CN118243594B - Flow cytometry-based sputum-induced eosinophil quantification method - Google Patents
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Abstract
The invention discloses a method for quantifying induced phlegm eosinophils based on flow cytometry. Mainly comprises a sputum liquefying step, a sputum cell suspension staining step and a door setting analysis step. The sputum liquefying step comprises the steps of weighing a sputum sample, adding DTE, vibrating and incubating, filtering, centrifuging and washing to obtain a cell suspension; the sputum cell suspension staining step comprises the steps of adding an antibody into quantitative cell suspension, adding sheath liquid for washing, retaining residual sheath liquid and adding 7-AAD for incubation; the gated assay step involves delineating leukocytes based on antibodies, removing dead cells, and then determining delineating granulocytes and eosinophils, and measuring eosinophil numbers. The method can effectively avoid errors of manual microscopic examination, reduce errors, improve the accuracy and the inspection efficiency of inspection results, and calculate the quantity of eosinophils in the induced sputum of unit mass so as to realize accurate quantification.
Description
Technical Field
The invention relates to the technical field of cell quantitative detection methods, in particular to a method for quantifying induced phlegm eosinophils based on flow cytometry.
Background
Asthma is a chronic disease characterized by chronic airway inflammation and causes various respiratory symptoms. The number of adult asthma patients in China is estimated to be up to 4570 ten thousand; asthma has a variety of clinical and inflammatory phenotypes, eosinophilic Asthma (EA) being the most common of these (sputum eosinophils (sEOS). Gtoreq.2.5%), which induces elevated EOS in sputum, eosinophil infiltration 2,3 seen in bronchial mucosa, submucosa and lung tissue. Higher levels of sputum eosinophils (sEOS) are associated with greater number of annual episodes, poor asthma control and quality of life. And is therefore particularly important for diagnosis of EA patients.
SEOS the proportion of specific cell types was assessed by induced sputum cell sorting. The sputum cell classification examination is finished by using an artificial microscope classification method in clinic in China. However, the manual microscope classification method has a plurality of defects. Firstly, the manual microscope cell classification method has high requirements on staff, and the manual microscope cell classification method is required to carry out classification judgment according to the cell morphology in sputum, but the individual difference of the cell volume in the sputum is large, the morphological structure is not as good as that in blood, and the accuracy of the result is greatly influenced by subjective factors. The manual microscope cell sorting method only picks part of sputum to test, and the homogeneity of the texture of the sputum is poor, so that the test result may be affected. The artificial microscope cell sorting method counts only 400 white blood cells to calculate the percentage of eosinophils, and the limited number of counted cells results in poor accuracy of results. The manual microscope cell classification method only counts the percentage of eosinophils, and can not truly reflect the true content of eosinophils in the induced sputum, so that the provided information is limited, and the requirements of diagnosis and treatment scheme guidance of EA patients can not be fully met. Manual microscopy cell sorting is time consuming and requires at least 10 minutes to read.
Therefore, there is a need in the clinic for a method for quantifying induced sputum eosinophils with more accurate test results and higher test efficiency.
Disclosure of Invention
The invention aims to provide a flow cytometry-based sputum eosinophil induction quantification method for solving the defects in the prior art, and the technical problems to be solved by the invention are realized by the following technical scheme, which comprises the following steps:
Sputum liquefying step: firstly, placing sputum into a plate, uniformly mixing, and taking viscous liquid in the sputum into a centrifuge tube for weighing to obtain the sputum mass M; adding 1, 4-Dithioerythritol (DTE) into the centrifuge tube, shaking, incubating in a shaking table in a dark place, filtering, centrifuging and washing to obtain a cell suspension;
Sputum cell suspension staining procedure: adding the cell suspension to an absolute counter tube having a total microsphere number Q 1; adding antibodies including CD16-FITC, CD66b-PE, siglec8 PE-Cy7, CD14 APC and CD45 APC-Cy7, then adding sheath fluid for washing, retaining residual sheath fluid and adding 7-AAD for incubation, and obtaining a sample volume V 1;
A door setting analysis step: taking sputum cell suspension of A sampling volume V 2, setting A threshold according to CD45 APC-Cy7 antibody, circling side scattering light range (SSC-A) increase and CD45 positive areA cells, and removing non-white blood cells and cell fragments; the area where the microsphere is located is defined, the microsphere number Q 2 is obtained, a gate is reversely arranged, the area where the microsphere is located is removed, and fragments are removed according to forward scattered light (FCS) and side scattered light (SSC); taking single core cells based on the forward scattered light range (FCS-A) and the side scattered light range (SSC-A); delineating leukocytes through CD45 positive regions; then dead cells are removed, and the survival rate of the white blood cells is judged through 7-AAD negative; eosinophils Q eos were measured by delineating granulocytes in the CD66b positive, CD14 negative region and finally eosinophils (EOS cells) in the siglec8 positive, CD16 negative region;
Further, the sputum mass M is at least 200mg.
Further, the volume ratio of the sputum mass M to the 1, 4-dithioerythritol is 1mg: 4. Mu.L.
Further, in the sputum liquefying step, the centrifuge tube was shaken with a shaker for 10 seconds after the centrifuge tube was added with 1, 4-Dithioerythritol (DTE), and then the centrifuge tube was laid flat on a shaker and incubated for 15 minutes at 80rpm/min in the dark.
Further, in the step of liquefying sputum, the filtration was performed by adding 5mL of the sheath solution, mixing the mixture upside down, filtering the mixture with a 70 μm cell filter, and collecting and filtering residual cells on a centrifuge tube and a filter screen with 15mL of the sheath solution.
Further, the centrifugation at the sputum liquefying step is performed including 2 times of centrifugation, and the supernatant is removed after the 1 st centrifugation and centrifuged again.
Further, after adding the antibody in the sputum cell suspension staining step, shaking for 2 seconds, mixing uniformly, and incubating for 30 minutes at 4 ℃ in a dark place.
Further, the gating analysis step is performed within 1 hour after completion of the sputum cell suspension staining step.
Further, the content C (unit: individual/mg) of induced sputum eosinophils was calculated according to the following formula:
；
the invention has the following advantages:
(1) The invention uses a 6-color antibody staining protocol (CD 16-FITC, CD66b-PE, siglec8 PE-Cy7, CD14 APC, CD45 APC-Cy 7). The scheme can detect on the 2-laser 6-color 8-channel flow cytometer, and reduces the configuration requirement of the flow cytometer.
(2) Avoiding manual errors and reducing errors: under specific gating analysis logic, the flow cytometry can identify different types of cells through simple fluorescence signal difference, and has low requirements on operators. In addition, the flow cytometry adopts a whole sputum analysis method, so that the content of eosinophils in more than 1 ten thousand white blood cells can be calculated, and the result difference caused by uneven sputum texture and less counting is avoided.
(3) Improving the clinical value: the flow cytometry can detect the percentage of eosinophils in white blood cells, calculate the quantity of eosinophils in the induced sputum of unit mass, realize accurate quantification and improve the clinical application value.
(4) And the inspection efficiency is improved: the time of an operator for analyzing one sample through a preset template can be controlled within 3 minutes, and compared with the manual microscope cell classification method, the time for testing is greatly shortened.
Drawings
FIG. 1 is a flow cytometry-based quantification method of induced sputum eosinophils of the present invention;
FIG. 2 is a chart showing the number of cells of the above-described type collected by the on-board flow cytometer of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The application will be described in detail below with reference to the drawings in connection with embodiments.
FIG. 1 is a flow cytometry-based quantitative method for inducing sputum eosinophils according to the present invention, which mainly comprises a sputum liquefying step, a sputum cell suspension staining step and a gate-setting analysis step. Wherein in the sputum liquefying step, the method specifically comprises the following steps:
(1) The sputum specimen is placed in a biosafety cabinet, the discharged sputum is placed in a plate, and the sputum specimen is dispersed and uniformly mixed by forceps, so that the sputum specimen is more beneficial to liquefaction.
(2) The viscous sputum is clamped into a centrifuge tube by forceps, the sputum is weighed by an analytical balance, the mass of the sputum is recorded, and the sputum can be added according to the mass of the sputum when the subsequent reagent is added. Meanwhile, the sputum quality is at least 200mg to be qualified, and in the embodiment, the sputum quality is 500mg.
(3) 1, 4-Dithioerythritol (DTE) was added to the centrifuge tube, and incubated in a shaker in the absence of light after shaking. A ratio of 4. Mu.L of 1, 4-Dithioerythritol (DTE) per 1mg of sputum is too high to cause cell damage due to excessive liquefaction, and a ratio of too low to cause insufficient liquefaction. And (5) oscillating the centrifugal tube for 10 seconds by using an oscillator, and shaking down sputum on the wall of the centrifugal tube. And then the centrifuge tube is laid on a shaking table horizontally and incubated for 15 minutes in a dark place at a speed of 80rpm/min, and the liquefaction condition of the sputum specimen is observed visually during the incubation period, so that the incubation time can be stopped in advance or prolonged according to the liquefaction condition. Too long an incubation time may destroy the cells.
Dithiothreitol (DTT) added by conventional liquefaction methods requires incubation in a 37 ℃ water bath to exert the maximum disulfide bond breaking effect. The 1, 4-Dithioerythritol (DTE) used in the present example showed a comparable disulfide bond-breaking effect at room temperature. The flow cytometry detection obtained by the 2 methods has similar eosinophil count results, and even more cells can be obtained by 1, 4-Dithioerythritol (DTE). The sputum liquefying device simplifies the equipment requirements for sputum liquefying, and simultaneously avoids the test problems of inaccurate temperature control, sample water inflow and the like possibly caused by water bath. In addition, the proportion of DTT added in the traditional liquefaction method is 2-4 times of the volume of sputum, and the proportion depends on the viscosity of the sputum. However, the viscosity is not well-defined. In addition, sputum is less homogeneous, so using volume to calculate the DTT addition ratio may cause errors. In this example, the amount of 1, 4-dithioerythritol is 4 times the amount of the sputum, and the occurrence of the above-mentioned errors can be avoided.
Table 1 shows eosinophil count results of the present example and conventional liquefaction method (DTT method) in the artificial microscope cell sorter. Classification and enumeration of such cells is particularly important because eosinophil elevation is associated with a variety of airway disease types. The results show that the present technology enables the discovery of more eosinophil-enhanced patients, thereby facilitating the diagnosis of disease typing in patients by clinicians.
TABLE 1 eosinophil count results of artificial microscope cell sorting method by 1, 4-dithioerythritol method and DTT method
(4) After adding 5mL of the sheath solution, the mixture was inverted and homogenized, and then filtered through a 70 μm cell strainer. In addition, the residual cells on the centrifuge tube and filter were collected with 15mL sheath fluid and filtered to prevent the cells from being missed. The conventional liquefaction method filters impurities only once by using a filter screen, and residual cells on the cell filter screen may be missed.
(5) After centrifugation and washing, the cells were resuspended for use. Centrifuging for 2 times, centrifuging 400g for 15 minutes at 1 st time, removing supernatant, adding 1mL of sheath fluid, and then centrifuging at 2000 rpm/min; centrifugation for 5 min at 2 nd, washing for 1-2 times, and finally resuspending the cells according to the ratio of 100mg sputum to 100 mu L sheath fluid to obtain cell suspension for standby. In this example, the supernatant was removed after the 1 st centrifugation and the supernatant was centrifuged again to remove as much impurities as possible from the sputum.
After obtaining the cell suspension, a sputum cell suspension staining step was performed, specifically as follows:
(1) 100. Mu.L of the cell suspension was added to an absolute counter tube, which is a flow tube in which an accurate quantitative number of microspheres (Beads) was previously added, and in this example, the total number of microspheres in the absolute counter tube was 68000. After the operation of incubating the sample is completed by using the flow tube, after the data is acquired by a flow cytometer, the relative content (%) is calculated from the number of each target cell group, and then the content (in/mg) of the cell group in the sputum is calculated from each target group, the number of microspheres, the sample adding volume, the cell suspension volume and the sputum mass;
(2) Antibodies CD16-FITC, CD66b-PE, siglec8 PE-Cy7, CD14 APC, CD45 APC-Cy7 were added. The antibody with fluorescent label can be specifically combined on the surface of target cell, so that the laser light source of the flow cytometer can be used for excitation to produce optical signal, and the optical signal can be converted and identified by the instrument;
(3) Shaking for 2 seconds, mixing, and incubating for 30 minutes at 4 ℃ in dark place. The purpose of shaking mixing is to avoid cell deposition. Incubation at 4 ℃ in the dark is to ensure cell activity and avoid photobleaching;
(4) Adding 2mL of sheath liquid for washing for 1 time;
(5) About 100. Mu.L of the residual sheath fluid was retained, and 5. Mu.L of 7-AAD was added. When removing the supernatant, a sample adding gun is needed;
(6) mu.L of 7-amino actinomycin D (7-AAD) was added and incubated for 15 minutes. 7-AAD is added to recognize living cells;
(7) Samples were taken at a volume of 200 μl and assayed on-press within 1 hour to preserve cellular activity as much as possible.
Finally, performing ring gate analysis, which comprises the following specific steps:
(1) CD45 negative (CD 45-) cells and some debris were removed by thresholding CD45 APC-Cy 7. This step requires observation of the side scatter light range (SSC-A) and CD45 signal. Circling SSC-A increased and CD45 positive (cd45+) cells, thereby excluding non-leukocytes and cell debris;
(2) And circling the area where the microsphere is located. Removing the fragment interference and circumscribing the microsphere. The reverse gating removes the microsphere areas and removes debris based on forward scattered light (FCS) and SSC. The above steps are to further exclude background debris and reduce noise signals. There is an increase in FCS and SSC in this region relative to the background debris region.
(3) Single core cells were taken according to the forward scattered light range (FCS-A) and SSC-A. This step is to exclude doublet cells, incorporating only single cells.
(4) White blood cells (cd45+) were delineated. This step is to delineate the white blood cells. Since the surface of leukocytes all have CD45 antigen, the cells in which the CD45 positive (cd45+) region is located can be identified as leukocytes.
(5) Dead cells were removed and the leukocyte viability (7-AAD-) was judged. This step is to exclude dead cells, thereby further reducing noise signals.
(6) Granulocytes (CD 14-, CD6b+) were trapped. This step is to outline granulocytes, which have more CD66b antigen on their surface and less CD14 antigen, so that cells in the CD66b positive (CD6b+), CD14 negative (CD 14-) region can be identified as granulocytes.
(7) And (3) circumscribing eosinophils (siglec8+, CD 16-). This step is to delineate eosinophils, where siglec8+ antigen is present on the surface of eosinophils, but less CD16 antigen, so that cells in the siglec8 positive (siglec8+) and CD16 negative (CD 16-) region are identified as eosinophils.
In this example, the sputum mass was 500mg, the total microsphere number of the absolute counter tube was 68000, the sample volume was 200. Mu.L, the number of microspheres collected this time was 2448, the eosinophil number was 3076, the sample addition volume was 50. Mu.L, the sputum mass was 500mg, and the eosinophil content in the sputum was 683.56/mg calculated according to the absolute counter formula.
FIG. 2 is a graph showing the number of the above-mentioned various types of cells collected by the on-line flow cytometer of the present invention, wherein the number of the collected white blood cells is 4458. Eosinophils were 3076 in number. Eosinophils were 69.00%. In accordance with the criteria described in the literature, a patient may be diagnosed with an eosinophil phenotype.
The invention was carried out by using a 6-color antibody staining protocol (CD 16-FITC, CD66b-PE, siglec8 PE-Cy7, CD14 APC, CD45 APC-Cy 7). The scheme of the invention can detect on the 2-laser 6-color 8-channel flow cytometer, and reduces the configuration requirement of the flow cytometer.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A flow cytometry-based method of quantifying induced sputum eosinophils, comprising:
Sputum liquefying step: firstly, placing sputum into a plate, uniformly mixing, taking viscous fluid in the sputum into a centrifuge tube, and weighing to obtain the mass M of the sputum, wherein the mass M of the sputum is at least 200mg; 1, 4-dithioerythritol is then added to the centrifuge tube, and the volume ratio of the sputum mass M to the 1, 4-dithioerythritol is 1mg:4 mu L, vibrating the centrifuge tube for 10 seconds by using a vibrator after adding 1, 4-dithioerythritol into the centrifuge tube, then placing the centrifuge tube on a shaking table in a lying manner, incubating for 15 minutes at a speed of 80rpm/min in a dark place, and filtering, centrifuging and washing to obtain a cell suspension;
Antibody staining: adding the cell suspension into an absolute counting tube, wherein the absolute counting tube has total microsphere number Q 1; adding antibodies including CD16-FITC, CD66b-PE, siglec8 PE-Cy7, CD14 APC and CD45 APC-Cy7, then adding sheath fluid for washing, retaining residual sheath fluid and adding 7-AAD for incubation, and obtaining a sample volume V 1;
A door setting analysis step: taking a sputum cell suspension of a sample adding volume V 2, setting a threshold according to a CD45 APC-Cy7 antibody, circling cells in a side scattering light range and CD45 positive area, and removing non-white blood cells and cell fragments; the area where the microsphere is located is defined, the microsphere number Q 2 is obtained, a gate is reversely arranged, the area where the microsphere is located is removed, and fragments are removed according to the forward scattering light range and the side scattering light range; taking a single core cell according to the forward scattered light range and the side scattered light range; delineating leukocytes through CD45 positive regions; then dead cells are removed, and the survival rate of the white blood cells is judged through 7-AAD negative; granulocytes were defined by CD66b positive and CD14 negative regions, and finally eosinophils were defined by siglec8 positive and CD16 negative regions, and eosinophil number Q eos was measured, and the content C of induced sputum eosinophils was calculated according to the following formula:
。
2. The method for quantifying the induced sputum eosinophils based on flow cytometry according to claim 1, wherein when the filtering is performed in the sputum liquefaction step, 5mL of sheath fluid is added first, and then the mixture is inverted and mixed, and filtered with a 70 μm cell filter, and then the residual cells on a centrifuge tube and a filter are collected with 15mL of sheath fluid and filtered.
3. The method for quantifying induced sputum eosinophils based on flow cytometry according to claim 1, wherein said centrifugation is performed in said sputum liquefaction step comprising 2 centrifugation steps, and the supernatant is removed after the 1 st centrifugation step and centrifuged again.
4. The method for quantifying induced sputum eosinophils based on flow cytometry according to claim 1, wherein the step of staining the sputum cell suspension is performed by adding the antibody, shaking for 2 seconds, mixing, and incubating at 4℃for 30 minutes in the absence of light.
5. The flow cytometry-based method of quantifying sputum eosinophil induction of claim 1, wherein said gating assay step is performed within 1 hour after completion of said sputum cell suspension staining step.
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| CN112986109A (en) * | 2021-04-16 | 2021-06-18 | 深圳市中西医结合医院 | Instrument for counting eosinophilic granulocytes in induced sputum |
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