CN107064085B - Method for measuring natural killer cell activity - Google Patents
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Abstract
A method for measuring the activity of natural killer cells features that the parameters of "NK cell ratio" and "the survival rate of nucleated cells" are introduced to calculate the ratio of effector cells to target cells, and the quality control is performed in each step to reduce the difference in detection.
Description
Technical Field
The invention relates to a method for measuring natural killer cell activity, belonging to the field of biological detection.
Background
NK cells (Natural killer cells, NK cells) were originally called innate immune lymphotoxic cells because they can kill autologous tumor cells without prior sensitization. Over the years of intensive research on the phenotype and function of NK cells, the main biological functions of NK cells are mainly to secrete cytokines to participate in immune regulation and directly kill target cells [1, 2 ]. NK cells are distributed in different tissues with different functions, so that the Natural Killer (NK) cells have the effects of resisting tumors, participating in various pathological processes, maintaining immune homeostasis and promoting the process of placental vascularization in the gestation period. Correspondingly, malignant tumors, various infections, autoimmune diseases, etc. are all involved in NK cell disorders [3-6 ].
NK cell killing activity is an important index for evaluating immune state, and can be used for monitoring the change of the immune state in the immune treatment process and the like. It has been reported in the literature to be also associated with the efficacy of chemotherapy for malignant cervical cancer, survival after chemotherapy for pancreatic cancer, and the rate of progression of Alzheimer's disease [7-9 ]. Impaired NK killing activity is one of the characteristics of familial or acquired hemophagocytic lymphohistiocytosis, inflammation, autoimmune diseases and immunodeficiency diseases, but because the laboratory results of the indexes cannot be compared, the indexes lack normal reference value ranges, and the indexes are often used as confirmation indexes of other diagnosis indexes in relevant disease diagnosis [10 ]. The NK killing activity detection needs a strict and standardized detection analysis method and a corresponding normal reference value range urgently, and the bottleneck problem of the application of the method is solved.
The "gold standard method" of the NK killing detection method is a 51Cr release test, but the application thereof in clinical detection is limited due to problems such as radioactive contamination [11, 12 ]. Other methods for detection of NK cell killing activity include the low toxicity dye Calcein-AM release assay, LDH release assay, flow cytometry or bioluminescence based detection methods [13-17 ]. The detection method based on flow cytometry has the advantages of simple and convenient operation, stability, less pollution, low baseline, good correlation with a 51Cr release experiment method and the like, and the method is mainly adopted for detecting the killing activity of NK cells clinically [14, 18 ].
However, the method in the prior art only calculates the number of nucleated cells and the number of target cells, which may cause deviation of detection results, and cannot realize strict evaluation on killing capacity of NK cells.
Disclosure of Invention
The invention provides a method for measuring natural killer cell activity, which is characterized by comprising the following steps:
1) subculturing the target cells;
2) analyzing the proportion of natural killer cells in effector cells;
3) culturing target cells and effector cells according to a set effective target ratio;
4) detecting the death rate of the target cells to be detected in the step 3);
5) substituting the death rate of the target cells in the step 4) into a formula to calculate the activity of the natural killer cells:
the natural killer cell activity is (target cell death rate of the test group-target cell death rate of the control group)/(1-target cell death rate of the control group) × 100%.
Further, the effective target ratio in step 3) is 4 or more.
Further, the passage cells are subjected to quality control of density and/or mortality in step 1).
Further, the effector cells are subjected to quality control of at least one of cell density, viable cell rate and natural killer cell ratio in step 2).
Further, the quality control method comprises dye labeling of the subcultured target cells.
Further, labeling natural killer cells with antibodies is included.
Further, the natural death rate of the target cells is controlled within 3 percent.
Further, the ratio of the target cell to the effector cell FH1 channel MIF (mean fluorescence intensity) values was 80-fold or more.
Further, in step 4), the following detection degrees are discarded: compared with the target cell only control tube, the target cell concentration in the effective target cell mixed incubation detection tube is lower than 85 percent.
Further, the effective target ratio is selected according to whether the detected object suffers from the tumor.
The invention establishes a brand-new NK cell killing activity detection method, introduces parameters of 'NK cell proportion' and 'nucleated cell activity rate' when calculating the proportion of effector cells and target cells, improves the detection rigidness, and does not have the problems of high NK cell magnetic bead separation cost, mechanical damage of flow cytometry separation and the like compared with the detection after NK cells are separated.
Compared with the traditional detection method, the method improves the comparability of the NK cell killing activity evaluation among samples from different sources, and has wider application prospect.
The invention firstly proposes to control the quality of each link of the detection, aims to reduce the difference of the detection among rooms and is expected to solve the current situation that the detection results of all laboratories cannot be mutually referred.
The invention firstly evaluates the normal reference value range of the killing activity of human peripheral blood N K cells which can be referred to by different laboratories.
Drawings
FIG. 1 shows the proportional distribution of NK cells in nucleated cells of a specimen to be examined
FIG. 2 peripheral blood NK cell killing Activity of tumor patient group and healthy volunteer group
Detailed Description
1 data and method
1.1 the study subjects selected 108 healthy volunteers and tumor patients who were treated in the affiliated hospital of the military medical academy of sciences from 6 months 2014 to 10 months 2016, and the relevant results of age, sex and NK killing activity detection were collected. And 158 cases of nucleated cell NK proportion information of other NK killing activity detection samples are collected. The reference values are taken into the selection criteria: firstly, healthy people; the hemogram is normal; and signing an informed consent. Reference value exclusion criteria: firstly, patients with immune-related diseases; ② there is history of application of taking hormone and immunomodulator; ③ 3 patients with definite infection within weeks; fourthly, patients with cardiovascular and cerebrovascular diseases; suspected or definite diagnosis is tumor patient; sixthly, other diseases or medical history influencing immune function.
1.2 reagent and apparatus PI fluorochromes, monoclonal antibodies for human NK cell subset analysis (CD45-APC, CD56-PE, CD3-PerCP) and their isotype control were purchased from American BD company, CSFE fluorochromes were purchased from Japan synneus company, human lymphocyte separation liquid was purchased from Tianjin negative-class Hitaceae Biotechnology Co., Ltd., and 1640 medium for cell culture and fetal bovine serum were purchased from Thremo Fisher company. The detection was performed using a BD Accuri C6 flow cytometer.
1.3 Experimental methods
1.3.1 subculture of target cells: k562 cells at 1-2X 105cells/ml density, inoculated in a T75 flask containing 20ml of 1640 complete medium, passaged every three days, maintaining its good proliferation status.
1.3.2 dye labeling of target cells: the K562 cell culture suspension was collected in a 15ml centrifuge tube and centrifuged at 1000rpm for 10 minutes in a horizontal centrifuge. After washing twice with physiological saline, the single cell state was resuspended in 500ul serum-free 1640 medium, and after mixing well, the sample was taken and counted using C6 for use. Diluting CSFE dye working solution with 1640 serum-free culture medium, mixing, rapidly adding into K562 cell suspension, and mixing to give CSFE dye final concentration of 5 μ M and cell suspension density of 1 × 107cells/ml. 5% CO at 37 deg.C2The incubator was shaken every 5 minutes. After incubation for 10 minutes, an equal volume of 4 ℃ precooled fetal bovine serum was added, and the staining reaction was terminated in a 4 ℃ freezer for 10 minutes. After supplementing physiological saline and mixing evenly, centrifuging at 1000rpm for 10 minutes, abandoning the supernatant, suspending in 1640 complete culture medium, and placing in an incubator for incubation for 1 hour. The cells were collected, washed once with physiological saline, 1640 the cells were resuspended in complete medium, and samples were counted with C6 for use.
1.3.3 isolation of Effector cells: if the sample is whole blood, Peripheral Blood Mononuclear Cells (PBMC) are separated by centrifuging the whole blood sample at 1800rpm for 10 minutes, removing plasma, re-separating the cells with an equal volume of saline, gently centrifuging the whole blood sample on a lymphocyte separation medium, and carefully removing the buffy coat. Washed twice with physiological saline, 1640 cells were resuspended in complete medium, sampled and diluted in leukocyte count solution, and counted with C6 until use. If the sample is an immune cell culture product, the sample is washed with physiological saline only twice. Samples were diluted in 1640 complete medium and counted with C6 until use.
1.3.4 labelling of NK cell subsets of effector cells: about 5X 105100ul of effector cell suspension of single nuclear cells (PBMC), CD3-CD56+ NK cell subgroup antibody labeling according to the flow detection antibody instruction, detecting by a BD FACS Calibur flow cytometer, and analyzing the NK cell proportion in the nuclear cells. The red blood cell lysis step is performed if PBMC is present, and not performed if the immune cell culture product is present.
1.3.5 detection of the Living cell Rate of Effector cells: if the sample is an immune cell culture product, the rate of viable cells of the nucleated cells needs to be detected. And (3) taking 100ul of cell suspension, adding the PI dye to the final concentration of 3.75ug/ml, balancing for 5-10 minutes, and detecting the death rate of effector cells by using a flow cytometer.
1.3.6 effector cells and target cells in mixed culture: setting effective target ratio (ratio of NK cells and target cells marked with CSFE) as 0: 1, 1: 1, 2: 1, 4: 1 and 6: 1, calculating required effector cell volume according to effector cell nucleated cell density, effector cell viability and NK cell ratio in the nucleated cells, mixing effector cells and target cells, and supplementing to 200ul co-culture system with 1640 complete culture medium. At least three wells of each group were mixed and incubated for 2.5 hours.
1.3.7 target cell mortality assay: resuspending the mixed cultured cells from step 1.3.6, adding PI dye to a final concentration of 3.75ug/ml, equilibrating for 5-10 min, and detecting the CSFE-positive target cell death rate of each tube by flow cytometry.
1.3.8 calculation of NK cell killing Activity: substituting the target cell death rate of each tube into a formula (the target cell death rate of an experimental group-the target cell death rate of a control group)/(the target cell death rate of a 1-the control group) multiplied by 100 percent, and calculating the average killing activity value of each effective target ratio NK cells.
1.4 quality control
1.4.1 quality control of target cells subcultured K562 cells were seeded at a fixed density, and growth state and rate were observed, and cell cycle and other indicators were examined as necessary. The balance time of the K562 cell line after being marked by CSFE in 1640 complete culture medium is controlled, the natural mortality of the marked K562 cell line is required to be less than 3%, and the ratio of the average Fluorescence Intensity (MIF) values of target cells and effector cells FH1 channel is more than 80 times, so that the consistency of the target cells used in each detection is guaranteed.
1.4.2 quality control of Effector cells EDTA anticoagulation tube peripheral blood sample collection, after the separation within 3 hours send to the laboratory, place in room temperature and place, the Effector cells for subsequent use are resuspended in 1640 complete medium. For example, non-fresh blood samples or long-term placement under other conditions, the viability rate of nucleated cells is detected, and the volume of PBMC suspension required for each effect target ratio is calculated according to the cell density, the viable cell rate and the NK ratio of the nucleated cells.
1.4.3 quality control of flow cytometers the instrument was calibrated for fixed maintenance, PI fluorochrome signals were detected using the FH3 channel, and the Absolute cell function of Accuri C6 was used instead of manual measurement of effector or target cell suspension density.
1.4.4 quality control of data analysis the same technician performs readings under the same standard, and if data is accepted or rejected and analyzed, the same technician performs readings together by three fixed technicians. Compared with the target cell control tube only, when the concentration of the target cells in the effective target cell mixed incubation detection tube is lower than 85 percent of the target cell mixed incubation detection tube, part of the target cells are considered to be completely broken after death, so that the detection result is lower than the actual target cell death rate, and the detection result is not credible.
1.5 statistical method SPSS21.0 software is used for statistical analysis, each index is expressed by X +/-s, multiple linear regression analysis is used for factor correlation analysis, and independent sample t test is used for mean comparison between two groups. P < 0.05 is statistically significant.
2 results
2.1 inclusion in the basic cases of the subjects
The normal reference group includes 47 healthy volunteers, 22 women [ 28-68 years old, mean (47.5 + -9.1) years old ], and 25 men [ 27-69 years old, mean (47.3 + -11.2) years old ]. The tumor group included 61 patients with various tumors [ 23 cases of liver cancer, 8 cases of pancreatic cancer, 5 cases of lung cancer and colon cancer, 3 cases of rectal cancer, stomach cancer, breast cancer and ovarian cancer, 2 cases of thyroid cancer, 1 case of kidney cancer, vaginal cancer, hypopharyngeal cancer, esophageal cancer, hepatobiliary cancer and bile duct cancer ], 18 women [ 36-84 years old, mean (60.2 ± 11.9) years old ], 43 men [ 17-85 years old, mean (53.9 ± 13.5) years old ], which are shown in Table 1.
TABLE 1 general situation table (example)
2.2NK proportion Difference
Analyzing the proportion distribution of NK cells in nucleated cells in various source samples in NK killing activity detection (see figure 1), and prompting that the parameter distribution in a tumor patient group is 10.8% +/-7.87% (3% -42%), the skewness is 1.714 and the kurtosis is 3.609; the distribution of this parameter in the group of healthy volunteers was 12.31% + -5.25% (4% -23%), skewness 0.576, kurtosis-0.860; the distribution of the parameters in other source groups is 13.48% ± 12.30% (0.11% -75.19%), the skewness is 3.095, and the kurtosis is 11.283, so that the NK cell proportion distribution rules in the nucleated cells of the sample to be detected with NK killing activity from different sources are different, for example, the huge difference of the parameters is not considered when the ratio of effector cells to target cells is calculated, only the number of the nucleated cells and the number of the target cells are calculated, the detection result may be deviated, and the strict evaluation on the NK cell killing capability cannot be realized.
2.3 analysis of factors involved in NK cell killing Activity
The ages were grouped under 44, 45-59, 60 and above, and the correlation between age, sex and whether tumor had been detected and NK killing activity was analyzed. The results suggest that there is no correlation between the age, sex and whether tumor has been detected when the effective target ratio is 1 and 2 and the NK killing activity (P > 0.05). When the effective target ratio is 4 and 6, the sex, age and the correlation have no statistical significance (P is more than 0.05), and whether the patient has the tumor or not and the correlation have statistical significance (P is less than 0.05). Based on the analysis of the data in this study, it was not found that age and sex influence the killing ability of NK cells in peripheral blood, but whether a tumor is affected will influence the index, and the effective-to-target ratio should be set to 4 or 6 or more.
2.4 Normal reference value Range of NK cell killing Activity
Considering that the healthy control group data of this study showed that both age and sex were independent of NK cell killing activity, they were not grouped according to age or sex when determining the reference value range. The reference range was determined by the percentile method, setting the two-sided 90% reference range of NK cell killing activity, and the results are shown in table 2.
TABLE 2 Normal reference value range of peripheral blood NK cell killing Activity (%)
2.5 NK killing Activity characteristics of tumor patients
The difference in killing activity of NK cells in peripheral blood between the tumor patient group and the healthy volunteer group is compared, and the result indicates that the difference between the two groups has statistical significance (P is more than 0.05) when the effective-target ratio is 4 and 6 respectively. When the effective target ratio is 1 to 2, the difference between the two groups is not statistically significant (P < 0.05). The tumor patients are prompted that the index is lower than that of healthy donors, and the detection items should consider setting the effective target ratio to be 4 and 6.
Reference to the literature
1.Cooper，M.A.，et al.，Human natural killer cells：a unique innate immunoregulatory role for the CD56(bright)subset.Blood，2001.97(10)：p.3146-51.
2.Cerwenka，A.and L.L.Lanier，Natural killer cells，viruses and cancer.Nat Rev Immunol，2001.1(1)：p.41-9.
3.Yu，J.，A.G.Freud，and M.A.Caligiuri，Location and cellular stages of natural killer cell development.Trends Immunol.34(12)：p.573-82.
4.Vivier，E.，et al.，Functions of natural killer cells.Nat Immunol，2008.9(5)：p.503-10.
5.Parham，P.and A.Moffett，Variable NK cell receptors and their MHC class I ligands in immunity，reproduction and human evolution.Nat Rev Immunol.13(2)：p.133-44.
6.Orange，J.S.，Natural killer cell deficiency.J Allergy Clin Immunol.132(3)：p.515-25；quiz 526.
7.Cosiski Marana，H.R.，.J.Santana da Silva，and J.Moreira de Andrade，NK cell activity in the presence of IL-12is a prognostic assay to neoadjuvant chemotherapy in cervical cancer.Gynecol Oncol，2000.78(3Pt 1)：p.318-23.
8.Prolo，P.，et al.，Physiologic modulation of natural killer cell activity as an index of Alzheimer′s disease progression.Bioinformation，2007.1(9)：p.363-6.
9.Karakhanova，S.，et al.，Prognostic and predictive value of immunological parameters for chemoradioimmunotherapy in patients with pancreatic adenocarcinoma.Br J Cancer.112(6)：p.1027-36.
10.Ishii，E.，Hemophagocytic Lymphohistiocytosis in Children：Pathogenesis and Treatment.Front Pediatr.4：p.47.
11.Brunner，K.T.，et al.，Quantitative assay of the lytic action of immune lymphoid cells on 51-Ct-labelled allogeneic target cells in vitro；inhibition by isoantibody and by drugs.Immunology，1968.14(2)：p.181-96.
12.Fehniger，T.A.，et al.，Potential mechanisms of human natural killer cell expansion in vivo during low-dose IL-2therapy.J Clin Invest，2000.106(1)：p.117-24.
13.Lichtenfels，R.，et al.，CARE-LASS(calcein-release-assay)，an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity.J Immunol Methods，1994.172(2)：p.227-39.
14.Valiathan，R.，et al.，Evaluation of a flow cytometry-based assay for natural killer cell activity in clinical settings.Scand J Immunol.75(4)：p.455-62.
15.Cho，D.，et al.，Cytotoxicity of activated natural killer cells against pediatric solid tumors.Clin Cancer Res.16(15)：p.3901-9.
16.Konjevic，G.，V.Jurisic，and I.Spuzic，Association of NK cell dysfunction with changes in LDH characteristics of peripheral blood lymphocytes(PBL)in breast cancer patients.Breast Cancer Res Treat，2001.66(3)：p.255-63.
17.Karimi，M.A.，et al.，Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.PLoS One.9(2)：p.e89357.
18.Kim，G.G.，et al.，A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells：comparisons to a 4 h 51Cr-release assay.J Immunol Methods，2007.325(1-2)：p.51-66.
19.Defining，establishing，and verifying RIs in the clinical aboratory.Approved guideline 3rd Ed.Clinical Laboratory and Standards Institute，2008.
20.Mace，E.M.，et al.，Cell biological steps and checkpoints in accessing NK cell cytotoxicity.Immunol Cell Biol.92(3)：p.245-55.
Claims (10)
1. A method for measuring natural killer cell activity, which is characterized by comprising the following steps:
1) subculturing the target cell K562;
2) analyzing the proportion of natural killer cells in effector cells;
3) setting an effective target ratio, wherein the effective target ratio is the ratio of NK cells to target cells, calculating the volume of the required effector cells according to the density and the survival rate of the effector cells nucleated cells and the ratio of the NK cells in the effector cells nucleated cells, and culturing the target cells and the effector cells according to the set effective target ratio;
4) detecting the mortality of the target cells cultured in step 3);
5) substituting the death rate of the target cells in the step 4) into a formula to calculate the activity of the natural killer cells:
the natural killer cell activity is (the death rate of target cells to be tested-the death rate of target cells in a control group)/(1-the death rate of target cells in a control group). gamma.100%.
2. The method for measuring natural killer cell activity according to claim 1, wherein the effective-to-target ratio in step 3) is 4 or more.
3. The method for measuring natural killer cell activity according to claim 1, wherein the mass control of density and/or mortality is performed on the passaged cells in step 1).
4. The method for measuring natural killer cell activity according to claim 1, wherein at least one of the cell density, the viable cell rate and the ratio of natural killer cells is controlled for the effector cells in step 2).
5. The method of measuring natural killer cell activity according to claim 3, wherein said quality control comprises dye labeling of subcultured target cells.
6. The method for measuring natural killer cell activity according to claim 4, comprising labeling natural killer cells with an antibody.
7. The method for measuring natural killer cell activity according to claim 3, wherein the natural death rate of the target cell is controlled within 3%.
8. The method of claim 3, wherein the ratio of the target cell to the effector cell FH1 channel mean fluorescence intensity values is 80 times or more.
9. The method for measuring natural killer cell activity according to any one of claims 1 to 8, wherein the result of measuring the concentration of target cells in the tube in which the effective target cell mixture is incubated is less than 85% compared with that in the control tube of target cells alone, is discarded in step 4).
10. The method for measuring natural killer cell activity according to claim 1, wherein the effective target ratio is selected depending on whether the subject has suffered from the tumor.
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