CN117805375A - Antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping and application thereof - Google Patents
Antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of antibody medicine, in particular to an antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping and application thereof. The antibody composition provided by the invention can be used for carrying out immunophenotype analysis on samples such as bone marrow fluid, hydrothorax and ascites, peripheral blood or lymph nodes of clinical patients, directly carrying out one-step detection on clinical suspected NK cell tumors by using the antibody composition, and carrying out the second-step detection on the clinical unknown patients suspected to be NK cell tumors after primary screen detection, carrying out comprehensive immunophenotyping, clonal identification, differentiation pathway identification, stage judgment of tumor cells and screening of MRD markers and treatment targets on the NK cells.
Description
Technical Field
The invention relates to the technical field of antibody medicine, in particular to an antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping and application thereof.
Background
NK (natural killer) cells are derived from bone marrow lymphoid stem cells and are predominantly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. NK cells, unlike T, B cells, are a class of lymphocytes that can nonspecifically kill tumor cells and virus-infected cells without prior sensitization.
NK recognition is largely based on the expression of membrane surface or intracellular molecules such as CD56, CD16, CD57, CD7, CD2, CD161, CD94, granzyme a/B and perforin, not expressing the T-line specific markers CD3 and T-line associated markers CD4, CD5, tcrαβ, or tcrγδ, but low levels of CD8, CD3, CD159a, CD159c and CD158 Series (KIRs). Generally using CD3 - CD56 + And/or CD16 + NK cells were gated. And dividing NK cells into CD56 st+ CD16 - And CD56 dim+ CD16 + Class 2, the former early NK cells, mainly with cytokine secretion function; the latter are late NK cells. Mainly performs the function of killing cells.
Tumors of NK include precursor NK cell tumors and NK cell lymphomas of mature NK cell origin. The former is difficult to define so far due to the great overlap of NK cell differentiation pathways with myeloid precursor cells and T-derived cells, and the literature report has great uncertainty. The entity, which is partially diagnosed as a myeloid/NK acute leukemia, is considered to be of precursor NK cell origin, but is indistinguishable from acute myeloid leukemia with the original immunophenotypic trait, micro-differentiation (AML-M0). Blast-like plasmacytoid dendritic cell tumors (BPDCN) were once classified as NK leukemias because of CD56 expression. Early in development, NK progenitor cell expression markers overlap with T-ALL, including CD7, CD2, even CD5 and cCD3 (e-chain); thus, it is difficult to distinguish between T-ALL and precursor NK cell tumors, and it is not clear which are NK cell specific markers. In 2008 WHO classification system, NK-lymphoblastic leukemia/lymphoma was classified under the series of non-classification leukemia directory, and 2016 under lymphoblastic leukemia/lymphoma directory as temporary classification. The difficulty of such tumor diagnosis is also illustrated.
Mature NK cell lymphomas (NHL-NK) are classified according to WHO of 2022 into invasive NK cell leukemia (ANKL), NK large granule lymphoblastic leukemia (NK-LGLL), extranodal NK/T cell lymphomas (ENKTL).
The overall incidence of NK cell tumors is low, and there is a lack of systematic understanding of them. The lack of unique histopathological patterns, overlapping cellular morphological features, immunophenotyping complexity, lack of specific genetic abnormalities and diverse clinical manifestations all make NK tumor diagnosis more difficult.
The difficulty in NK cell lymphoma diagnosis is determining its clonality. Determination of clonality is a prerequisite for diagnosing tumors. The clonality of T cells can be determined by detecting tcrvβ receptor library or TRBC 1. NK cells do not possess such receptors, however, and therefore clonality is difficult to determine. The literature reports the use of KIR (CD 158) series receptors to help determine NK cell clonality. The CD158 series has a certain expression frequency in normal people, and can be abnormally high or not expressed in NK cell tumors. However, since NK cell tumors have low incidence, few studies have been reported, and the positive rate is not high, the expression in NK cell lymphomas needs further study.
Flow Cytometry (FCM) plays an important role in the diagnosis of leukemia and lymphoma, and FCM has the advantages of express delivery, high throughput, and sensitivity. Tens of thousands of cells are detected per second, while a variety of cellular markers, including membrane surface and intracellular protein molecules, are detected. With some series of specific markers, lymphocytes, granulocytes, monocytes and other cells can be distinguished in complex blood cells or Bone Marrow (BM) or tissue specimens, and then the different cells can be subjected to a fine analysis.
Since neoplastic NK cells often undergo abnormal changes in antigen expression, such as non-or under-expression of CD56, CD16, CD57, CD7, etc., gating NK cells with these antigens is difficult in this case. The remaining NK cell-associated markers such as CD161, CD94, granzyme and perforin are not only expressed in NK cells but also in part of T cells and therefore are not very specific, whether these antibodies can help gate NK cells and determine the NK cell origin is still uncertain. In addition, since the incidence of NK cell tumors is low, the use frequency of NK related antibodies in a general immunophenotyping antibody combination is low, so that the normal expression profile of some antibodies is not clear, and the effect of the markers in identifying neoplastic or cloned NK cells is not clear.
Disclosure of Invention
In order to solve the problems, the invention provides an antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping and application thereof. The antibody composition provided by the invention can be used for carrying out comprehensive immune typing, cloning identification, differentiation pathway identification, stage judgment of tumor cells and screening of MRD marks and treatment targets on NK tumor cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which comprises a membrane-labeled antibody composition and an intracellular-labeled antibody composition;
the membrane-labeled antibody composition comprises: anti-CD 3 antibodies, anti-CD 2 antibodies, anti-CD 5 antibodies, anti-CD 7 antibodies, anti-CD 8 antibodies, anti-CD 56 antibodies, anti-CD 57 antibodies, anti-CD 16 antibodies, anti-CD 161 antibodies, anti-CD 94 antibodies, anti-CD 159a antibodies, anti-CD 159c antibodies, anti-CD 158a antibodies, anti-CD 158b antibodies, anti-CD 158e antibodies, anti-NKp 30 antibodies, anti-NKp 46 antibodies, anti-HLA-DR antibodies, and anti-CD 45 antibodies;
the intracellular marker antibody composition comprises: anti-granzyme B antibodies and anti-perforin antibodies.
Preferably, the antibodies in the antibody composition are fluorescein-labeled antibodies;
In the membrane-labeled antibody composition, the fluorescent labels of the anti-CD 3 antibody, the anti-CD 2 antibody, the anti-CD 5 antibody, the anti-CD 7 antibody, the anti-CD 8 antibody, the anti-CD 56 antibody, the anti-CD 57 antibody, the anti-CD 16 antibody, the anti-CD 161 antibody, the anti-CD 94 antibody, the anti-CD 159a antibody, the anti-CD 159c antibody, the anti-CD 158a antibody, the anti-CD 158b antibody, the anti-CD 158e antibody, the anti-NKp 30 antibody, the anti-NKp 46 antibody, the anti-HLA-DR antibody, and the anti-CD 45 antibody are respectively in order: cFluor668, BV510, BV650, BV480, cFluorB548, PE-Fire700, BV711, cFluorV450, PE-Cy7, PE-Dazz1e594, PE-Cy5, PE, APC-Fire750, FITC, APC, BV785, BV605, APC-Fire810 and cFluorV547;
in the intracellular marker antibody composition, the fluorescein marker of the anti-granzyme B antibody is BV421, and the fluorescein marker of the anti-perforin antibody is cFluor720.
Preferably, the membrane-labeled antibody composition and the intracellular-labeled antibody composition are each used in 1 flow tube at the time of detection.
The invention provides an application of the antibody composition in preparation of NK cell detection related products, wherein the NK cell detection comprises one or more of (1) - (6):
(1) Tumor immunophenotyping of NK cells;
(2) Carrying out cloning identification on NK cells;
(3) Identifying the differentiation pathway of NK cells;
(4) Carrying out differentiation stage on NK cells;
(5) Screening MRD markers;
(6) And screening NK tumor treatment targets.
The invention provides a kit for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which comprises the antibody composition according to the technical scheme; the membrane-labeled antibody composition and the intracellular-labeled antibody composition in the antibody composition are packaged separately.
Preferably, the kit further comprises: erythrocyte lysate, membrane breaker and buffer PBS.
The invention provides a system for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which comprises a detection module and an analysis module;
the detection module is used for detecting the antigen expression level of a sample to be detected through flow cytometry, and comprises the antibody composition or the kit according to the technical scheme;
the analysis module is used for analyzing the detection result of the detection module.
The invention provides an application of the kit or the system in the technical scheme in preparing products related to NK tumor cell detection, wherein the NK cell detection comprises one or more of (1) - (6):
(1) Tumor immunophenotyping of NK cells;
(2) Carrying out cloning identification on NK cells;
(3) Identifying the differentiation pathway of NK cells;
(4) Carrying out differentiation stage on NK cells;
(5) Screening MRD markers;
(6) And screening NK tumor treatment targets.
The beneficial effects are that:
the antibody composition provided by the invention can be used for carrying out immunophenotype analysis on samples such as bone marrow fluid, hydrothorax and ascites, peripheral blood or lymph nodes of clinical patients, the invention is directly used for carrying out one-step detection on clinical suspected NK cell tumors, and when the clinical patients with unknown clinical diagnosis need to be screened and detected through a primary screen pipe (see Chinese patent CN 202310744220.4) for first-step screening and detecting the suspected NK cell tumors, the invention is used for carrying out second-step detection, carrying out comprehensive immunophenotyping, clonal identification, differentiation path identification, stage judgment on tumor cells and screening MRD marks and treatment targets on the NK cells.
Furthermore, the antibody composition provided by the invention can be matched with a primary screen pipe for use, so that the phenotype of NK cells can be comprehensively analyzed, and specific effects include:
1. understanding NK cell differentiation rules, CD3, CD56, CD7, CD5 vs. CD3 in the primary screen - CD56 + CD7 + CD5 - NK cells were gated and analyzed for the proportion of NK cells (Normal<40%). When NK cell proportion is increased and abnormal phenotype of weak expression (dim) or non-expression of CD56 and CD7 occurs, the antibody composition provided by the invention is used for further detection of NK cells in the second step.
2. When normal NK cells were analyzed by using the primary screen, the present invention found that few early NK cells were present, and the phenotype was CD117 + CD45 dim+ CD56 is strongly expressed (st). While the primary screen contains antibodies of nuclei (n) TdT, CD34, CD38, CD33, MPO and cCD3, so that the invention can simultaneously observe the expression of antigens of early NK cells, and find that the early NK cells are nTDT - CD34 - CD38 - CD33 - MPO - Cd3 may appear to be a few positive. When the proportion of NK cells is increased in early stage and the antigen has abnormal expression, such as negative change-positive or positive change-negative, the phenotype is prompted to be abnormal, and the antibody composition provided by the invention is required to be used for further detection of NK cells in the second step.
3. For samples suspected of NK cell tumors in clinical diagnosis, the antibody composition provided by the invention can be directly used for NK cell detection.
4. When the 1-3 conditions occur, the 21 antibody combinations are utilized to further and comprehensively detect NK cells so as to determine whether the NK cells are abnormal, whether the phenotypes are clonality and determine the proportion and abnormality of the neoplastic NK cells, and can judge the differentiation stage of the NK tumor cells. Markers for determining NK cell tumors, immunophenotyping, tumor staging identification, and determining Minimal Residual Disease (MRD) monitoring after treatment: leukemia-associated immunophenotyping (LAIP), screening for therapeutic targets, and MRD detection after treatment.
More importantly, the antibody composition provided by the invention can be used for flexibly setting the NK cells by utilizing various markers, comprehensively analyzing the expression rules of antigens in different differentiation stages of the NK cells, helping to determine which markers are NK cell specific markers, especially early NK differentiation markers, and has an important effect on determining whether the NK cells are tumor cells derived from NK cell series. At present, NK cells are mainly divided into CD56 st+ CD16 -/dim+ And CD56 + CD16 + The antibody composition provided by the invention can carry out finer staging on NK cells according to the CD159 and CD158 series and the expression of NKp46, perforin and granzyme at the same time, and carry out fine analysis on NK cells in different stages and different subgroups so as to determine the expression of 21 antibodies in the combination on the NK cells in different stagesThe characteristics provide basis for analyzing the differentiation path of NK cells, especially for identifying precursor NK cell tumors, and solve a great difficulty in clinical diagnosis of the tumors.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows an example of the results of normal NK cell detection in normal bone marrow specimens;
FIG. 2 shows an example of detection results of NK tumor cells at stage 1;
FIG. 3 shows an example of CD5 + NK tumor cell detection results;
FIGS. 4 and 5 show an example of CD7 - NK tumor cell detection results;
FIG. 6 shows an example of CD56 - NK tumor cell detection results;
FIG. 7 shows the results of detection of NK tumor cells, a precursor for the presence of bone marrow specimens.
Detailed Description
The invention provides an antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which comprises a membrane-labeled antibody composition and an intracellular-labeled antibody composition;
the membrane-labeled antibody composition comprises: anti-CD 3 antibodies, anti-CD 2 antibodies, anti-CD 5 antibodies, anti-CD 7 antibodies, anti-CD 8 antibodies, anti-CD 56 antibodies, anti-CD 57 antibodies, anti-CD 16 antibodies, anti-CD 161 antibodies, anti-CD 94 antibodies, anti-CD 159a antibodies, anti-CD 159c antibodies, anti-CD 158a antibodies, anti-CD 158b antibodies, anti-CD 158e antibodies, anti-NKp 30 antibodies, anti-NKp 46 antibodies, anti-HLA-DR antibodies, and anti-CD 45 antibodies;
the intracellular marker antibody composition comprises: anti-granzyme B antibodies and anti-perforin antibodies.
In the present invention, the antibody in the antibody composition is preferably a fluorescein-labeled antibody;
in the membrane-labeled antibody composition, the fluorescent labels of the anti-CD 3 antibody, the anti-CD 2 antibody, the anti-CD 5 antibody, the anti-CD 7 antibody, the anti-CD 8 antibody, the anti-CD 56 antibody, the anti-CD 57 antibody, the anti-CD 16 antibody, the anti-CD 161 antibody, the anti-CD 94 antibody, the anti-CD 159a antibody, the anti-CD 159c antibody, the anti-CD 158a antibody, the anti-CD 158b antibody, the anti-CD 158e antibody, the anti-NKp 30 antibody, the anti-NKp 46 antibody, the anti-HLA-DR antibody, and the anti-CD 45 antibody are preferably respectively in order: cFluor668, BV510, BV650, BV480, cFluorB548, PE-Fire700, BV711, cFluorV450, PE-Cy7, PE-Dazz1e594, PE-Cy5, PE, APC-Fire750, FITC, APC, BV785, BV605, APC-Fire810 and cFluorV547;
In the intracellular marker antibody composition, the fluorescein marker of the anti-granzyme B antibody is preferably BV421, and the fluorescein marker of the anti-perforin antibody is preferably cfluor 720; the membrane-labeled antibody composition and the intracellular-labeled antibody composition are preferably used in 1 flow tube, respectively, at the time of detection.
The invention also provides application of the antibody composition in preparation of NK cell detection related products, wherein the NK cell detection comprises one or more of (1) - (6):
(1) Tumor immunophenotyping of NK cells;
(2) Carrying out cloning identification on NK cells;
(3) Identifying the differentiation pathway of NK cells;
(4) Carrying out differentiation stage on NK cells;
(5) Screening MRD markers;
(6) And screening NK tumor treatment targets.
The invention also provides a kit for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which comprises the antibody composition according to the technical scheme; the membrane-labeled antibody composition and the intracellular-labeled antibody composition in the antibody composition are packaged separately.
In the present invention, the kit preferably further comprises: erythrocyte lysate, membrane breaker and buffer PBS.
The invention also provides a system for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which comprises a detection module and an analysis module;
the detection module is used for detecting the antigen expression level of a sample to be detected through flow cytometry, and comprises the antibody composition or the kit according to the technical scheme;
the analysis module is used for analyzing the detection result of the detection module.
The invention also provides an application of the kit or the system in the technical scheme in preparing products related to NK tumor cell detection, wherein the NK cell detection comprises one or more of (1) - (6):
(1) Tumor immunophenotyping of NK cells;
(2) Carrying out cloning identification on NK cells;
(3) Identifying the differentiation pathway of NK cells;
(4) Carrying out differentiation stage on NK cells;
(5) Screening MRD markers;
(6) Screening NK tumor treatment target
The antibody composition provided by the invention can be used for carrying out immunophenotype analysis on samples such as bone marrow fluid, hydrothorax and ascites, peripheral blood or lymph nodes of clinical patients, the invention is directly used for carrying out one-step detection on clinical suspected NK cell tumors, and when the clinical patients with unknown clinical diagnosis need to be detected by a primary screen pipe (see Chinese patent CN 202310744220.4) and suspected NK cell tumors, the invention is used for carrying out second-step detection, and carrying out comprehensive immunophenotyping, clonal identification, differentiation path identification, stage judgment on tumor cells and screening of MRD marks and treatment targets on NK cells.
The antibodies of the embodiments of the present invention are preferably purchased from BD company, biolegend, cytek company, beckmann company; the experimental methods used in the embodiment of the invention are all conventional methods unless specified otherwise; all materials, reagents, etc. in the examples of the invention are commercially available unless otherwise specified.
For further explanation of the present invention, an antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping and its application, which are provided in the present invention, will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1 formulation of reagents
The final antibody combinations and compatible luciferins used in this example are shown in table 1, for a total of 19 membrane labeled antibodies, including: anti-CD 16-eFlun 450, anti-CD 7-BV480, anti-CD 2-BV510, anti-CD 45-cFluorV547, anti-NKp 46-BV605, anti-CD 5-BV650, anti-CD 57-BV711, anti-NKp 30-BV785, anti-CD 158b-FITC, anti-CD 8-cFluorB548, anti-CD 159c-PE, anti-CD 94-PE-dazle 594, anti-CD 159a-PE-Cy5, anti-CD 56-PE-Fire700, anti-CD 161-PE-Cy7, anti-159 e-APC, anti-CD 3-cFluor668, anti-158 a-APC-Fire750, anti-HLA-DR-APC-Fire 810. An intracellular marker antibody 2, comprising: anti-granzyme B-BV421 and perforin-cFluorR 720.
Table 1 antibody combinations and fluorescein compatibility
Configuration of antibody combinations: the two groups of antibodies (membrane-labeled antibody and intracellular-labeled antibody) were mixed and placed in 2 containers according to the amount determined in advance, to obtain an antibody premix for membrane labeling and an antibody premix for intracellular labeling. The above antibodies were purchased from BD company, biolegend, cytek company and beckmann company.
The antibodies are combined together to prepare the detection kit for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping. The kit also comprises a red blood cell dissolving solution, a membrane breaker and a buffer solution PBS, wherein the red blood cell dissolving solution can be self-configured and can be purchased commercially.
EXAMPLE 2 antibody combination flow cytometry analysis of immunophenotype of NK cell tumors
1. Experimental main material and instrument
1. Materials: 10 XPBS buffer, flow cytometer specific hemolysin (BD Co.);
2. instrument: NL-3000 model full spectrum flow cytometer (Cytek company) is equipped with 405nm,488nm, 635nm three lasers, 38 fluorescence detectors. A desk type low-speed centrifuge and a vortex mixer.
2. Method of
1. Sample collection:
the obtained human bone marrow fluid or 1-3 mL of peripheral blood is immediately placed in a heparin anticoagulation tube and rapidly inverted for several times to prevent the sample from coagulating. Various cells such as hydrothorax and ascites, lymph nodes, lavage liquid and the like are collected and sent to a laboratory as soon as possible, and specimens are placed at 4 ℃ for cold storage. Flow Cytometry (FCM) detection must be completed within 48 hours, operating in accordance with instructions.
2. Sample preparation process:
(1) Cell count: adding 150 mu PBS into 10 mu l of bone marrow, mixing, counting each microliter of cell number by using Michaelis FCM (model mindray), and adjusting the cell concentration to 5-10×10 according to the detection result 6 And (5) taking 50-100 μl of cells and adding the cells into the test tube. Lymph node specimens were prepared as single cells using a tissue grinder and then counted.
(2) Antigen staining:
a) Each tube is added with the antibody premix for membrane marking in the example 1 and the cells, and the mixture is fully and uniformly mixed, and incubated for 15min at room temperature in a dark place;
b) Film permeation: 100 μl of the film-penetrating agent A solution is added, and the mixture is allowed to act at room temperature for 5min.
c) Hemolysis: and adding 2ml of 1 XFACS hemolysin, uniformly mixing by low-speed vortex, and standing at room temperature in a dark place for 8-10 min. Centrifuge wash at 300g for 5min and discard supernatant.
d) Washing: 1ml PBS wash was added, and the supernatant was discarded by centrifugation at 300g for 5min.
e) Fixing: 100 μl of permeabilizer B was added to fix and permeabilize the cell membrane, and the antibody premix for intracellular labeling of example 1 was added, mixed well and incubated at room temperature in the dark for 15min.
f) Washing: 1ml of a solution containing 0.1% NaN was added 3 And PBS wash with 1% -2% BSA, 300g centrifugally washed for 5min, and the supernatant is discarded. 200 μl of PBS was added to the suspension cells and the suspension cells were checked on-press.
(3) And (3) detecting:
a) Determining an optimum voltage and compensation: the voltage was set according to the usual procedure for a spectroflow cytometer and single dye samples were prepared for instrument setting with reference to the fluorescent color matching of the kit described in example 1.
b) Instrument setup, calibration and quality control: and starting the preheating machine for more than 20min by Cytek NL-3000, washing by deionized water, detecting an internal quality control product, and ensuring that each detection value is within a control range. And (5) calling AL-PANAL for loading and collecting data.
c) And (3) detecting: and obtaining 5-10 ten thousand cells per tube according to the set instrument conditions. If the detection can not be carried out on the machine in time, 0.5ml of 1% paraformaldehyde is added, and the mixture is uniformly mixed and then is stored in a refrigerator at 4 ℃ for 24 hours to finish the detection.
3. Analysis of results: data were analyzed using Kaluza software:
case analysis
(1) The FSC-A/FSC-H set was used to remove the double-body cell gate A, SSC-A/SSC-B was continued to set gate B to remove adhesion, the FSC-A/SSC-A pattern removed debris and dead cells, and the living single cells were set to gate R1.
(2) R1 portal cells are displayed, a CD45/SSC map is established, and according to the distribution of the cells, lymphocytes (strangles), monocytes (single cells), granulocytes (granules), nucleated erythrocytes (red) and naive cells (young) are gated and different colors are set. The cell proportion of each group was observed to be normal. Whether the proportion of naive cells is increased, etc. In normal bone marrow, each population of cells has a normal range of proportions: 20-40% of gonococcal cells, 2-8% of monocytes, 40-60% of granulocytes, 2-15% of nucleated erythrocytes and less than 5% of naive cells.
(3) Displaying lymphocytic portal cells, creating a CD3/CD56 map for CD3 + CD56-T cells, CD3 + CD56 + NKT cells, CD3 - CD56 +/- NK cells were gated (NK 1 gate) and the proportion of cells in each population was observed.
(4) Determining the proportion of NK cells in lymphocytes or nucleated cells: NK1 gated cells were shown, a series of two-dimensional dot patterns of 2 antibodies were created, and NK cells were further gated to remove non-NK cell interference. For normal NK cells, CD2/CD7, CD5/CD161 vs. CD3 - NK1 intramolecular CD2 + CD7 + CD5 -/dim NK cells were gated (NK) to remove CD2 - CD7 - B cells and CD5 st+ Residual T cells. Accurate gating is a prerequisite for accurate analysis of NK cell phenotype. However, tumorous NK may exhibit CD7 - 、CD56 - 、CD16 - 、CD161 - Such abnormal phenotypes require specific expression of neoplastic NK cells in a particular sample, e.g., most NK cells express granzyme B, CD, NKp46, and CD3 when CD5 is not expressed - NK1 in-gate utilization of granzyme B positive, CD94 positive, NKp46 positive and CD5 negative for CD7 - CD56 - CD16 - The NK cells of the (2) are flexibly gated, the proportion of the NK cells in the lymphocytes or the nucleated cells is finally determined, and the expression condition of other antigens in the tumor NK cell combination is analyzed to judge the abnormality of the tumor NK cells.
(5) Analyzing the differentiation rule of NK cells and judging whether phenotypic abnormality occurs or not: a series of two-dimensional plots of 2 antibodies were created showing NK-portal cells and all expression of the remaining markers in the combination were observed. Mainly comprises CD56/CD16, CD56/CD94, CD56/NKp46, CD94/C159a, CD94/C159C, CD 56/perforin, CD 56/granzyme B, CD e/CD158a, CD158b/CD158a, CD159C/CD159a, NKp30/NKp46, CD5/CD7, CD16/CD57, CD16/CD161, CD8/HLA-DR, CD94/CD161 and the like.
(6) In the invention, firstly, normal specimens are detected, normal reference values (tables 2-3) are established, and average number + -3 XSD is used as a standard for judging abnormality. When tumor specimens were analyzed, the clonality of NK cells was determined (table 4).
(II) judging LAIP markers, wherein the LAIP for NK tumors mainly comprises:
1. cross-series antigen expression or cross-line antigen expression, note whether myeloid and T-line related antigens are expressed: CD33, CD5, etc.
2. And judging whether the NK cell antigen expression intensity is abnormal or not in the sample by taking the normal NK cell antigen expression intensity as a standard, such as CD56, CD16, CD161, CD94, CD7 and the like.
3. The clonality abnormality, abnormally high expression (restriction or clonality) of CD158a/b/e and CD159a/c, is shown in the threshold value in Table 5.
And (III) providing a basis for screening the treatment target related markers: the targeted drugs aiming at NK tumors are few at present, and through the detection of the invention, the markers with higher NK tumor expression rate and higher specificity to NK cells can be screened, for example, the invention screens the NK tumor occurrence rate with higher CD159a positive and more than 90% of tumor cells express the antigen, while NKp46 is only expressed in NK cells and not expressed in T cells, thus being very specific NK cell markers and being applicable to the development of therapeutic targets.
(IV) MRD detection: the combination is used for MRD detection after treatment, abnormal NK cells are detected according to LAIP of a patient, and meanwhile, the clonality of the abnormal NK cells can be analyzed to find out neoplastic NK cells.
4. Results:
a total of 20 normal human Peripheral Blood (PB) were examined, of which 11 men and 9 women, the median age was 42 years (22-65 years). Establishment of normal human lymphocytes, CD3 - CD56 + NK cells, CD3 + CD56 - T cells and CD3 + CD56 + The reference ranges for NKT cells and primary antibodies in this combination are shown in tables 2 and 3. As can be seen from table 3, antigens frequently used in NK cell analysis are expressed on NK cells, mostly also on NKT or T cells, such as CD94, CD161, CD159a, granzyme B, perforin; CD159c is expressed predominantly in NK and NKT; CD158 series antibodies are mainly expressed in NK cells, with minor expression in NKT; while CD16, NKp30 and NKp46 are expressed only in NK cells, the specificity for identifying NK cells is strong.
TABLE 2 results of subpopulations of normal human PB lymphocytes
TABLE 3 parameter values (%)
48 cases of specimens were examined from 8 months in 2021 to 10 months in 2023, 27 cases of men and 21 cases of women, and the median age was 58 years (6 to 84). Among them 31 NK tumors, 4 unidentified clonality NK (NK-Cus) and 11 normal or 1 reactive NK cytopenia specimens were included. The 11 normal NK specimens included: 4 myeloproliferative neoplasms, 2T cell proliferative neoplasms, 1 plasma cell leukemia, 1 thrombocytopenia, and 1 granulocytic ischemia (see table 4 for basic information). 1 case of reactive NK cell increase is a virus infected person, male, 68 years old, WBC 36.07×10 9 /L，HB 138×10 9 g/L，PLT 202×10 9 Lymphocytes account for 80.84% of L, where Nk cells account for 91.47%. Three lines of 1 reduced patient appearance of abnormal precursor NK cells, men, 65 years old, WBC2.times.10 9 /L，HB 46×10 9 g/L，PLT 16×10 9 Lymphocytes account for 25.31% of L, where Nk cells account for 5.31%. From Table 4, it can be seen that NK tumor samples have significantly increased lymphocyte% and NK%.
Table 4 basic information of 48 examples of specimens
Note that: NK% is the percentage of NK in lymphocytes.
According to the reference value without antigen expression, whether the antigen is abnormally expressed is mainly determined by taking mean ± 3×sd as a standard, and individual markers are finely adjusted according to the results of normal bone marrow specimens. Reference is made mainly to 14 markers: CD56 hi >26%、CD5>19%、CD7<60%、CD16<47%、CD56<80%、CD161<47.39%、NKp30>91%、CD94<10%、CD158a>50%、CD158b>88%、CD158e>44%、CD159a>89%、CD159c>50% of granzyme B<16.83% perforin<60%. Each parameter anomaly was counted as 1 minute and three groups of samples were subjected to integral analysis (table 5). As can be seen from Table 5, the markers with the highest frequency of abnormal expression are the clonality (high expression or restriction expression) of CD159a, CD159c and the low expression of CD161, whereas the clonality expression rate of the CD158 series markers is low.
Table 5 number of cases of antigen expression abnormality and percentage thereof in each group
NK tumors can be identified from normal NK or responsive NK cells by integral analysis. All scores of 31 NK tumor specimens were <2, 11 normal NK cells and 1 virus infected reactive specimens were all <2, 3 of which were 1 and the rest were 0. The 4 samples of NK-Cus are patients with follicular lymphoma, thrombocytosis, anemia and thrombocytopenia, and NK cell integration of 2 appears in the samples, so that the standard of NK tumor patient integration is achieved, but the NK is not high, the significance is unknown, and the samples are separately classified.
According to the differentiation rule of NK cells and the expression characteristics of NK tumor cell antigens, NK cell tumors are divided into 1-4 total 4 phases according to 6 antigens of CD159a, CD159c, CD158a/b/e and CD57, and the 1 st phase: only CD159a clonality occurs; phase 2: clonality of 159a and CD159c occurs; stage 3: any one of the 3 antibodies to CD158a/b/e exhibits clonality, and may exhibit CD159c clonality at the same time, or both CD158 and CD159a are negative; stage 4: high expression (> 70%) of CD57 occurs on the basis of phase III phenotype.
31 NK tumors, 13 cases, 6 cases, 7 cases and 5 cases, respectively, were found in stages 1 to 4 (Table 5). The number and percentage of abnormal expression of different antigens in 1-2 and 3-4 stages are analyzed, so that the strong expression of CD56, the low expression of CD16, perforin, CD161 and CD7 can be all found in NK tumors in 1-2 stages. Wherein, the strong expression of CD56, the low expression of CD16 and perforin accords with the differentiation rule of early NK cells, but the low expression of CD161 and CD7 is not consistent with the differentiation stage, and the tumor abnormality is caused. CD158 series expression in CD56 in normal NK cells dim CD16 + CD57 is also expressed in the most advanced stages. The 1-4 phases represent the low-to-high change process of NK tumor cell differentiation. The invention discovers that 3 cases of CD5 expression abnormality only occur in NK tumor stage 4, which indicates that CD5 expression occurs in advanced NK cell differentiation stage. The invention can stage NK tumors according to the parameters of CD159a, CD159c, CD158a/b/e and CD57, and can reflect NK cell differentiationStage. Although normal early NK cells strongly express CD56, CD94 and NKp46, and do not express CD16, NK tumors cannot be staged completely according to these markers because tumor NK does not develop according to normal differentiation rules, and the results of the present invention demonstrate that early NK can be identified according to CD159a and CD159 c. It was also demonstrated that CD159a was expressed earlier than CD159c, and that more NK stage 1-2 tumors demonstrated that CD159a and CD159c had a stronger role in NK cell clonality identification than the CD158 series. NK tumors were judged only according to the clonality of the CD158 series antigen, and most NK tumors were missed.
Precursor NK cell tumor: the present invention found a patient with a three-line decrease in the proportion of lymphocytes in the bone marrow, but a small amount of CD5 was found by the detection of the screening tube - CD7 + CD56 + CD117 p+ cCD3 - CD33 p+ CD34 - Abnormal naive cells, CD45 of this population of cells dim+ Whether it is precursor NK cells cannot be determined. Further detection with the combination of the invention found CD3 - CD7 + There are three sub-populations in the cell, 1, CD7 respectively + CD94 - CD56 -/dim+ ；2、CD7 st+ CD94 + CD56 st+ ；3、CD7 d+ CD94 + CD56 dim+ CD16 + . Group 1 cells do not express CD94 and other NK-associated markers, strongly express CD117, do not express CD34 and CD38, CD56 goes from negative to weak positive, and a few cells express CD33 and MPO, resembling NK progenitor cells. Group 2 cells expressing early NK cell-related markers: CD159a, NKp46, a minority of cells weakly express CD16, conforming to the early NK cell phenotype. The 3 rd group of cells simultaneously express granzyme B, conforming to late NK cells. CD117 + NK cells are present in a small proportion in normal bone marrow, only in part CD56 hi CD16 - Early NK cells. CD117 expression was not seen in 31 NK tumors. Patient CD117 + Up to 63% in NK, the proportion is markedly increased and 17% earlier NK progenitor cells also appear, which the present invention is considered to belong to as precursor NK cells. Although the proportion in nucleated cells was 1.0%, it was suggested that there was a possibility of occurrence of acute NK cell leukemia. Whereas acute NK cell leukemia is rarely seen, how to diagnose it consistently is an unprecedented problem. This example illustrates that NKp46, CD159a and CD94 play an important role in the identification of precursor NK, providing an important basis for the diagnosis of precursor NK tumors. Because the incidence rate of the tumor is too low, only 1 case is seen within 2 years, and further accumulated cases are needed to be verified, but the invention provides important basis for diagnosing the tumor.
Case detection results:
FIG. 1 shows an example of the results of normal NK cell detection in normal bone marrow specimens. Lymphocytes (strangles), monocytes (mono), granulocytes (granulocytes) and nucleated erythrocytes (red) were gated and ratios shown in the CD45/SSC plot. CD56 by CD56/CD3 + CD3 - NK cell (NK 1) CD56 + CD3 + (NKT) cells, CD56 - CD3 + (T) cells are gated. The establishment of a CD2/CD7 and CD5/CD161 map shows NK 1-gated cells, selection of CD2 + CD7 + And CD5 - CD161 + The cells further gate NK cells (NK). Establishment of CD56/CD16 map shows NK door inner cells, for CD56 hi CD16 - Early stage (56) hi ) NK and CD56 dim+ CD16 + Late stage (56) d ) NK cells are gated. A series of 2-dimensional dot diagrams are established, all of which show NK-in-gate cells, and the expression characteristics of different antigens in early and late NK cells are analyzed. Early NK cell phenotype is shown to be CD94 st+ NKp46 + CD159 + CD161 + CD7 st+ While none of the remaining markers are expressed; antigens expressed by late NK cells are granzyme, perforin, CD159c, CD158 series, CD57. HLA-DR is not expressed.
FIG. 2 shows the results of cell detection of an NK tumor sample at stage 1. Lymphocytes (strangles), monocytes (mono), granulocytes (granulocytes) and nucleated erythrocytes (red) were gated and ratios shown in the CD45/SSC plot. Suggesting CD56/CD3 diagram for CD56 + CD3 + (NKT) cells, CD56 - CD3 + (T) cells are gated. CD 56-was selected because NK tumors can develop CD56-, CD 56-was selected +/- CD3 - (NK 1) preliminary gating of NK cells. Establishment of CD2/CD7 and granzyme/CD 94 charts shows NK 1-gated cells, selection of CD2 + CD7 + And granzyme + The cells further gate NK cells (NK). Establishment of CD56/CD16 map shows NK door inner cells, for CD56 hi CD16 -/dim+ Early NK (56) hi ) And CD56 dim+ CD16 dim+ Advanced NK (56) d ) A door is arranged. A series of 2-dimensional dot diagrams are established, all of which show NK portal cells, and the expression characteristics of different antigens in early and late NK cells are analyzed. Few cell CD56 are shown from the CD94/CD56 plot - CD94 + CD56 + CD94 - NK cells, CD56 from other figures + CD94 - NK cells simultaneously expressed granzyme B, perforin, CD159c, CD158, CD57, CD161, CD16 and CD5, polyclonality expressed the CD158 series, but did not express CD159a, indicating normal late NK cells. CD94 + CD56 +/- The cells expressed all CD159a, CD94st and NKp46, expressed low granzyme B, perforin, CD16, HLA-DR, CD7, rarely expressed CD158e, did not express CD158a, CD158B, NKp30, CD159c, CD161, CD5 as clonal NK cells. Belonging to NK tumor of stage 1. The sample had CD56 - Clonal NK cells, therefore, cannot select only CD56 + CD3 - NK cells, gating methods need to be varied.
FIG. 3 shows an example of CD5 + NK tumor cell detection results. Lymphocytes (strangles), monocytes (mono), granulocytes (granulocytes) and nucleated erythrocytes (red) were gated and ratios shown in the CD45/SSC plot. CD56 by CD56/CD3 +/- CD3 - (NK 1) preliminary gating of NK cells (NK 1) with CD56 + CD3 + (NKT) cells, CD56 - CD3 + (T) cells are gated. The establishment of a CD2/CD7 and CD56/CD161 map shows NK 1-gated cells, selection of CD2 + CD7 + And CD56 +/- CD161 + The cells further gate NK cells (NK). Establishment of CD56/CD16 map shows NK door inner cells, for CD56 hi CD16 - Early stage (56) hi ) NK and CD56 dim+ CD16 + Late stage (56) d ) NK cells are gated. A series of 2-dimensional dot patterns are established, all of which show NK-in-door cells, and different antigens are analyzed in the morningExpression profile in phase and late NK cells. Shown at 56 hi Early NK cell phenotype was not seen with significant abnormalities. But CD5 + NK cells account for 55.54% of NK cells, the proportion is obviously increased, and the NK cells are used for CD5 + NK cells were gated showing CD5 + The cell phenotype is CD159c + CD158a + CD16 st+ CD161 + CD57 + CD7 dim+ Perforin element + Granzyme B + CD94, NKp46, CD158b, CD158e, CD159a, CD8, HLA-DR were not expressed, and cloned NK cells were classified as stage 4.
FIGS. 4 and 5 show an example of CD7 - NK tumor cell detection results. For lymphocyte (stranguria), monocyte (mononucleocyte), granulocyte (granulocyte) and nucleated erythrocyte (erythrocyte), CD56 +/- CD3 - （NK1）、CD56 + CD3 + (NKT) cells, CD56 - CD3 + (T) cells are gated on. Establishment of CD94/NKp46 and CD56/CD94 panels shows NK 1-gated cells, selection of CD94 +/dim+ CD 94-cells were removed (NK). The CD5/CD7 graph was constructed to show cells within the NK gate, and it was seen that the vast majority of cells did not express CD7 (7-), and only a few cells expressed CD7 (7+), gating was performed on the 2 population of cells. A series of 2-dimensional dot patterns were created, all showing NK-in-gate cells, and the remaining antigens were analyzed for CD7 + And CD7 - Expression profile in group 2 cells. FIG. 4 shows CD7 - NK cell phenotype, cell population CD159a + 90% of the total weight of the product, CD56 from strong to negative, CD56 hi And NKp46 ratio was increased, CD16, CD161 and perforin were not expressed, score 6 points, belonging to the first stage NK tumor. FIG. 5 shows CD7 + NK cells, the population of cells expressing CD16, CD56 dim CD94, perforin, granzyme B, all expressed in the reference range, normal NK cells.
FIG. 6 shows an example of CD56 - NK tumor cell detection results. For lymphocyte (stranguria), monocyte (mononucleocyte), granulocyte (granulocyte) and nucleated erythrocyte (erythrocyte), CD56 +/- CD3 - （NK1）、CD56 + CD3 + (NKT) cells, CD56 - CD3 + (T) cells are gated on. Establishment of perforin/granzyme B and CD94/CD7 mapIndicating NK1 portal cells, perforin selection + And CD94 + Accurate gating (NK) of cells on NK cells. Establishment of CD56/CD16 map shows NK door inner cells, for CD56 hi CD16 - Early stage (56) hi ) NK and CD56 dim+ CD16 + Late stage (56) d ) NK cells gating, see>90% of the cells do not express CD56. A series of 2-dimensional dot diagrams are established, all of which show NK-in-door cells, and the expression characteristics of different antigens in three groups of NK cells are analyzed. Visible CD56 hi NK cell antigen expression was not seen as a clear abnormality, and was normal early NK cells. While CD56 d And CD56 - NK cell antigen expression is substantially consistent, and this population of cells strongly expressed perforin, granzyme B, expressed CD7 and CD16, weakly expressed CD94, did not express CD159a, CD159c, NKp46, CD158B, CD158e, CD159a, were late stage NK cells, but were negative in both the CD56 and CD158 series, were abnormal clonal manifestations. NK tumors of stage 3 are classified.
FIG. 7 shows the results of detection of NK tumor cells, a precursor for the presence of bone marrow specimens. FIG. 7A shows the combination of antibodies of example 1 of the present invention, FIG. 7B shows the result of screening with Chinese patent CN202310744220.4, and FIG. 7B shows the presence of CD5 - CD7 + CD56 + CD117 p+ cCD3 - CD33 p+ CD34 - Whether the abnormal naive cells are NK cells cannot be determined. Further detection with the combination of the invention (A in FIG. 7) was carried out using CD45/CD7 (7 + ) CD56/CD3 (NK 1) and SSC low NKp46 + (NK) gating of this population of cells. There are three sub-populations of cells, 1.CD7 respectively + CD94 - CD56 - ；2.CD7 st+ CD94 + CD56 st+ ；3.CD7 d+ CD94 + CD56 dim+ CD16 + . Group 1 cells do not express CD94 and other NK-associated markers, strongly express CD117, do not express CD34 and CD38, CD56 goes from negative to weak positive, and a few cells express CD33 and MPO, resembling NK progenitor cells. Group 2 cells expressing NK early-related markers: CD159a, NKp46, a few cells weakly expressed CD16, conforming to early NK cells. Group 3 cells simultaneously expressed granzyme B, and the phenotype conformed to late NK cells. CD117 + The cells reached 63% in NK, ratioThe examples were markedly elevated, with earlier NK progenitor cells, precursor NK cells, also included. Although the proportion in nucleated cells was 1.0%, it was suggested that there was a possibility of occurrence of acute NK cell leukemia.
For screening residual leukemia markers (LAIP): after finding the differentiation rule of NK cells, the differentiation rule is easy to identify, the phenotype abnormality can be judged according to the stage of tumors, for example, the stage 1 tumors show weak expression of CD56 and CD7, and the cloning expression of CD159a can be used as LAIP for MRD detection. For stage 2 tumors, double-cloning expression of CD159a and CD159c occurred, as was the simultaneous presence of CD161 dim Or CD7 dim Or CD56 hi Is a good detection of the LAIP of the MRD. Stage 3-4 tumors are mainly based on the clonality of CD158 and CD159c or NKp30, and may have the simultaneous appearance of CD94 dim Granzyme B is under-expressed, or expresses CD5, and can be used as a marker for identifying neoplastic NK cells, as a LAIP for detecting MRD.
Screening of treatment targets: through comprehensive analysis of antigen expression specificity of normal NK cells and tumor NK cells, antigens with high occurrence rate of tumor NK cells such as CD159a or CD159c and antigens with high specificity such as NKp46 can be selected as candidate target antigens for tumor treatment.
Comparative example 1
The difficulty in designing more than 20 antibody combinations is that the number of fluorescein is too much, which is one time more (8-10) than the current traditional flow cytometry, and more than half of fluorescein is not used clinically. The intensity of each fluorescein is different, interference exists between spectrums, and proper antigens need to be selected for compatibility, so that the interference between the luciferins is as low as possible. In addition, the antibodies used need to be purchased from companies, and different companies have different antibody qualities, which are good and which are poor, and verification is needed. The available fluorescence limits for different companies for a certain antibody are limited. The choice of which antibodies to combine also requires a rich clinical immunophenotyping test. These all present great difficulties in designing more than 20 antibody combinations. The development of the antibody combination of the invention also goes through a plurality of stages, and satisfactory results are achieved in more than 1 year, and the development process is as follows:
The first version of the antibody combination contained 20 antibodies, excluding anti-CD 56, anti-CD 2 and anti-HLA-DR antibodies relative to the antibody combination in Table 1, but including NKp44-PE-CY7 and CD4-APC-Fire810, and the anti-CD 161 antibody was BV510 from BD company.
In specific case implementation, CD3 is adopted - CD16 + ，CD7+CD5 - After 6 cases of detection, CD16 and CD7 are frequently expressed weakly or negatively, and the NK cells are difficult to accurately gate, so that CD56-PE-Fire700 is added into a first-version combination, which is a second-version antibody combination, and total of 21 antibodies are added. After 11 consecutive cases of detection, CD161-BV510 was found to be all negative, and different from the theoretical result, CD161 antibodies of a plurality of companies were tried, and finally Beckmann company CD161 was selected, but only FITC, PE, PE-CY7 was selected because of limited available fluorescence, and in 11 cases of samples which have been detected, NKp44-PE-CY7 was all negative, CD161-PE-CY7 was selected, original NKp44 was removed, and the combination was the third edition, and 20 antibodies were total. When 31 samples were continuously tested and the results were summarized, the CD158b-PerCP-Cy5.5 results were found to be very different from the conventional 10-color results, and the conventional test of CD158b was clonally expressed, while the present invention was negative. After repeated validation with normal specimens, it was considered that there was a significant interference between the spectra of CD158b-PerCP-Cy5.5 and PE-Dazz1e594, PE-Cy5, PE-Fire700 in the third plate combination, affecting the detection of CD158b, which had to be adjusted. Since there are fewer antibodies available for CD158b and more antibodies available for CD7, CD158b-FITC was replaced, and original CD7-FITC was replaced with BV480. Meanwhile, when the result analysis shows that the NK tumor does not express CD4, but HLA-DR is frequently expressed when the NK tumor is performed, so that the CD4 is removed, the original channel is changed into HLA-DR, and CD2-BV 510 is added, so that the capacity of gating NK cells is enhanced. Form 4 th edition, the final antibody combination (table 1). The above antibodies were purchased from BD company, biolegend, cytek company and beckmann company.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. An antibody composition for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, characterized by comprising a membrane-labeled antibody composition and an intracellular-labeled antibody composition;
the membrane-labeled antibody composition comprises: anti-CD 3 antibodies, anti-CD 2 antibodies, anti-CD 5 antibodies, anti-CD 7 antibodies, anti-CD 8 antibodies, anti-CD 56 antibodies, anti-CD 57 antibodies, anti-CD 16 antibodies, anti-CD 161 antibodies, anti-CD 94 antibodies, anti-CD 159a antibodies, anti-CD 159c antibodies, anti-CD 158a antibodies, anti-CD 158b antibodies, anti-CD 158e antibodies, anti-NKp 30 antibodies, anti-NKp 46 antibodies, anti-HLA-DR antibodies, and anti-CD 45 antibodies;
the intracellular marker antibody composition comprises: anti-granzyme B antibodies and anti-perforin antibodies.
2. The antibody composition of claim 1, wherein the antibodies in the antibody composition are fluorescein-labeled antibodies;
in the membrane-labeled antibody composition, the fluorescent labels of the anti-CD 3 antibody, the anti-CD 2 antibody, the anti-CD 5 antibody, the anti-CD 7 antibody, the anti-CD 8 antibody, the anti-CD 56 antibody, the anti-CD 57 antibody, the anti-CD 16 antibody, the anti-CD 161 antibody, the anti-CD 94 antibody, the anti-CD 159a antibody, the anti-CD 159c antibody, the anti-CD 158a antibody, the anti-CD 158b antibody, the anti-CD 158e antibody, the anti-NKp 30 antibody, the anti-NKp 46 antibody, the anti-HLA-DR antibody, and the anti-CD 45 antibody are respectively in order: cFluor668, BV510, BV650, BV480, cFluorB548, PE-Fire700, BV711, cFluorV450, PE-Cy7, PE-Dazz1e594, PE-Cy5, PE, APC-Fire750, FITC, APC, BV785, BV605, APC-Fire810 and cFluorV547;
In the intracellular marker antibody composition, the fluorescein marker of the anti-granzyme B antibody is BV421, and the fluorescein marker of the anti-perforin antibody is cFluor720.
3. The antibody composition according to claim 1 or 2, wherein the membrane-labeled antibody composition and the intracellular-labeled antibody composition are each used in 1 flow tube at the time of detection.
4. Use of the antibody composition of any one of claims 1-3 in the preparation of a product related to NK cell detection comprising one or more of (1) - (6):
(1) Tumor immunophenotyping of NK cells;
(2) Carrying out cloning identification on NK cells;
(3) Identifying the differentiation pathway of NK cells;
(4) Carrying out differentiation stage on NK cells;
(5) Screening MRD markers;
(6) And screening NK tumor treatment targets.
5. A kit for analyzing NK cell differentiation stages and NK cell tumor immunophenotyping, comprising the antibody composition of any one of claims 1 to 3; the membrane-labeled antibody composition and the intracellular-labeled antibody composition in the antibody composition are packaged separately.
6. The kit of claim 5, further comprising: erythrocyte lysate, membrane breaker and buffer PBS.
7. A system for analyzing NK cell differentiation stage and NK cell tumor immunophenotyping, which is characterized by comprising a detection module and an analysis module;
the detection module is used for detecting the antigen expression level of a sample to be detected through flow cytometry, and comprises the antibody composition according to any one of claims 1-3 or the kit according to claim 5 or 6;
the analysis module is used for analyzing the detection result of the detection module.
8. Use of the kit of claim 5 or 6 or the system of claim 7 for the preparation of a product related to NK tumor cell detection comprising one or more of (1) - (6):
(1) Tumor immunophenotyping of NK cells;
(2) Carrying out cloning identification on NK cells;
(3) Identifying the differentiation pathway of NK cells;
(4) Carrying out differentiation stage on NK cells;
(5) Screening MRD markers;
(6) And screening NK tumor treatment targets.
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