plot of immunophenotype CD56-CD159c expression using a first set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 37 is a scatter plot of KIR negative NK cells logically gated by CD158a-CD158e, CD158b-CD158e using a first panel of antibodies for detection of aberrant NK lymphocytes to be tested in example 3 of the present invention.
FIG. 38 is a plot of immunophenotype CD3-CD56 expression using a second set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 39 is a diagram showing the use of a second set of antibodies to detect immunophenotype CD3-CD8 expression of NK lymphocytes to be tested in example 3 of the present invention.
FIG. 40 shows the immunophenotype CD56-CD16 expression of NK lymphocytes to be tested for the second group of antibodies in example 3 of the present invention.
FIG. 41 is a diagram showing the use of a second set of antibodies to detect immunophenotype CD56-CD94 expression of NK lymphocytes to be tested in example 3 of the present invention.
FIG. 42 shows the immunophenotype CD56-CD161 expression of NK lymphocytes to be tested using the second set of antibodies in example 3 of the present invention.
FIG. 43 is a diagram showing the detection of immunophenotype CD56-CD45RO expression of NK lymphocytes to be tested using the second set of antibodies in example 3 of the present invention.
FIG. 44 is a plot of immunophenotype CD56-CD45RA expression using a second set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 45 is a scatter plot of immunophenotype CD56-CD57 expression of NK lymphocytes to be tested for the second group of antibodies in example 3 of the present invention.
FIG. 46 is a plot of immunophenotype CD3-CD56 expression using a third set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 47 is a plot of immunophenotype CD3-CD2 expression using a third set of antibodies to detect aberrant NK-lymphocytes to be tested in example 3 of the present invention.
FIG. 48 is a plot of immunophenotype CD56-Granzyme B expression using a third set of antibodies to detect aberrant NK lymphocytes to be tested in example 3 of the present invention.
FIG. 49 is a plot of immunophenotype CD56-Perforin expression using a third set of antibodies to detect aberrant NK lymphocytes to be tested in example 3 of the present invention.
FIG. 50 is a plot of immunophenotype CD56-Ki67 expression scattergrams of NK lymphocytes to be tested for the third group of antibodies in example 3 of the present invention.
FIG. 51 is a plot of immunophenotype CD45-SSC expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 52 is a plot of immunophenotype CD3-CD56 expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 53 is a plot of immunophenotype CD3-CD7 expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 54 is a scatter plot of immunophenotype CD56-CD7 expression of NK lymphocytes to be tested for the use of the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 55 is a plot of immunophenotype CD56-FS expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 56 is a scatter plot of immunophenotype CD56-CD158a expression of NK lymphocytes to be tested for the use of the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 57 is a scatter plot of immunophenotype CD56-CD158b expression of NK lymphocytes tested for the detection of NCCN using the combination of antibodies recommended in example 4 of the present invention.
FIG. 58 is a scatter plot of immunophenotype CD56-CD158e expression of NK lymphocytes to be tested for the use of the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 59 is a scatter plot of NK cells negative for KIR using the combination of antibodies proposed in NCCN for detection of immunophenotype CD158a-CD158e, CD158b-CD158e logic gate of NK lymphocytes tested in example 4 of the present invention.
FIG. 60 is a plot of immunophenotype CD56-CD16 expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 61 is a plot of immunophenotype CD56-CD94 expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 62 is a scatter plot of immunophenotype CD56-CD161 expression of NK lymphocytes to be tested for the use of the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 63 is a scatter plot of immunophenotype CD56-CD57 expression of NK lymphocytes tested for the detection of NCCN using the combination of antibodies proposed in example 4 of the present invention.
FIG. 64 is a scatter plot of immunophenotype CD3-CD2 expression of NK lymphocytes to be tested for the use of the combination of antibodies recommended in NCCN in example 4 of the present invention.
FIG. 65 is a scatter plot of immunophenotype CD56-Granzyme B expression of NK lymphocytes tested for the detection of NCCN using the combination of antibodies recommended in example 4 of the present invention.
FIG. 66 is a plot of immunophenotype CD56-Perforin expression using the combination of antibodies recommended in NCCN for detection of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 67 is a plot of immunophenotype CD45-SS expression scattergrams of NK lymphocytes to be tested for the detection of the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 68 is a plot of immunophenotype CD3-CD56 expression of NK lymphocytes to be tested for the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 69 is a plot of immunophenotype CD3-CD7 expression of NK lymphocytes to be tested for the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 70 is a plot of immunophenotype CD56-CD7 expression scattergrams of NK lymphocytes to be tested for the detection of the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 71 is a plot of immunophenotype CD56-FS expression scatter of NK lymphocytes to be tested for the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 72 is a plot of immunophenotype CD5-CD7 expression of NK lymphocytes to be tested for the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 73 is a plot of immunophenotype CD56-CD158a expression scattergrams (in CD56+CD5+NK) of NK lymphocytes to be tested for using the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 74 is a plot of immunophenotype CD56-CD158b expression scattergrams (in CD56+CD5+NK) of NK lymphocytes to be tested for using the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 75 is a plot of immunophenotype CD56-CD158e expression scatter (in CD56+CD5+NK) of NK lymphocytes to be tested using the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 76 is a plot of immunophenotype CD56-CD159a expression scatter (in CD56+CD5+NK) of NK lymphocytes to be tested for using the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 77 is a plot of immunophenotype CD56-CD159c expression scatter (in CD56+CD5+NK) of NK lymphocytes to be tested for using the first set of antibodies of the present invention in example 4 of the present invention.
FIG. 78 is a scatter plot of NK cells negative for KIR using the second set of antibodies of the present invention for the detection of immunophenotype CD158a-CD158e, CD158b-CD158e logic gate of NK lymphocytes to be tested in example 4 of the present invention.
FIG. 79 is a plot of immunophenotype CD3-CD8 expression of NK lymphocytes to be tested for the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 80 is a plot of immunophenotype CD56-CD16 expression of NK lymphocytes to be tested for the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 81 is a plot of immunophenotype CD56-CD8 expression of NK lymphocytes to be tested for the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 82 is a plot of immunophenotype CD56-CD94 expression scatter (in CD56+CD8+NK) of NK lymphocytes to be tested for using the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 83 is a plot of immunophenotype CD56-CD161 expression scatter (in CD56+CD8+NK) of NK lymphocytes to be tested using the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 84 is a plot of immunophenotype CD56-CD45RO expression scattergrams (in CD56+CD8+NK) of NK lymphocytes to be tested for using the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 85 is a plot of immunophenotype CD56-CD45RA expression scattergrams (in CD56+CD8+NK) of NK lymphocytes to be tested for using the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 86 is a plot of immunophenotype CD56-CD57 expression scattergrams (in CD56+CD8+NK) of NK lymphocytes to be tested for using the second set of antibodies of the present invention in example 4 of the present invention.
FIG. 87 is a plot of immunophenotype CD3-CD2 expression of NK lymphocytes to be tested for the third group of antibodies of the present invention in example 4 of the present invention.
FIG. 88 is a plot of immunophenotype CD56-Granzyme B expression using the third set of antibodies of the present invention to detect NK lymphocytes to be tested in example 4 of the present invention.
FIG. 89 is a plot of immunophenotype CD56-Perforin expression using the third set of antibodies of the present invention to detect NK lymphocytes to be tested in example 4 of the present invention.
FIG. 90 is a plot of immunophenotype CD56-Ki67 expression scattergrams of NK lymphocytes to be tested for the detection of the third set of antibodies of the present invention in example 4 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: ALaboratory Manual, 2013), or by the manufacturer's recommendations. The various reagents commonly used in the examples are all commercially available products.
In the invention, aiming at the defects of the prior art, an antibody combination, a kit and a system for rapidly detecting NK large-particle lymphocytes are provided. The antibody combination comprises three groups of antibodies, uses a CD3-CD56+ region as a main target cell group, covers a plurality of antibodies recognizing NK large-particle lymphocytes such as KIR series (CD158a、CD158b、CD158e)、CD159a(NKG2A)、CD159c(NKG2C)、CD94、CD161、Granzyme B、Perforin、CD45RA、CD45RO、Ki67 and the like, and can better recognize neoplastic NK large-particle lymphocytes by comprehensive logic analysis strategies. The invention reasonably applies various antibody indexes, has wide coverage range, and can rapidly and simply (only 2-3 hours are needed for receiving the detection result from a sample) detect NK large-particle lymphocytes with high sensitivity by adopting the antibody combination through a multiparameter flow detection technology. In addition, the invention optimizes the template of result analysis, and only uses a single loop gate (CD 3-CD56+) to loop out NK lymphocytes before optimization to see whether KIR series (CD 158a, CD158b and CD158 e) are expressed or not, but because neoplastic NK cells CD56 can be lost sometimes, (CD 3-CD56+) loop gate inaccuracy leads to deviation of analysis results, the method can be used for combining CD3-CD56+ and CD3-CD5+ to circle suspicious NK cells, judging whether the NK lymphocytes have clonality or not by looking at CD56-CD158b,CD56-CD158e,CD56-CD159a,CD56-CD159c,CD56-CD94,CD56-CD161,CD56-CD45RO,CD56-CD45RA,CD56-CD57,CD3-CD2,CD56-Granzyme B,CD56-Perforin and the like, and judging the proliferation index of the neoplastic NK cells by looking at CD56-Ki67, if the expression of Ki67 is higher, the possibility of invasive NK lymphocyte leukemia is eliminated due to higher mortality, and clinical prompt is required. In addition, the invention fixes the analysis template, can directly analyze the dragged data, has simple operation, reduces the requirement of the expertise level of the analyst, and makes up the limitation of the existing detection technology.
In some embodiments, an antibody combination for detecting NK large granule lymphocytes is contemplated comprising:
1. The first group of antibodies, including antibodies CD158a, CD159C (NKG 2C), CD3, CD56, CD159a (NKG 2A), CD158b, CD7, CD5, CD158e and CD45, recognize suspected NK cells (CD 3-CD56+), by using the framework antibodies CD3, CD56, in addition to CD45 as a leukocyte gating antibody, and loop out NK lymphocytes expressed by CD3-CD56+, and then look at the presence or absence of CD3-CD5+CD7+ abnormal cells from CD3-CD5 and CD56-CD7, can be used to preliminarily suspect abnormal NK lymphocytes, and then bind to indicators such as CD158a, CD159C (NKG 2C), CD159a (NKG 2A), CD158b, CD158e and the like to confirm the clonality of the group of NK lymphocytes. The first group of antibodies increased CD159C (NKG 2C), CD159a (NKG 2A), which complements the cloning of KIR cell negative NK.
2. A second set of antibodies, including CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57, and CD45 antibodies. NK large granular lymphocytic leukemia was determined by using CD3-CD56+ circle to give NK lymphocytes, and then looking at whether CD94, CD161, CD16 and CD57 were expressed, and then combining CD45RA homogeneous expression, CD45RO not expression, and the first tube combination antibody expression.
3. A third group of antibodies, including Granzyme B, perforin, CD3, CD56, ki67, CD2 and CD45 antibodies. The CD3-CD56+ ring is used for marking NK lymphocyte, and then whether Granzyme B and Perforin are expressed is checked to determine whether the NK lymphocyte is cytotoxic, in addition, the CD56-Ki67 can be combined to judge the proliferation index of the neoplastic NK cell, if the Ki67 is expressed more, the possibility of invasive NK lymphocyte leukemia is eliminated, and if the Ki67 is expressed less, the NK large granular lymphocyte leukemia is indicated.
In some embodiments, each of the antibodies is a monoclonal antibody labeled with a detection label.
In some embodiments, the monoclonal antibody is a fluorescein-labeled antibody, so that the indicators can be detected simultaneously in a ten-color or more flow cytometry, and the method has certain convenience, wherein the fluorescein is selected from FITC, PE, ECD, PE-Cy TM 5.5.5, PE-Cy7, APC-700, APC-750, PB and KO.
In some embodiments, the CD158a, CD94, and Granzyme B antibodies label fluorescein FITC, the CD159C (NKG 2C), CD161, and Perforin antibodies label fluorescein PE, the CD3 and CD16 antibodies label fluorescein ECD, the CD56 antibodies label fluorescein PE-Cy TM 5.5.5, the CD159a (NKG 2A), CD45RO, and Ki67 antibodies label fluorescein PE-Cy7, the CD158B and CD3 antibodies label fluorescein APC, the CD7 and CD8 antibodies label fluorescein APC-700, the CD5, CD45RA, and CD2 antibodies label fluorescein APC-750, the CD158e and CD57 antibodies label fluorescein PB, and the CD45 antibodies label fluorescein KO.
The embodiment of the invention also provides application of the antibody combination in auxiliary detection of NK large granular lymphocytes.
The embodiment of the invention also provides a kit for detecting NK large granule lymphocytes, which comprises the antibody combination for detecting NK large granule lymphocytes.
The embodiment of the invention also provides application of the kit for detecting NK cell large granule lymphocytes in auxiliary detection of NK cell large granule lymphocytes.
In particular, in applications that aid in the detection of NK cell large granule lymphocytes, the detection is specifically performed by employing flow cytometry.
The embodiment of the invention also provides a method for detecting NK large granular lymphocytes, which comprises the following steps:
Preparing a sample single cell suspension;
dividing single cell suspension of a sample to be detected into three parts, and respectively adding the first group of antibodies, the second group of antibodies and the third group of antibodies for incubation;
And performing flow cytometry detection on the cells of the sample to be detected after incubation.
The embodiment of the invention also provides a system for detecting NK large granular lymphocytes, which comprises:
the detection module is used for carrying out flow cytometry detection on cells to be detected and comprises a single cell suspension module, an incubation module, a resuspension module, a measurement module and a detection module, wherein the single cell suspension module is used for preparing single cell suspension;
the data acquisition module is used for acquiring data of a flow cytometry detection result of the antibody combination-stained cells to be detected;
The data analysis module is used for analyzing the acquired data and judging whether the acquired NK lymphocytes are neoplastic NK large-particle lymphocytes according to preset analysis logic and judgment standards, wherein the preset judgment standards comprise a normal control crowd antibody expression pattern template and a test cell antibody expression pattern, the preset judgment standards are that the test cell is judged to be an immunophenotype normal NK lymphocyte group if the test cell antibody expression pattern falls into the normal control crowd antibody expression pattern template, and the test cell is judged to be a suspected tumor cell group if the test cell antibody expression pattern does not fall into the normal control crowd antibody expression pattern template.
The technical process of the system for detecting NK large-particle lymphocytes in the invention is just to refer to the conventional technology of flow cytometry, and equipment and consumable materials for flow cytometry analysis are selected from the equipment and consumable materials such as a flow special pipe, an oscillator, a liquid transfer device and the like.
In some embodiments, the normal control population antibody expression pattern template is established by obtaining flow cytometry detection result data of a normal control population cell population, grouping nucleated cells by gating, circling a target cell population (namely NK lymphocytes), and analyzing the expression condition of each fluorescent antibody in the target cell population to obtain the normal control population antibody expression pattern template.
In some embodiments, the test cell antibody expression pattern is established by obtaining flow cytometry detection result data of the test cell, grouping nucleated cells according to the mode in the normal control crowd antibody expression pattern, circling a target cell group (namely NK lymphocyte), and analyzing each fluorescent antibody expression condition in the target cell group according to the mode in the normal control crowd antibody expression pattern to obtain the test cell antibody expression pattern.
In some embodiments, the gating uses CD45-SSC (side scatter light) gating, and divides the cell population into granulocyte population, monocyte population, lymphocyte population (CD 45 is leukocyte common antigen, so that CD45 is used as gating antibody, the CD45 of normal blood cell is expressed with the intensity of lymphocyte > monocyte > granulocyte, SSC is used as side scatter light, the normal expression intensity of side scatter light is related to the granularity of the cell, granulocyte > monocyte > lymphocyte, the combined expression characteristics of CD45 and SSC can be used for dividing the lymphocyte, monocyte and granulocyte into the partition, wherein the lymphocyte is positioned at the strongest and the lowest position of CD45, the monocyte is slightly weaker than lymphocyte and SSC is slightly higher than lymphocyte, and the signal of granulocyte is the weakest than monocyte, so that the cell population is divided into granulocyte, monocyte and lymphocyte according to the expression pattern, the cell population is further analyzed for the purpose of lymphocyte, the antibody corresponding to the fluorescence expression intensity in the purpose cell is analyzed, the antibody pair comprises ：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.
Specific sources of some of the antibodies used in the examples below are CD3ECD (accession number A07748), CD56PE-Cy TM 5.5 (accession number B49189), CD7 APC700 (accession number B36290), CD5 APC750 (accession number B92413), CD45KO (accession number B36294), CD16ECD (accession number B49216), CD45 RO PECY7 (accession number B13648), CD3 APC (accession number IM 2467), CD45 RA APC750 (accession number B49194), granzymeB FITC (accession number B46038), CD2APC750 (accession number B01681), manufacturer BeckmanCoulter;
CD158a FITC (cat No. 339504), CD159C (NKG 2C) PE (cat No. 375004), CD159a (NKG 2C) PECY (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;
ki67APC (cat 561283), manufacturer BD;
perforin PE (cat. No. 1P-765-T100), vendor Exbio.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 antibody combinations for detection of NK macrogranulosa lymphocytes
The antibody combination for detecting NK large particle lymphocytes of the present embodiment comprises a first group of antibodies, a second group of antibodies and a third group of antibodies, wherein,
The first set of antibodies includes CD158a, CD159C (NKG 2C), CD3, CD56, CD159a (NKG 2A), CD158b, CD7, CD5, CD158e, and CD45 antibodies;
the second set of antibodies includes CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57 and CD45 antibodies;
The third set of antibodies includes Granzyme B, perforin, CD3, CD56, ki67, CD2 and CD45 antibodies.
The fluorescent labeling and the amount of the related monoclonal antibody in the above antibodies are shown in Table 1.
TABLE 1
Note that the commercial antibodies were subjected to concentration gradient verification to determine the optimal amounts, and the amounts of monoclonal antibodies were taken and placed in flow-type tubes numbered 1, 2, and 3, respectively.
Example 2 System and method for detecting NK macrogranulosa lymphocytes
The embodiment provides a system for rapidly detecting NK large-particle lymphocytes, which comprises a detection module, a data acquisition module and a data analysis module. The detection module performs flow cytometry detection on the to-be-detected cells, the data acquisition module acquires flow cytometry detection result data of the to-be-detected cells dyed with the detection reagent composition described in embodiment 1, and the data analysis module analyzes the acquired data and judges whether the to-be-detected cells are neoplastic NK lymphocytes according to a preset judgment standard.
By adopting the system of the embodiment, the specific workflow for detecting NK large-particle lymphocytes is as follows:
1. Each antibody in the antibody combination of example 1 was formulated as shown in table 1.
2. Sample processing.
And (3) regulating the concentration of the sample to be tested to 1X 10 6/ml according to the cell number to prepare single-cell suspension.
3. And (5) detecting a sample.
(1) The first group of antibodies, the second group of antibodies and the third group of antibodies (cell membrane antibody CD3/CD56/CD2/CD45 are firstly added) in the embodiment 1 are respectively added into the flow tube and marked 1,2 and 3, the adding amount is respectively 35.5 mu l, 31 mu l and 11 mu l, then 100 mu l of the suspension in the step 2 are respectively added, vortex vibration and mixing are carried out, and the incubation is carried out for 15min at room temperature and in a dark place.
(2) 400 Μl Bc hemolysin is added to each of the incubated flow tubes 1,2, and 3, vortex shaking is performed, and the mixture is left to stand until hemolysis is clear. After the hemolysis is transparent, the flow tubes 1 and 2 are centrifuged at 1500r/min for 5min, the supernatant is discarded, the 3 rd tube is used for detecting the intracellular antibodies according to the following steps, 2ml calf serum is added into the tubes 1 and 2, vortex vibration is carried out, the centrifugation is carried out at 1500r/min for 5min, the supernatant is discarded, and 400 mu l 1% paraformaldehyde is added for resuspension.
(3) And (3) after the cell surface markers are dyed according to the detection of the detection method (1) and the detection method (2), adding 450uL of a 1X FACS membrane breaker, uniformly mixing, incubating for 5min in a dark place, adding PBS, washing once, discarding supernatant, adding anti-intracellular fluorescent antibodies (Granzyme B, perforin and Ki 67) according to the antibody amount, incubating for 30min at a dark place at room temperature, adding 2ml of calf serum, vortex shaking, centrifuging for 5min at 1500r/min, discarding supernatant, and adding 400 mu l of 1% paraformaldehyde for resuspension.
(4) The flow tubes 1, 2, 3 were examined by BeckmanCoulter Navios ten-color flow cytometer and analyzed for immunophenotype.
4. And (5) data analysis.
(1) Building a healthy human antibody expression pattern template
According to the flow cytometry detection result data of 40 normal control population cell groups, dividing the cell groups by gating, preferably using CD45-SSC (side scatter light), gating to circle out target cell groups, analyzing the cell groups for expression of each fluorescent antibody, selecting 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.
(2) Establishing antibody expression mode of cell to be tested
Obtaining flow cytometry detection result data of the cells to be detected, setting a gate to divide the cells into groups according to the mode in the normal control crowd antibody expression mode, circling a target cell group, and analyzing the expression condition of each fluorescent antibody in the target cell group according to the mode in the normal control crowd antibody expression mode to obtain the antibody expression mode of the cells to be detected.
(3) Analysis of antibody expression patterns in test cells
If the antibody expression pattern of the cells to be detected falls into the antibody expression pattern template of the normal control population, judging the cells to be detected as NK lymphocyte populations with normal immunophenotype;
And if the antibody expression pattern of the cell to be detected does not fall into the antibody expression pattern template of the normal control population, judging the cell to be detected as NK large-particle lymphocytes.
Example 3 verification of the accuracy of the detection method of the present invention
By adopting the detection method of example 2, NK large granule lymphocytes of two samples of cells to be detected (1 sample of normal cells+1 sample of abnormal cells) are detected, and antibody expression pattern analysis is performed to verify the accuracy of the detection method of the invention.
1. Normal sample
The sample was normal for physical examination and was analyzed using peripheral blood smear morphology, no clinical samples of significantly large granular lymphocytes were seen. The antibody expression pattern analysis was performed as follows:
And obtaining flow cytometry detection result data of the sample to be detected. The cell population was divided into 3 regions, i.e., granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) 3 regions (as shown in fig. 1), respectively, using a gate for CD45-SSC (side scattered light), and the lymphocyte region was the target cell population, and it was found that the proportion of lymphocytes was 22.03% of the total number of nuclear cells, and the immunophenotype analysis was performed on this cell population as follows.
FIG. 2 is a plot of CD3-CD56 immunophenotype scattergrams of lymphocytes, from which it can be seen that the NK lymphocytes of CD3-CD56+ account for 14.80% of lymphocytes, in normal proportion.
FIG. 3 is a scatter plot of CD3-CD7 expression of lymphocyte populations, showing that CD3-CD7+ NK lymphocytes account for 14.58% of lymphocytes, substantially equal to the proportion of NK cells shown in FIG. 2, without obvious abnormal NK cells of CD 7-.
FIG. 4 is a scatter plot of CD3-CD5 expression of lymphocyte populations, focusing on the presence or absence of CD3-CD5+ NK lymphocytes, where CD3-CD5+ free NK lymphocytes are shown.
FIG. 5-FIG. 12 are graphs showing the expression of CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, CD56-CD159c, respectively, of NK lymphocyte populations, and the normal expression of NK lymphocyte CD7 of the CD3-CD56+ population can be checked, and the cloning index (CD 158a, CD158b, CD158, CD159a, CD159 c) of the CD3-CD56+ population (the normal NK lymphocytes are small or partially expressed, and the judgment standard of the cloning NK lymphocytes is that CD159a >88% or CD158a >53% or CD158b >72% or CD158e >54% or KIR negative (CD 158a, CD158b, CD158 e) 72%). As shown, NK cells were found to be normal NK lymphocytes from the above clonality index expression cases, with a CD158a expression rate of 17.11% (< 53%) being normal, a CD158b expression rate of 39.72% (< 72%), a CD158e expression rate of 29.11% (< 54%), a CD159a expression rate of 10.15% (< 88%), and KIR negative (CD 158a, CD158b, CD158 e) cells of 40.16% (< 72%).
FIG. 13 is CD3-CD56+ in a second set of antibodies, circling NK lymphocytes.
FIGS. 14-20 are scatter plots of CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, CD56-CD57 expression of CD3-CD56+ NK lymphocytes, showing that NK cells were partially positive and CD94/CD161/CD57 expression was not abnormal, and that partial expression of both binding CD45RA and CD45RO suggests that NK cells of this example were normal NK lymphocytes.
FIG. 21 is a CD3-CD56+ in a third set of antibodies, circling NK lymphocytes.
FIGS. 22 to 25 are scatter plots of CD2, granzyme B, performin, ki67 expression of CD3-CD56+ NK lymphocytes, showing that NK cells were all expressed, ki67 was negative, and no abnormality was observed in expression, suggesting that NK cells of this example were normal NK lymphocytes.
In the sample to be tested, the cell population is judged to be the cell population with normal immunophenotype in the normal control crowd antibody expression pattern template, and the cell population is consistent with morphological analysis results and clinical symptoms.
2. Abnormal sample
The sample is a sample for determining clinical diagnosis of NK large granular lymphocytic leukemia with obvious large granular lymphocytes by adopting peripheral blood smear morphological analysis.
The antibody expression pattern analysis was performed as follows:
And obtaining flow cytometry detection result data of the sample to be detected. The cell population was divided into 3 regions based on the expression of CD45-SSC using a gate for CD45-SSC (side scattered light), and as shown in FIG. 26, granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) were each divided into 3 regions, and the lymphocyte regions were the target cell population of the present embodiment, and the proportion of lymphocytes occupying 15.12% of the total number of nuclear cells was normal, and immunophenotype analysis was performed on the cell population.
FIG. 27 is a plot of CD3-CD56 immunophenotype scattergrams of lymphocytes, from which it can be seen that the NK lymphocytes of CD3-CD56+ account for 32.93% of lymphocytes, with a significant increase in proportion.
FIG. 28 is a scatter plot of CD3-CD7 expression of lymphocyte populations, showing that CD3-CD7+ NK lymphocytes account for 32.10% of lymphocytes, substantially equal to the proportion of NK cells shown in FIG. 2, without obvious abnormal NK cells of CD 7-.
FIG. 29 is a scatter plot of CD3-CD5 expression of lymphocyte populations, from which NK lymphocytes with CD3-CD5+ are seen, suggesting an abnormal NK cell phenotype.
FIGS. 30 to 37 are scatter plots of the NK lymphocyte clone expression levels of CD56-CD7, CD56-FS, CD56-CD158a, CD56-CD158b, CD56-CD158e, CD56-CD159a, CD56-CD159c, respectively, and as shown in FIGS. 32 to 37, analysis of NK lymphocyte clonality index (CD 158a, CD158b, CD158, CD159a, CD159 c) revealed that the NK cell clone expression level was 2.56% (< 53%) and CD158b expression level was 86.46% (> 72%) for the cloned NK lymphocyte, the CD158e expression level was 1.76% (< 54%), the CD159a expression level was 12.26% (< 88%), and the KIR negative (CD 158a, CD158b, CD158 e) cell clone expression level was 5.58% (< 72%), from which was judged to be the cloned NK lymphocyte.
FIG. 38 is CD3-CD56+ in a second set of antibodies, circling NK lymphocytes.
FIGS. 39-45 are scatter plots of CD3-CD8, CD56-CD16, CD56-CD94, CD56-CD161, CD56-CD45RO, CD56-CD45RA, CD56-CD57 expression of CD3-CD56+ NK lymphocytes, showing that NK cells were positive for homogeneity as abnormal (normal NK lymphocytes were partially positive and non-aggregated), CD16 and CD161 were not abnormal, but that CD94 expression was reduced, additionally binding CD45RA homogeneity, CD45RO was not expressed, and CD57 expression was enhanced, suggesting that NK cells of this example were abnormal NK lymphocytes.
FIG. 46 is CD3-CD56+ in a third set of antibodies, circling NK lymphocytes.
FIGS. 47-50 are scatter plots of CD2, granzyme B, performance, ki67 expression of CD3-CD56+ NK lymphocytes, showing that a small portion of CD2 of NK cells was lost, granzyme B expression was not abnormal, performance expression was enhanced, ki67 was negative, suggesting that NK cells of this example were inert abnormal NK lymphocytes.
The immunophenotype indicates that the cell population of the sample to be detected is not in the antibody expression pattern template of the normal control population, is inert NK large-particle lymphocytes with tumor, and accords with morphological analysis results and clinical diagnosis.
Example 4 comparison of detection accuracy of antibody combinations of the invention with antibody combinations of prior art methods
Comparing the antibody combination of example 1 of the present invention with the fluorescein labels of the antibodies of the recommended test antibody combinations (CD 158a, CD3, CD56, CD158B, CD7, CD158e, CD94, CD161, CD16, CD57, granzyme B, performin, CD 2) in NCCN, reference table 1, the amounts also referring to table 1), the accuracy in testing the same test cells.
1. Flow cytometry detection data of the test samples were obtained using the detection antibodies recommended in NCCN (CD 158a, CD3, CD56, CD158B, CD7, CD158e, CD94, CD161, CD16, CD57, granzyme B, performin, CD 2).
The cell population was divided into 3 regions, i.e., granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) 3 regions (as shown in fig. 51), respectively, using a gate for CD45-SSC (side scattered light), and the lymphocyte region was the target cell population, and it was found that 13.68% of the total number of nuclear cells was occupied by lymphocytes in a normal proportion, and the immunophenotype analysis was performed on this cell population.
FIG. 52 is a plot of CD3-CD56 immunophenotype scattergrams of lymphocytes, from which it can be seen that CD3-CD56+ NK lymphocytes account for 13.69% of lymphocytes, in normal proportion.
FIGS. 53-66 are scatter plots of NK lymphocyte populations for the expression of CD7, CD56-FS, CD158a, CD158B, CD158e, CD16, CD94, CD161, CD57, CD2, granzyme B, and Perforin, showing that no significant abnormalities were seen in NK lymphocyte expression of CD7, CD16, CD94, CD161, CD57, CD2, granzyme B, and Perforin, and that the NK lymphocyte clonality index was analyzed to have a CD158a expression rate of 8.54% (< 53%), a CD158B expression rate of 36.25% (< 72), a CD158e expression rate of 11.17% (< 54%), KIR negative (CD 158a, CD158B, and CD158 e) cells of 46.39% (< 72%), and was considered polyclonal normal NK lymphocytes.
2. The antibody combination of the embodiment 1 of the invention is used for detecting the sample to be detected, and the flow cytometry detection result data of the sample to be detected is obtained.
Similarly, a cell population was classified into 3 regions, i.e., granulocytes (upper middle region in the figure), monocytes (upper right region in the figure), and lymphocytes (lower right region in the figure) 3 regions (as shown in fig. 67), based on the expression of CD45-SSC, by gating the cell population with CD45-SSC, and the lymphocyte region was 13.80% of the total number of nuclear cells, and the cell population was analyzed by immunophenotyping as follows.
FIG. 68 is a plot of CD3-CD56 immunophenotype scattergrams of lymphocytes, from which it can be seen that the NK lymphocytes of CD3-CD56+ account for 14.21% of lymphocytes, in normal proportion.
Fig. 69-90 show graphs of the scatter plots of the expression profiles of CD7, CD56-FS, CD5-CD7, CD158a, CD158B, CD158e, CD16, CD161, CD57, CD2, granzyme B, performin for NK lymphocyte populations, showing that there was a CD5 positive expression of 48.21% NK lymphocytes, circling the population of cd5+ NK lymphocytes, analyzing the expression of CD16, CD94, CD161, CD57, CD2, granzyme B, performin for them without significant abnormalities (no significant difference from the detection protocol recommended by NCCN described above), but with loss of CD94, CD8 homogeneity expression, CD45RO negative, CD45RA homogeneity expression all suggesting an abnormality for the population, further analyzing the clonality index CD158a expression rate of the population of cd5+ NK lymphocytes of 0.19% (< 53%), the expression rate of CD158B of 0.19% (< 72%), the expression rate of CD158e of 0.38% (< 54%), the NK 158B, CD 158% of 6% by NK cells, and the population of 6% (-6%) of the lymphocytes with a high-quality as a cell count of the group of 6% > (NK 158) and the cell population of the particles of the same were the same, as those with a large-quality as those of the group of the NK lymphocytes.
The results of this example show that the antibody combination designed by the invention uses CD3-CD56+ region as main target cell group, covers various NK large granule lymphocyte-recognizing antibody combinations such as KIR series (CD158a、CD158b、CD158e)、CD159a(NKG2A)、CD159c(NKG2C)、CD5、CD8、CD94、CD161、Granzyme B、Perforin、CD45RA、CD45RO、Ki67, and the like, and can better recognize neoplastic NK large granule lymphocyte by comprehensive logic analysis strategy, and has better accuracy than the recommended detection antibody in NCCN.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. An antibody combination for detecting NK large-particle lymphocytes, characterized in that said antibody combination consists of a first set of antibodies, a second set of antibodies and a third set of antibodies;
The first set of antibodies are CD158a, CD159C (NKG 2C), CD3, CD56, CD159a (NKG 2A), CD158b, CD7, CD5, CD158e, and CD45 antibodies;
The second set of antibodies is CD94, CD161, CD16, CD56, CD45RO, CD3, CD8, CD45RA, CD57, and CD45 antibodies;
the third group of antibodies are Granzyme B, perforin, CD3, CD56, ki67, CD2 and CD45 antibodies, and each of the antibodies is a monoclonal antibody marked with a detection marker.
2. The antibody combination of claim 1, wherein the CD158a, CD94 and Granzyme B antibodies label the same fluorescein;
The CD159C (NKG 2C), CD161 and Perforin antibodies label the same fluorescein;
The CD3 antibodies in the first and third sets of antibodies and the CD16 antibodies in the second set of antibodies label the same fluorescein;
The CD56 antibody labels a fluorescein;
The CD159a (NKG 2A), CD45RO and Ki67 antibodies label the same fluorescein;
The CD158b antibody and the CD3 antibody of the second set of antibodies label the same fluorescein;
The CD7 and CD8 antibodies label the same fluorescein;
The CD5, CD45RA and CD2 antibodies label the same fluorescein;
The CD158e and CD57 antibodies label the same fluorescein;
The CD45 antibody labels a fluorescein;
the fluorescein is selected from FITC, PE, ECD, PE-Cy TM 5.5.5, PE-Cy7, APC-700, APC-750, PB, KO.
3. The antibody combination of claim 2, wherein the CD158a, CD94 and Granzyme B antibodies label fluorescein FITC, the CD159C (NKG 2C), CD161 and performin antibodies label fluorescein PE, the CD3 antibodies in the first and third sets of antibodies and CD16 antibodies in the second set of antibodies label fluorescein ECD, the CD56 antibodies label fluorescein PE-Cy TM 5.5.5, the CD159a (NKG 2A), CD45RO and Ki67 antibodies label fluorescein PE-Cy7, the CD158B antibodies and CD3 antibodies in the second set of antibodies label fluorescein APC, the CD7 and CD8 antibodies label fluorescein APC-700, the CD5, CD45RA and CD2 antibodies label fluorescein APC-750, the CD158e and CD57 antibodies label fluorescein PB, and the CD45 antibodies label fluorescein KO.
4. A kit for detecting NK macrogranulosa lymphocytes, characterized in that it comprises the antibody combination for detecting NK macrogranulosa lymphocytes according to any one of claims 1-3.
5. The kit of claim 4, wherein, the kit also includes reagents for flow cytometry detection.
6. The kit according to claim 5, wherein 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 respectively added for incubation, and then the flow cytometry detection is carried out.
7. The kit according to claim 6, wherein the volume ratio of the single cell suspension to the first group of antibodies is 100. Mu.l to 36. Mu.l, wherein the amount of each antibody in the first group of antibodies is 1.5. Mu.l to 5. Mu.l, the cell number concentration in the single cell suspension is 1X 10 5 cells/ml to 1X 10 6 cells/ml, and/or,
The volume ratio of the single cell suspension to the second group of antibodies is 100 mu l to 33 mu l, wherein the dosage of each antibody in the second group of antibodies is 1 mu l to 5 mu l, and/or,
The volume ratio of the single cell suspension to the third group of antibodies is 100 mu l to 32 mu l, wherein the dosage of each antibody in the third group of antibodies is 1 mu l to 10 mu l;
The cell number concentration in the single cell suspension is 1X 10 5/ml to 1X 10 6/ml.
8. Use of the antibody combination of any one of claims 1-3 or the kit of any one of claims 4-7 for the assisted detection of NK large granule lymphocytes for non-diagnostic purposes.
9. A method for detecting NK large granule lymphocytes for non-diagnostic purposes, said method comprising the steps of:
Preparing a sample single cell suspension;
Dividing the single cell suspension of the sample to be tested into three parts, and respectively adding the first group of antibodies, the second group of antibodies and the third group of antibodies according to any one of claims 1-3 for incubation;
And performing flow cytometry detection on the cells of the sample to be detected after incubation.
10. The method of claim 9, further comprising, after performing the flow cytometry detection on the sample cells to be detected:
analyzing the data obtained after the flow cytometry detection, and judging whether the antibody expression pattern of the cells of the sample to be detected falls into an expression pattern template of the antibodies of the normal control population, wherein the antibody expression pattern of the cells of the sample to be detected and the expression pattern of the antibodies of the normal control population are established through the following steps:
and (3) clustering the nucleated cells by using the data of the detection result of the flow cytometry, circling the target cell population, and analyzing the expression condition of each fluorescent antibody pair in the target cell population.
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