Method for detecting systemic lupus erythematosus immune spectrum based on full-spectrum flow cytometry

Abstract

The invention discloses a detection method of a systemic lupus erythematosus immune spectrum based on full spectrum flow cytometry, and relates to the technical field of cell detection, and the detection method comprises the following steps: providing a fluorescein coupling antibody of a detection index; carrying out antibody titration on the fluorescein coupling antibody to obtain a dyeing concentration; the method comprises the following steps: carrying out surface staining on peripheral blood mononuclear cells of an SLE patient by adopting a fluorescein coupled antibody, carrying out intracellular mitochondrial superoxide staining, and resuspending in a detection solution to obtain a test sample; carrying out sample loading test on the test sample and the prepared single staining reference sample and negative sample, and collecting and analyzing particle information; and performing enclosure and dimension reduction analysis on the analyzed data to obtain a systemic lupus erythematosus immune spectrum and a cell activation state analysis result. According to the application, dozens of immune cell subtypes and activation states thereof in SLE can be detected under the condition of a single-tube sample, an SLE patient immune map is constructed, and a reference is provided for evaluating disease progression and formulating a treatment scheme.

Description

Technical Field

[0001] The present application relates to the field of cell detection technology, and in particular to a method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry. Background Art

[0002] Systemic lupus erythematosus (SLE) is a typical autoimmune disease characterized by abnormal activation of the immune system, which leads to the production of autoantibodies and attacks on multiple organs and tissues. The pathogenesis of SLE is affected by a variety of immune cells and immune factors, including B cells and T lymphocyte subsets. These immune cells play an important role in different stages of the disease and are closely related to the severity, disease activity and clinical manifestations of SLE. In recent years, research on immune cell subsets in the peripheral blood of SLE patients has attracted widespread attention. The levels of these immune cell subsets are closely related to disease activity, and monitoring them is helpful in evaluating disease progression and formulating treatment plans.

[0003] Currently, the commonly used methods for immune cell clustering detection in blood samples are traditional flow cytometry and single-cell sequencing analysis. Due to the interference caused by fluorescence spillover, traditional flow cytometry requires simultaneous labeling of multiple tubes of cells to complete the detection of more than 30 antibodies. In addition, the clustering antibodies need to be reused in different detection tubes, which consumes a large amount of samples for detection, uses a large number of antibodies, and increases the cost. In addition, the differences in the types of antibodies, clone numbers, and fluorescein used in different detection tubes will cause large errors in detection, which is not suitable for practical applications. Single-cell sequencing analysis requires firstly to perform flow sorting of each immune cell in the blood sample. The operation is complicated, the detection time is long, the subsequent data analysis is difficult, and the cost of use is too high. The test results also need to be verified at the protein level, which is difficult to be widely promoted in clinical practice. Mass spectrometry flow cytometry uses metal isotope labeling instead of fluorescent labeling in traditional flow cytometry. Although it avoids fluorescence crosstalk, it can detect hundreds of markers at the same time and has high detection sensitivity. However, all detection antibodies need to be customized again, the instruments and equipment are expensive, and the detection cost is high. In addition, the data acquisition speed is slow, and real-time live cell analysis cannot be performed, so it has certain limitations in clinical application.

[0004] Spectral flow cytometry is a recently developed technology that enables high-dimensional flow cytometric analysis of cells and particles in suspension. This single-cell technology is becoming increasingly popular in scientific research because it can conservatively detect 35 or more antigens simultaneously in a single-tube detection format. Spectral flow cytometry is compatible with traditional flow cytometry for detecting antibodies, the technology is easier to promote, and it can analyze living cells, with fast data acquisition and easy operation. Although spectral flow cytometry also has the disadvantages of complex detection scheme design and difficult data processing, it is generally most appropriate to use this technology for the detection of patient immune cells and the construction of immune maps. Recently, spectral flow cytometry has obtained regulatory approval for in vitro diagnostic devices in China and Europe, enabling this technology to be used in some clinical flow cytometry laboratories.

[0005] At present, there are also research results that provide a method for lymphocyte immunophenotyping. The kit detects 14 indicators, which can divide lymphocytes into natural killer (NK) cells, T cells, B cells and their subpopulations, but they need to be detected in multiple test tubes. In addition, the number of cell subpopulations detected is relatively small, and many important immune cell subpopulations are not involved. There are also research results that provide antibody compositions and their applications for detecting acute leukemia based on full-spectrum flow cytometry, detection methods for lung cancer tumor-infiltrating lymphocyte subpopulations, and methods for detecting pancreatic cancer immune microenvironment analysis. The above two results detect 36 and 21 indicators for disease characteristics, respectively. And most of the experimental materials studied are experimental mice.

[0006] However, there is no method to use full-spectrum flow cytometry to analyze the immune microenvironment of SLE patients and construct an immune map. Summary of the invention

[0007] In order to fill the gap in this field, the present application has developed a detection method for the systemic lupus erythematosus immune map based on full-spectrum flow cytometry, which detects the immune environment of SLE patients based on full-spectrum flow cytometry and forms a set of efficient, reliable and complete methods, which can perform 39-color staining on SLE patients, analyze and distinguish dozens of immune cell subtypes such as neutrophils, eosinophils, basophils, dendritic cells, monocytes, natural killer cells, T cells and their subpopulations, and B cells, detect their activation status, and form an immune map through in-depth data analysis.

[0008] To achieve the above objectives, this application provides the following technical solutions: The present application provides a method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry, comprising: providing fluorescein-coupled antibodies for the following detection indicators according to a color scheme: CD57, L / D, CD3, CD21, CD19, CCR6, IgM, CD45, CD95, CD28, CD11c, CD56, CCR7, CD69, CD25, CCR4, CXCR5, IgA, CD14, CD8, CD4, mitochondrial superoxide, CD27, IgG, TCR gd, CD127, TIGIT, IgD, CD366, CD20, CD123, CXCR3, CD24, CD16, CD279, HLA-DR, CD39, CD45RA, CD38; titrate the fluorescein-coupled antibody to obtain the staining concentration of the fluorescein-coupled antibody; use the fluorescein-coupled antibody to perform surface staining on peripheral blood mononuclear cells in blood samples of patients with systemic lupus erythematosus, and perform intracellular mitochondrial superoxide staining, resuspend in the detection solution to obtain a test sample; load the test sample and the prepared single-stained reference sample and negative sample on a spectral flow cytometer in a predetermined order, collect particle information and perform analysis; perform gating and dimensionality reduction analysis on the analyzed data to obtain the systemic lupus erythematosus immune map and cell activation state analysis results.

[0009] In some embodiments of the present application, the color scheme is: the fluorescein of CD57 is BUV395, the fluorescein of L / D is FVS440, the fluorescein of CD3 is BUV496, the fluorescein of CD21 is BUV563, the fluorescein of CD19 is BUV615, the fluorescein of CCR6 is BUV661, the fluorescein of IgM is BUV737, the fluorescein of CD45 is BUV805, the fluorescein of CD95 is BV421, the fluorescein of CD28 is BUV496, the fluorescein of CD47 is BUV563, the fluorescein of CD57 is BUV395 ... The fluorescent dye for CD11c is BV510, the fluorescent dye for CD56 is BV570, the fluorescent dye for CD197 is BV605, the fluorescent dye for CD69 is BV650, the fluorescent dye for CD25 is BV711, the fluorescent dye for CCR4 is BV786, the fluorescent dye for CXCR5 is BB515, the fluorescent dye for IgA is FITC, the fluorescent dye for CD14 is RB545, the fluorescent dye for CD8 is NovaFluorBlue 585, the fluorescent dye for CD4 is BB630, the fluorescent dye for mitochondrial superoxide is MitoSOX Red, the fluorescent dye for CD27 is BB660, the fluorescent dye for IgG is BB700, the fluorescent dye for TCR gd is RB705, the fluorescent dye for CD127 is RB744, the fluorescent dye for TIGIT is RB780, and the fluorescent dye for IgD is PerCP / Fire TM806, the fluorescent dye for CD366 is PE, and the fluorescent dye for CD20 is Spark YG TM 593, the fluorescent dye for CD123 is PE-CF594, and the fluorescent dye for CXCR3 is PE / Fire TM 640, the fluorescent dye for CD24 is PE / Cyanine5, the fluorescent dye for CD16 is PE-AF 700, the fluorescent dye for CD279 is PE-CY7, the fluorescent dye for HLA-DR is PE / Fire 810, the fluorescent dye for CD39 is R718, the fluorescent dye for CD45RA is APC-H7, and the fluorescent dye for CD38 is APC / Fire TM 810.

[0010] In some embodiments of the present application, healthy human peripheral blood mononuclear cells are used for antibody titration before the formal experiment. The essence of antibody titration is to find the best balance between the specific and nonspecific binding of antibodies. Too low a concentration of antibody cannot saturate or effectively bind to the antigen site, resulting in uneven staining or weak signal; while too high a concentration of antibody causes an increase in low affinity and nonspecific binding efficiency. At this time, both negative and positive group signals increase, but the grouping effect decreases. It is reflected by the antibody titration working curve. Selecting staining conditions that do not cause a significant increase in background and have the best signal-to-noise ratio can reduce background interference while saving antibody usage and improve data quality. The method for antibody titration of the fluorescein-coupled antibody includes: creating an initial titration concentration of twice the recommended amount of fluorescein-coupled antibody, setting several concentration gradients by multiple dilution; adding healthy human peripheral blood mononuclear cells blocked by FC to each tube of diluted fluorescein-coupled antibody and mixing; performing conventional staining steps, performing on-machine detection and collecting data; determining the optimal staining concentration of the fluorescein-coupled antibody by calculating the staining index: ; Among them, Stain Index represents the staining index, MFI positive population Represents the mean fluorescence intensity of the positive population, MFI negative population Represents the mean fluorescence intensity of the negative population, rSD negative population Represents the relative standard deviation of the negative population.

[0011] In some embodiments, a single tube sample is generally 1×10 6 As the upper limit, the dilution was performed from 2 times the recommended amount, including 6 gradients of negative control. Through calculation, the specific dilution ratio of each fluorescent antibody was clarified, and the accuracy of the detection was improved. The application also used healthy human peripheral blood mononuclear cells to test the repeatability and reliability of the scheme. The CV value of the cell clustering ratio data obtained by the detection method of the application was less than 10%, indicating that the detection method of the application has good repeatability and reliability.

[0012] In some embodiments of the present application, the peripheral blood mononuclear cells are obtained by processing a blood sample from a patient with systemic lupus erythematosus, and the processing method includes: adding an equal volume of the blood sample and a lymphocyte separation solution preheated to room temperature into a first centrifuge tube, centrifuging at a temperature of 20°C~25°C for 25min~35min, a speed of 2000rpm~2500rpm, an increase speed set to 1~3, and a decrease speed set to 0; placing the white film layer cells separated by centrifugation into a second centrifuge tube, adding a phosphate buffered saline solution and centrifuging for 3min~10min, a speed of 1000rpm~1500rpm, and resuspending and counting the white film layer cells with a phosphate buffered saline solution to obtain the peripheral blood mononuclear cells.

[0013] In some embodiments of the present application, the surface staining method comprises: resuspending the target number of peripheral blood mononuclear cells using Hank's balanced salt solution to obtain a resuspended cell solution; adding a monocyte blocking agent and a fixable live and dead cell identification dye to the resuspended cell solution, and incubating at room temperature in the dark for 10 min to 20 min, wherein the volume ratio of the Hank's balanced salt solution, the monocyte blocking agent and the fixable live and dead cell identification dye is (15 to 30): 1: (0.05 to 0.2); adding the continuously expressed or difficult to stain detection indicators CCR6, I Fluorescently conjugated antibodies of gM, CD28, CCR7, CCR4, CXCR5, IgA, IgG, CD127 and CXCR3 were filled up to the first target volume with Brilliant staining buffer and added to the blocked resuspended cell solution, and pre-stained at room temperature in the dark for 10 min to 20 min; fluorescein-conjugated antibodies of the remaining detection indicators were added to the pre-stained system, filled up to the second target volume with flow staining solution, and stained at room temperature in the dark for 20 min to 40 min; the surface-stained peripheral blood mononuclear cells were resuspended in the flow staining solution, centrifuged and the supernatant was discarded.

[0014] In some embodiments of the present application, the method for staining intracellular mitochondrial superoxide includes: adding MitoSox Red dye to surface-stained peripheral blood mononuclear cells, incubating for 20 min to 40 min at 30°C to 40°C and 2% to 10% volume concentration of carbon dioxide, adding flow staining solution to resuspend, centrifuging and discarding the supernatant; resuspending the peripheral blood mononuclear cells stained with intracellular mitochondrial superoxide in the detection solution.

[0015] In some embodiments of the present application, the single-stained reference samples used to detect cell life and death and intracellular reactive oxygen levels are prepared using the same cells as the test samples and are labeled with corresponding fluorescein-conjugated antibodies, respectively, and the remaining single-stained reference samples are prepared using compensation microspheres.

[0016] In some embodiments of the present application, the negative samples used to detect cell life and death and intracellular reactive oxygen levels are prepared using the same cells as the test samples, but are not labeled with fluorescein-conjugated antibodies, and the remaining negative samples are prepared using compensation microspheres or unstained cells.

[0017] In some embodiments of the present application, the method of performing gating and dimensionality reduction analysis on the parsed data includes: using FlowJo software to perform detailed gating analysis on the data, using the Workspace column tSNE plug-in to perform dimensionality reduction analysis on the data, and displaying the expression of activated molecules in each subgroup.

[0018] In some embodiments of the present application, the dimensionality reduction analysis method includes: detecting whether the instrument state is stable when collecting data by Time / FSC H, removing adhesions by FSC A / FSC H, and marking live cells with a dye-negative group for fixable dead and live cells identification; distinguishing lymphocytes from monocytes by CD45 / SSC-A, and distinguishing granulocytes by CD45 / SSC-BA; distinguishing different differentiation stages of monocytes by different expressions of CD14 and CD16: CD14 hi CD16 − Represents classical monocytes, CD14 hi CD16 + Represents intermediate monocytes, CD14 + CD16 hi Represents nonclassical monocytes; in granulocytes, CD16 - Markers: eosinophils, CD16 + Neutrophil marker, CD123 + 、CD38 + , HLA-DR - Marks basophils; in lymphocytes, it is expressed by CD 3- 、CD56 + , HLA-DR - Mark NK cells and further distinguish NK cells based on the different expressions of CD56 and CD16: CD56+CD16- represents Early NK, CD56 + CD16 + Represents Mature NK, CD56 - CD16 +Stands for terminal NK; in lymphocytes, CD3 - 、CD19 - 、CD56 low , HLA-DR + 、CD14 - Mark DC cells and further distinguish DC cells based on the different expressions of CD11C and CD123: CD11C + CD123 + Represents myeloid DC, CD123 + CD11C - Represents conventional DC; lymphocytes are identified by CD3 - , TCRγδ - 、CD19 + 、CD20 low 、CD38 + 、CD24 - Plasmablast markers, identified by CD3 - , TCRγδ - 、CD19 + 、CD20 + Marks B cells and further differentiates them based on their expression of CD24 and CD38: CD38 + CD24 - Represents plasmablasts, CD38 + CD24 + Represents transitional B cells, CD38 - CD24 + Represents memory B cells, CD38 - CD24 - Represents mature naive B cells; distinguishes memory B cells based on the different expression of CD27 and CD21: CD27 + CD21 - Represents activated memory B cells, CD27 + CD21 + Represents resting memory B cells, CD27 - CD21 - Stands for tissue-like memory B cells, CD27 - CD21 + Represents naive B cells; based on CD21 - CD38 - Differentiate effector B cells, further through IgD + IgM + / - Represents unconverted effector B cells, expressed through IgD - IgM + / - Represents converted effector B cells, through CD21 + CD38 + / -Represents the non-effector B cell population; within the non-effector B cell population, it is further differentiated based on the different expressions of IgD and CD27: CD27 - IgD - Represents double negative B cells, CD27 - IgD + Represents transitional B cells and naive B cells, CD27 + IgD + Represents unconverted memory B cells, CD27 + IgD - Stands for converted memory B cells; converted memory B cells are distinguished based on the different expressions of IgA and IgG: IgA - IgG - Represents Ig-negative memory B cells, IgA + IgG - Represents memory B cells expressing IgA, IgA - IgG + Represents memory B cells expressing IgG; or based on IgM and IgG, the converted memory B cells are divided into memory B cells that only express IgM, and IgM + IgG - Representative; in non-converted memory B cells, further differentiated according to the different expression of IgM and CD27: IgM + CD27 + Stands for marginal zone B cells, IgM - CD27 + Represents memory B cells that only express IgD; in lymphocytes, CD3 - , TCRγδ - 、CD19 - 、CD20 - 、CD123 - 、CD14 - 、CD127 + , HLA-DR - Marks ILC cells; in lymphocytes, CD3 + , TCRγδ - 、CD56 - Mark T cells and further distinguish CD4 T cells and CD8 T cells based on CD4 / CD8. Among them, the subtypes of CD4 T cells are distinguished based on CXCR5, CXCR3, CXCR4, and CXCR6: CXCR5 - CXCR4 + stands for helper T cell 2, CXCR3 + CCR6 - stands for helper T cell 1, CXCR3 - CCR6+ Represents helper T cell 17, CXCR5 + CXCR3 + / - stands for follicular helper T cell, CCR4 - CCR6 + Stands for helper T cell 9; CD4 T cells distinguish memory cell subsets based on CCR7 and CD45RA: CD45RA + CCR7 + Represents naive CD4 T cells, CD45RA - CCR7 + Represents central memory CD4 T cells, CD45RA - CCR7 - Represents effector memory CD4 T cells, CD45RA + CCR7 - Represents terminally differentiated effector memory CD4 T cells that re-express CD45RA. The activation status of each subtype is further detected based on the expression of CD27 and CD28. Regulatory T cells are detected by CD4 + CD25 + CD127 - Marker; CD8 T cells distinguish memory cell subsets based on CXCR7 and CD45RA: CD45RA + CCR7 + Represents naive CD8 T cells, CD45RA - CCR7 + Represents central memory CD8 T cells, CD45RA - CCR7 - Represents effector memory CD8 T cells, CD45RA + CCR7 - Represents terminally differentiated effector memory CD8 T cells that re-express CD45RA, and the activation status of each subtype is detected based on the expression of CD27 and CD28.

[0019] In some embodiments of the present application, the method for performing a sample loading test and collecting particle information for analysis includes: loading the negative sample, adjusting the FSC and SSC voltages of the spectral flow cytometer, displaying all target cell populations, and after the setting is completed, checking whether the fluorescence intensity of the test sample exceeds the maximum detection range under this setting; loading the single-stained reference samples in turn, and collecting particle information, and observing whether there is positive expression and whether the spectrum that appears is correct during the process of collecting particle information; after the particle information of all the single-stained reference samples is collected, adjusting the position of the gate in the analysis window of the spectral flow cytometer, selecting the population to be analyzed, moving the negative gate and the positive gate to the appropriate position in the histogram, and checking to ensure that the spectrum of the positive area is correct and the background of the negative area is clean; roughly drawing a circle gate, and then loading the test sample, saving the analysis data after the test is completed.

[0020] Compared with the prior art, the detection method of systemic lupus erythematosus immune profile based on full-spectrum flow cytometry in the present application has the following beneficial effects: The detection method of the present application can detect dozens of immune cell subtypes and their activation states in SLE under the condition of a single tube sample, construct an immune map of SLE patients, and provide a reference for evaluating disease progression and formulating treatment plans. Compared with the detection of traditional flow cytometry, the number of markers that can be detected simultaneously is greatly increased, which saves the amount of samples and antibodies, improves accuracy, and through the spectral scheme design and the data analysis method in the spectral flow technology, the problems of fluorescence leakage and complex compensation adjustment in the traditional flow can be well solved. Compared with single-cell sequencing analysis, spectral flow cytometry has lower cost and stronger clinical applicability, and can provide cell typing information equivalent to single-cell sequencing data. The CV value of the cell clustering ratio data obtained using the detection method of the present application is less than 10%, indicating that the detection method has good repeatability and reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings describe in detail the exemplary embodiments disclosed in this application. The same reference numerals represent similar structures in several views of the drawings. Those skilled in the art will understand that these embodiments are non-limiting, exemplary embodiments, and the drawings are only used for the purpose of illustration and description, and are not intended to limit the scope of this application. Other embodiments may also accomplish the inventive intent in this application. It should be understood that the drawings are not drawn to scale. Among them: Figure 1 The distribution of the detection antibody and the corresponding fluorescein in the laser of the spectroscopy instrument of Example 1 of the present application; Figure 2 This is a schematic diagram of the spectral similarity analysis results of the flow cytometry antibody fluorescein in Example 1 of the present application; Figure 3The flow cytometry antibody titration result of Example 1 of the present application; Figure 4 This is a schematic diagram of the gate logic setting of the main cell subsets in Example 1 of the present application; Figure 5 This is a schematic diagram of the immune profile and cell activation state analysis after dimensionality reduction analysis in Example 1 of the present application; Figure 6 This is the repeatability reliability test data of Example 1 of the present application. DETAILED DESCRIPTION

[0022] For ease of understanding of the present invention, the application is described in more detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the application is not limited to the ad hoc methods, schemes, cell lines, constructs and reagents described herein, and is equally changeable. Unless otherwise defined, all technical and scientific terms used in this specification are identical with the meanings generally understood by those skilled in the art of the present invention. The terms used in this specification in the specification of the application are only for the purpose of describing specific embodiments, and are not intended to limit the application.

[0023] There are studies on the detection of immune cell clustering in blood samples of healthy people. Since blood samples of healthy people are relatively uniform, it is relatively easy to detect them using full-spectrum flow cytometry. However, unlike blood samples of healthy people, the microenvironment of blood samples of SLE patients is extremely complex, which makes it very challenging to detect immune cell clustering in blood samples of SLE patients. So far, there is no technology that can detect blood samples of SLE patients using full-spectrum flow cytometry.

[0024] Based on this, this application has developed for the first time a detection method for the systemic lupus erythematosus immune profile based on full-spectrum flow cytometry to assist in related clinical and basic research on disease diagnosis and treatment.

[0025] The method for detecting the systemic lupus erythematosus immune profile based on full-spectrum flow cytometry in the embodiment of the present application comprises the following steps: S1: Fluorescently conjugated antibodies for the following detection indicators are provided according to the color scheme; S2: performing antibody titration on the fluorescein-conjugated antibody; S3: using the fluorescein-conjugated antibody to perform surface staining on peripheral blood mononuclear cells (PBMCs) in a blood sample of a patient with systemic lupus erythematosus, and to perform intracellular mitochondrial superoxide staining, and resuspending in a detection solution to obtain a test sample; S4: loading the test sample and the prepared single-stained reference sample and negative sample on a spectral flow cytometer in a predetermined order for testing, and collecting particle information for analysis; S5: Perform gating and dimensionality reduction analysis on the parsed data to obtain the immune profile of systemic lupus erythematosus and the analysis results of cell activation status.

[0026] In step S1, the detection indicators are selected according to the immune characteristics of SLE patients, and the detection indicators include CD57, L / D, CD3, CD21, CD19, CCR6, IgM, CD45, CD95, CD28, CD11c, CD56, CCR7, CD69, CD25, CCR4, CXCR5, IgA, CD14, CD8, CD4, mitochondrial superoxide, CD27, IgG, TCR gd, CD127, TIGIT, IgD, CD366, CD20, CD123, CXCR3, CD24, CD16, CD279, HLA-DR, CD39, CD45RA, and CD38.

[0027] The color scheme is based on the color matching principle to distribute the fluorescein to the detection index. The color matching principle adopted in this application is briefly described as follows: First, collect as much biological information as possible around the detection index, and clearly detect the expression of the antigen on the cells to be detected. Generally, highly expressed antigens are called Class I antigens, moderately expressed or continuously expressed antigens are called Class II antigens, and antigens with low expression or unknown expression information are called Class III antigens, and the co-expression information of antigens must be clarified. Secondly, in the selection of fluorescein, it is necessary to follow that in the same experiment, all fluoresceins have a unique spectrum; highly expressed antigens are paired with weak fluorescein, and low expressed antigens are paired with strong fluorescein. Specifically, for Class I antigens, there are relatively abundant fluorophores to choose from, and weaker fluorophores can be used to minimize fluorescence spillover to other fluorescent channels. For Class II antigens, consider whether there is co-expression of medium-expressing antigens. If not, any other brighter fluorophores can be used. If co-expression occurs, try to use brighter fluorophores that will not spread to Class III antigens. If the only fluorophore that can be selected has a relatively large spread, use a weaker fluorophore to reduce the impact of the spread. For Class III antigens, assign the brightest fluorophore and evaluate the availability of reagents (there are usually not many commercial antibodies to choose from).

[0028] It should be noted that the above color matching principles can only provide a general idea of ​​color matching, and it is extremely difficult to obtain the final color matching scheme, and each additional color will multiply the difficulty of distributing fluorescein. It can be seen that it is very difficult to obtain a color matching scheme for 39-color dyeing. After a lot of research, the inventors of this application finally determined the following preferred color matching scheme: The fluorescein of CD57 is BUV395, the fluorescein of L / D is FVS440, the fluorescein of CD3 is BUV496, the fluorescein of CD21 is BUV563, the fluorescein of CD19 is BUV615, the fluorescein of CCR6 is BUV661, the fluorescein of IgM is BUV737, the fluorescein of CD45 is BUV805, the fluorescein of CD95 is BV421, and the fluorescein of CD28 is BV580 , the fluorescein of CD11c is BV510, the fluorescein of CD56 is BV570, the fluorescein of CD197 is BV605, the fluorescein of CD69 is BV650, the fluorescein of CD25 is BV711, the fluorescein of CCR4 is BV786, the fluorescein of CXCR5 is BB515, the fluorescein of IgA is FITC, the fluorescein of CD14 is RB545, the fluorescein of CD8 is NovaFluor Blue 585, the fluorescein of CD4 is BB630, the fluorescein of mitochondrial superoxide is MitoSOX Red, the fluorescein of CD27 is BB660, the fluorescein of IgG is BB700, the fluorescein of TCR gd is RB705, the fluorescein of CD127 is RB744, the fluorescein of TIGIT is RB780, and the fluorescein of IgD is PerCP / Fire TM 806, the fluorescent dye for CD366 is PE, and the fluorescent dye for CD20 is Spark YG TM 593, the fluorescent dye for CD123 is PE-CF594, and the fluorescent dye for CXCR3 is PE / Fire TM 640, the fluorescent dye for CD24 is PE / Cyanine5, the fluorescent dye for CD16 is PE-AF 700, the fluorescent dye for CD279 is PE-CY7, the fluorescent dye for HLA-DR is PE / Fire 810, the fluorescent dye for CD39 is R718, the fluorescent dye for CD45RA is APC-H7, and the fluorescent dye for CD38 is APC / Fire TM 810.

[0029] In step S2, the method for titrating the fluorescein-coupled antibody comprises: creating an initial titration concentration as twice the concentration recommended in the fluorescein-coupled antibody manual, and setting a number of concentration gradients by multiple dilution. Then, healthy human peripheral blood mononuclear cells blocked by FC are added to each tube of diluted fluorescein-coupled antibody and mixed. Then, conventional staining steps are performed, and on-machine detection and data collection are performed. Finally, the optimal staining concentration of the fluorescein-coupled antibody is determined by calculating the staining index.

[0030] In some embodiments, in step S3, the peripheral blood mononuclear cells are obtained by processing a blood sample of a patient with systemic lupus erythematosus, and the processing method comprises the following steps: S31: Add equal volumes of the blood sample and lymphocyte separation solution preheated to room temperature to the first centrifuge tube, centrifuge at 20°C to 25°C for 25min to 35min, with a rotation speed of 2000rpm to 2500rpm, and set the acceleration speed to 1 to 3 and the deceleration speed to 0: S32: placing the buffy coat cells separated by centrifugation in a second centrifuge tube, adding phosphate buffered saline (PBS) and centrifuging for 3 min to 10 min at a speed of 1000 rpm to 1500 rpm, and resuspending and counting the buffy coat cells with phosphate buffered saline (PBS) to obtain the peripheral blood mononuclear cells.

[0031] In some embodiments, in step S2, the method for performing surface staining comprises the following steps: S33: resuspending the target number of peripheral blood mononuclear cells using Hank's Balanced Salt Solution (HBSS) to obtain a resuspended cell solution; S34: adding a true-stain monocyte blocker and a fixable viability dye to the resuspended cell solution, and incubating at room temperature in the dark for 10 min to 20 min, wherein the volume ratio of the Hank's balanced salt solution, the monocyte blocker and the fixable viability dye is (15-30):1:(0.05-0.2); S35: Use Brilliant stain buffer to make up the volume to the first target volume with fluorescein-conjugated antibodies of the continuously expressed or difficult-to-stain detection indicators CCR6, IgM, CD28, CCR7, CCR4, CXCR5, IgA, IgG, CD127 and CXCR3, and add them to the blocked resuspended cell solution. Pre-stain at room temperature in the dark for 10 min to 20 min. S36: Add the remaining fluorescent conjugated antibodies of the detection indicators to the pre-stained system, fill up to the second target volume with staining buffer, and stain at room temperature in the dark for 20 min to 40 min; S37: Resuspend the surface-stained peripheral blood mononuclear cells in flow cytometry staining solution, centrifuge and discard the supernatant.

[0032] In some embodiments, in step S3, the method for staining intracellular mitochondrial superoxide comprises: S38: Add MitoSox Red dye to the surface-stained peripheral blood mononuclear cells, incubate at 30°C-40°C and 2%-10% volume concentration of carbon dioxide for 20-40 minutes, add flow staining solution to resuspend, centrifuge and discard the supernatant; S39: resuspending the peripheral blood mononuclear cells stained with intracellular mitochondrial superoxide in the detection solution.

[0033] In step S4, the single-stained reference sample, that is, the reference sample with only one fluorescent antibody added, is used to adjust the compensation between the fluorescent channels to eliminate the interference caused by the overlap of the fluorescence spectrum. It can also be used to verify the specificity and sensitivity of the antibody to ensure the accuracy of the experimental results, and is generally placed in a single-stained reference tube for sample testing. Since the present application is a 39-color staining, 39 single-stained reference samples are set accordingly. Among them, the single-stained reference samples used to detect cell life and death and intracellular reactive oxygen levels are prepared using the same cells as the test samples, and are respectively labeled with corresponding fluorescein-coupled antibodies, and the remaining single-stained reference samples are prepared using compensation microspheres.

[0034] The negative sample refers to a sample that has not been treated in any way. It is used as a control group in the experiment to determine the normal cell state and exclude false positive results, and is generally placed in a negative tube for sample testing. The negative sample used to detect cell death and intracellular reactive oxygen levels is prepared using the same cells as the test sample, but without fluorescent conjugated antibody labeling. The remaining negative samples are prepared using compensation microspheres or unstained cells.

[0035] In some embodiments, the method of performing a sample test and collecting particle information for analysis includes the following steps: S41: loading the negative sample, adjusting the FSC and SSC voltages of the spectral flow cytometer to display all target cell populations, and after the setting is completed, checking whether the fluorescence intensity of the test sample exceeds the maximum detection range under this setting; S42: loading the single-stained reference samples in sequence and collecting particle information, and in the process of collecting the particle information, observing whether there is positive expression and whether the spectrum that appears is correct; S43: After all the particle information of the single-stained reference samples is collected, the gate position is adjusted in the analysis window of the spectral flow cytometer, the population to be analyzed is selected, the negative gate and the positive gate are moved to appropriate positions in the histogram, and the spectrum of the positive area is checked to ensure that the spectrum of the positive area is correct and the background of the negative area is clean; S44: Draw a rough circle and then load the test sample. After the test is completed, save the analysis data.

[0036] In step S5, the method of performing gating and dimensionality reduction analysis on the parsed data includes: performing gating analysis on the data using FlowJo software, performing dimensionality reduction analysis on the data using the tSNE plug-in in the Workspace column, and displaying the expression of activated molecules in each subgroup.

[0037] In some preferred embodiments, the dimensionality reduction analysis method includes: detecting whether the instrument state is stable when collecting data by Time / FSC H, removing adhesions by FSC A / FSC H, marking live cells with Fixable Viability Dye negative population, distinguishing lymphocytes from monocytes by CD45 / SSC-A, and distinguishing granulocytes by CD45 / SSC-BA. Monocytes are distinguished by the different expression of CD14 and CD16 at different differentiation stages of monocytes: classical monocytes (CD14 hi CD16 − ), intermediate monocytes (CD14 hi CD16 + ) and nonclassical monocytes (CD14 + CD16 hi ). In granulocytes, CD16 - Markers: eosinophils, CD16 + Neutrophil marker, CD123 + 、CD38 + , HLA-DR - Marks basophils.

[0038] CD3 - 、CD56 + , HLA - DR -Mark NK cells and further distinguish NK cells into Early NK (CD56 + CD16 - )、Mature NK(CD56 + CD16 + )、terminal NK(CD56 - CD16 + ).

[0039] CD3 - 、CD19 - 、CD56 low , HLA-DR + 、CD14 - DC cells were labeled and further classified into myeloid DC (mDC, CD11C + CD123 + ) and conventional DCs (cDCs, CD123 + CD11C - ).

[0040] CD3 - , TCRγδ - 、CD19 + 、CD20 low 、CD38 + 、CD24 - Plasmablast markers, identified by CD3 - , TCRγδ - 、CD19 + 、CD20 + B cells are marked and further differentiated into plasmablasts based on CD24 and CD38. + CD24 - ), transitional B cells (CD38 + CD24 + ), memory B cells (memory B cells, CD38 - CD24 + ), mature naive B cells (CD38 - CD24 - ); Based on CD27 and CD21, the types of memory B cells include: activated memory B cells (Activated memory B cells, CD27 + CD21 -), resting memory B cells (CD27 + CD21 + ), tissue-like memory B cells (CD27 - CD21 - ) and naive B cells (naive B cells, CD27 _ CD21 + ); According to CD21 - CD38 - Differentiate effector B cells, and further differentiate non-switched effector B cells (IgD + IgM + / - ) and switched effector B cells (IgD - IgM + / - ); in non-effector B cell populations (CD21 + CD38 + / - ) were further distinguished based on the expression of IgD and CD27. - IgD - ), transitional B cells and naive B cells (transitional+naive B cells, CD27 - IgD + ), non-switched memory B cells (CD27 + IgD + ), switched memory B cells (CD27 + IgD - ). Based on IgA and IgG, converted memory B cells are divided into surface Ig negative memory B cells (IgA - IgG - ), IgA memory B cells (IgA memory B cells, IgA + IgG - ) and IgG memory B cells (IgA - IgG +), can also be divided into IgM-only memory B cells (IgM-only memory B cells, IgM + IgG - Unconverted memory B cells can be further divided into marginal zone B cells (IgM + CD27 + ) and IgD-only memory B cells (IgM - CD27 + ).

[0041] CD3 - , TCRγδ - 、CD19 - 、CD20 - 、CD123 - 、CD14 - 、CD127 + , HLA-DR - Labeling of ILC cells.

[0042] CD3 + , TCRγδ - 、CD56 - T cells are marked and further distinguished into CD4 T cells and CD8 T cells based on CD4 / CD8. Among them, CD4 T cells are distinguished into subtypes based on CXCR5, CXCR3, CXCR4, and CXCR6, including helper T cells 2 (th2, CXCR5 - CXCR4 + ), helper T cells 1 (th1, CXCR3 + CCR6 - ), helper T cells 17 (th17, CXCR3 - CCR6 + ), follicular helper T cells (tfh, CXCR5 + CXCR3 + / - ), helper T cells 9 (th9, CCR4 - CCR6 + CD4T cells are divided into memory cell subsets based on CCR7 and CD45RA, and are specifically divided into naive CD4 T cells (naive CD4 T cells, CD45RA + CCR7 + )、central memory CD4 T cells(central memory CD4 T cell,CD45RA- CCR7 + ), effector memory CD4 T cells (effector memory CD4 T cells, CD45RA - CCR7 - ) and terminally differentiated effector memory CD4 T cells that re-express CD45RA + CCR7 - ), and then further detect the activation status of each subtype based on the expression of CD27 and CD28. + CD25 + CD127 - CD8 T cells are classified into memory cell subsets based on CXCR7 CD45RA, which are specifically divided into naive CD8 T cells (naive CD8 T cells, CD45RA + CCR7 + )、central memory CD8 T cells(central memory CD8 T cell,CD45RA - CCR7 + ), effector memory CD8 T cells (effector memory CD8 T cells, CD45RA - CCR7 - ) and terminally differentiated effector memory CD8 T cells that re-express CD45RA + CCR7 - ), and the activation status of each subtype was detected according to the expression of CD27 and CD28.

[0043] The technical scheme of the present application will be described clearly and completely below in conjunction with the specific embodiments of the present application. Unless otherwise specified, the reagents and raw materials used can be purchased through commercial channels. The experimental methods for which specific conditions are not specified in the following specific embodiments are selected according to conventional methods and conditions, or according to the product specifications.

[0044] In the following examples, the spectral flow cytometer used was purchased from Cytek, model Aurora; the lymphocyte separation solution used was purchased from Stemcell, model Lymphoprep™ 07861; PBS was purchased from Source Culture, model B210KJ; HBSS was purchased from Source Culture, model B410KJ; true-stain monocyte blocker was purchased from BioLegend, model 426103; Fixable Viability Dye was purchased from BD Pharmingen, model 566332; Brilliant staining buffer was purchased from BD Pharmingen, model 566385; MitoSox Red dye was purchased from Invitrogen, model M36007; the sources of the fluorescein-conjugated antibodies used are shown in Table 1.

[0045] Table 1 Sources of fluorescein-conjugated antibodies

[0046] Example 1 This embodiment provides a method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry, comprising the following steps: 1. Perform spectral flow color matching According to the immune characteristics of SLE patients, the detection index is selected and the fluorescein is allocated to the detection index. Figure 1 The distribution of the detection antibody and the corresponding fluorochrome in the spectroscopy instrument laser is shown.

[0047] 2. Fluorescent antibody titration Take the initial titration concentration as 10µg / mL as an example: the initial concentration of an antibody is 0.2µg / µL, the experimental system is 100µL, and 6 concentration gradients are made (including the negative control group): Tube 1: 10µL antibody + 90µL diluent; Tube 2: 50µL antibody + 50µL diluent from tube 1; Tube 3: 50µL antibody + 50µL diluent from tube 2; Tube 4: 50µL antibody + 50µL diluent from tube 3; Tube 5: 50µL antibody + 50µL diluent from tube 4, mix well, take out 50µL antibody and discard it; Tube 6: only add 50µL diluent as a blank control.

[0048] The peripheral blood mononuclear cells of healthy people to be labeled were blocked by FC, and then 50 μL of about 1×10 6 cells, mix well, perform routine staining steps, and finally perform on-machine testing, collect data, and calculate according to Stain Index (SI)-staining index to find the optimal value. Figure 2 The results of the analysis of the spectral similarity of flow cytometry antibody and fluorescein are shown. Figure 3 The results are flow cytometry fluorescence antibody titration.

[0049] 3. Flow Cytometry (1) Before separating lymphocytes, preheat the centrifuge to 23°C and preheat the lymphocyte separation solution to room temperature. Add 4-5 mL of preheated lymphocyte separation solution to a 15 mL centrifuge tube, with the volume ratio of the solution to the fresh blood sample being approximately 1:1.

[0050] (2) Tilt the centrifuge tube and slowly drop the blood sample into it using a dropper so that the blood sample is on top of the lymphocyte separation fluid. The two fluids should be clearly layered, with the blood sample on the top and the lymphocyte separation fluid on the bottom.

[0051] (3) Centrifuge the above samples at 23°C for 30 min, with the speed set to 2200 rpm, the acceleration set to 2, and the deceleration set to 0.

[0052] (4) After centrifugation, carefully aspirate the buffy coat cells with a pipette, leaving as few as possible. Place the aspirated cells in a clean 15 mL centrifuge tube.

[0053] (5) Fill the centrifuge tube with PBS, wash the separated cells, and centrifuge at 1500 rpm for 5 minutes. After washing, resuspend the cells in an appropriate volume of PBS and count them for subsequent experimental staining. The separation of PBMC is complete.

[0054] (6) Surface staining: After counting, 1×10 6 PBMCs were resuspended in 20 μL HBSS, and 1 μL true-stain monocyte blocker and 0.1 μL Fixable Viability Dye were added to each tube. The cells were incubated at room temperature in the dark for 15 min.

[0055] Fluorescently conjugated antibodies of the continuously expressed or difficult-to-stain detection indicators CCR6, IgM, CD28, CCR7, CCR4, CXCR5, IgA, IgG, CD127 and CXCR3 were made up to 10 μL / tube using Brilliant stain buffer, and added to the blocked resuspended cell solution for pre-staining at room temperature in the dark for 15 minutes.

[0056] After pre-staining, add the remaining fluorescent conjugated antibodies for the detection indicators, fill up to 20 μL / tube with Staining buffer, and stain for 30 minutes at room temperature in the dark. Add 1 mL of Staining buffer to resuspend, centrifuge, 800g, 4°C, 5 minutes, and discard the supernatant.

[0057] (7) Mitochondrial superoxide staining: In this example, MitoSox Red dye was used to detect intracellular reactive oxygen species, and the working concentration for staining was 5 μM. Add 100 μL of MitoSox Red dye at a working concentration to the cells in the previous step, and incubate for 30 min at 37°C and 5% volume concentration of carbon dioxide. Add 1 mL of Staining buffer to resuspend, centrifuge, 800 g, 4°C, 5 min, and discard the supernatant. Add 150 μL of detection solution to resuspend the cells and wait for loading.

[0058] 4. On-machine testing (1) Experimental plan creation: In the Acquisition interface, create a new experiment and select the fluorescent label used in this experiment. Then set the single-stained reference tube and sample tube, and define the sample type of the single-stained reference tube in turn. The default is cells. In the next step, add the antibody label, and finally set the stop condition for data acquisition. After the settings are completed, click Save and Open to complete the creation.

[0059] (2) Adjust the collection conditions and instrument settings: First, select Cytek AssaySetting in User setting, load the negative tube at a low speed, adjust the voltage of FSC and SSC, and display all the target cell populations. After the settings are completed, load the sample tube at a low speed and check whether the fluorescence intensity of the sample exceeds the maximum detection range under this setting.

[0060] (3) Collect single-stained reference tube information: Load and collect samples in the order of the single-stained reference tubes. During the collection process, you can observe whether the single-stained reference tubes have positive expression and whether the spectrum is correct.

[0061] (4) Data analysis: After all single-stained reference tube data are collected, click Unmix above the experiment. In the pop-up analysis window, adjust the gate position and select the population to be analyzed. Move the positions of the negative gate and the positive gate in the histogram so that the negative gate contains most of the negative population and the positive gate is at the position with the highest fluorescence intensity. Then check whether the spectrum of the positive area is correct and the background of the negative area is clean. After the adjustment is completed, click Live Unmix in the lower right corner.

[0062] (5) Gate setting: After Live unmixing, create a new Unmixed worksheet and roughly draw the gate. You can refer to Figure 4Then, the sample tube is loaded with sample at a low speed, and the data is saved after the experiment.

[0063] 5. Data Analysis FlowJo software was used to perform detailed gate analysis on the parsed data, and the tSNE plug-in in the Workspace column was used to perform dimensionality reduction analysis on the parsed data, and the expression of activation molecules (CD69, PD-1, CD39, IgA, MitoSox Red, Tim3, FAS, TIGHT, CD57, CD127) in each subgroup was displayed, such as Figure 5 shown.

[0064] Reliability Verification In order to test the repeatability and reliability of this protocol, peripheral blood from healthy subjects was used in the experiment, and two experimenters conducted the experiment independently.

[0065] Design experimental groups: In order to ensure the accuracy and reliability of experimental data, provide more accurate fluorescence compensation and verify antibody specificity, set up blank groups, single staining groups, and sample groups in the flow cytometry experimental plan. For each experiment, the samples were repeated twice or three times.

[0066] like Figure 6 As shown, the CV values ​​of the cell population ratio data obtained by the detection method of the present application are all less than 10%, indicating that the detection method of the present application has good repeatability and reliability. The CV values ​​of the data between the two experimenters are all less than 10%, indicating that the detection method of the present application has good intermediate precision.

[0067] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry, characterized in that: include: Fluorescently conjugated antibodies for the following detection indicators are provided according to the color scheme: CD57, L / D, CD3, CD21, CD19, CCR6, IgM, CD45, CD95, CD28, CD11c, CD56, CCR7, CD69, CD25, CCR4, CXCR5, IgA, CD14, CD8, CD4, mitochondrial superoxide, CD27, IgG, TCR gd, CD127, TIGIT, IgD, CD366, CD20, CD123, CXCR3, CD24, CD16, CD279, HLA-DR, CD39, CD45RA, CD38; performing antibody titration on the fluorescein-coupled antibody to obtain the staining concentration of the fluorescein-coupled antibody; The fluorescein-coupled antibody is used to perform surface staining on peripheral blood mononuclear cells in a blood sample of a patient with systemic lupus erythematosus, and intracellular mitochondrial superoxide staining is performed, and the cells are resuspended in a detection solution to obtain a test sample; The test sample and the prepared single-stained reference sample and negative sample are loaded on the spectral flow cytometer for testing in a predetermined order, and particle information is collected for analysis; The analyzed data were gated and dimensionally reduced to obtain the immune profile of systemic lupus erythematosus and the analysis results of cell activation status.

2. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The color scheme is as follows: the fluorescein of CD57 is BUV395, the fluorescein of L / D is FVS440, the fluorescein of CD3 is BUV496, the fluorescein of CD21 is BUV563, the fluorescein of CD19 is BUV615, the fluorescein of CCR6 is BUV661, the fluorescein of IgM is BUV737, the fluorescein of CD45 is BUV805, the fluorescein of CD95 is BV421, the fluorescein of CD28 is BUV496, the fluorescein of CD57 is BUV495, the fluorescein of CD21 is BUV563, the fluorescein of CD19 is BUV615, the fluorescein of CCR6 is BUV661, the fluorescein of IgM is BUV737, the fluorescein of CD45 is BUV805, the fluorescein of CD95 is BV421, the fluorescein of CD28 is BUV496, the fluorescein of CD31 is BUV563, the fluorescein of CD19 is BUV615, the fluorescein of CCR6 is BUV661, the fluorescein of IgM is BUV737, the fluorescein of CD45 is BUV805, the fluorescein of CD95 is BV421, the fluorescein of CD28 is BUV496, the fluorescein of V580, the fluorescein of CD11c is BV510, the fluorescein of CD56 is BV570, the fluorescein of CD197 is BV605, the fluorescein of CD69 is BV650, the fluorescein of CD25 is BV711, the fluorescein of CCR4 is BV786, the fluorescein of CXCR5 is BB515, the fluorescein of IgA is FITC, the fluorescein of CD14 is RB545, the fluorescein of CD8 is NovaFluor Blue 585, the fluorescein of CD4 is BB630, the fluorescein of mitochondrial superoxide is MitoSOX Red, the fluorescein of CD27 is BB660, the fluorescein of IgG is BB700, the fluorescein of TCR gd is RB705, the fluorescein of CD127 is RB744, the fluorescein of TIGIT is RB780, and the fluorescein of IgD is PerCP / Fire TM 806, the fluorescent dye for CD366 is PE, and the fluorescent dye for CD20 is Spark YG TM 593, the fluorescent dye for CD123 is PE-CF594, and the fluorescent dye for CXCR3 is PE / Fire TM 640, the fluorescent dye for CD24 is PE / Cyanine5, the fluorescent dye for CD16 is PE-AF 700, the fluorescent dye for CD279 is PE-CY7, the fluorescent dye for HLA-DR is PE / Fire 810, the fluorescent dye for CD39 is R718, the fluorescent dye for CD45RA is APC-H7, and the fluorescent dye for CD38 is APC / Fire TM 810.

3. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The method for performing antibody titration on the fluorescein-coupled antibody comprises: Create a starting titration concentration that is twice the recommended amount of fluorescein-conjugated antibody, and set up several concentration gradients by serial dilutions; Add healthy human peripheral blood mononuclear cells blocked by FC to each tube of diluted fluorescein-conjugated antibody and mix well; Perform routine staining steps, conduct on-machine testing and collect data; The optimal staining concentration of the fluorescein-conjugated antibody was determined by calculating the staining index: ； Among them, Stain Index represents the staining index, MFI positive population Represents the mean fluorescence intensity of the positive population, MFI negative population Represents the mean fluorescence intensity of the negative population, rSD negative population Represents the relative standard deviation of the negative population.

4. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The peripheral blood mononuclear cells are obtained by processing a blood sample of a patient with systemic lupus erythematosus, and the processing method includes: Add equal volumes of the blood sample and lymphocyte separation solution preheated to room temperature into the first centrifuge tube, centrifuge at 20°C to 25°C for 25min to 35min, with a rotation speed of 2000rpm to 2500rpm, with the acceleration setting at 1 to 3 and the deceleration setting at 0; The buffy coat cells separated by centrifugation are placed in a second centrifuge tube, and phosphate buffered saline is added and centrifuged for 3min to 10min at a speed of 1000rpm to 1500rpm. The buffy coat cells are resuspended and counted with phosphate buffered saline to obtain the peripheral blood mononuclear cells.

5. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The surface dyeing method comprises: Resuspending the target number of peripheral blood mononuclear cells using Hank's balanced salt solution to obtain a resuspended cell solution; Adding a monocyte blocking agent and a fixable live or dead cell identification dye to the resuspended cell solution, and incubating in the dark at room temperature for 10 min to 20 min, wherein the volume ratio of the Hank's balanced salt solution, the monocyte blocking agent and the fixable live or dead cell identification dye is (15-30):1:(0.05-0.2); Use Brilliant staining buffer to make up the volume to the first target volume with fluorescein-conjugated antibodies of the continuously expressed or difficult-to-stain detection indicators CCR6, IgM, CD28, CCR7, CCR4, CXCR5, IgA, IgG, CD127 and CXCR3, and add them to the blocked resuspended cell solution. Pre-stain at room temperature in the dark for 10-20 minutes. Add the remaining fluorescent conjugated antibodies of the detection indicators to the pre-stained system, fill up to the second target volume with flow staining solution, and stain at room temperature in the dark for 20min~40min; The surface-stained peripheral blood mononuclear cells were resuspended in flow cytometry staining solution, centrifuged and the supernatant was discarded.

6. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The method for staining intracellular mitochondrial superoxide comprises: Add MitoSox Red dye to the surface-stained peripheral blood mononuclear cells, incubate for 20 min to 40 min at 30°C to 40°C and 2% to 10% volume concentration of carbon dioxide, add flow staining solution to resuspend, centrifuge and discard the supernatant; The peripheral blood mononuclear cells stained with intracellular mitochondrial superoxide were resuspended in the detection solution.

7. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The single-stained reference samples used to detect cell life and death and intracellular reactive oxygen levels were prepared using the same cells as the test samples and labeled with corresponding fluorescein-coupled antibodies, and the remaining single-stained reference samples were prepared using compensation microspheres.

8. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The negative samples used to detect cell life and death and intracellular reactive oxygen levels were prepared using the same cells as the test samples, but without fluorescein-conjugated antibody labeling, and the remaining negative samples were prepared using compensation microspheres or unstained cells.

9. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: The method of performing gating and dimensionality reduction analysis on the parsed data includes: performing detailed gating analysis on the data using FlowJo software, performing dimensionality reduction analysis on the data using the tSNE plug-in in the Workspace column, and displaying the expression of activated molecules in each subgroup.

10. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 9, characterized in that: The dimensionality reduction analysis method includes: detecting whether the instrument state is stable when collecting data by Time / FSC H, removing adhesions by FSC A / FSC H, and identifying the live and dead cells by a dye-negative group that can be fixed to mark live cells; Lymphocytes and monocytes were distinguished by CD45 / SSC-A, and granulocytes were distinguished by CD45 / SSC-BA; Monocytes can be distinguished into different differentiation stages by the different expressions of CD14 and CD16: CD14 hi CD16 − Represents classical monocytes, CD14 hi CD16 + Represents intermediate monocytes, CD14 + CD16 hi represents nonclassical monocytes; CD16 - Markers: eosinophils, CD16 + Neutrophil marker, CD123 + 、CD38 + , HLA-DR - Marking of basophils; CD4+ 3- 、CD56 + , HLA-DR - Mark NK cells and further distinguish NK cells based on the different expressions of CD56 and CD16: CD56+CD16- represents Early NK, CD56 + CD16 + Represents Mature NK, CD56 - CD16 + It stands for terminal NK; CD3 - 、CD19 - 、CD56 low , HLA-DR + 、CD14 - Mark DC cells and further distinguish DC cells based on the different expressions of CD11C and CD123: CD11C + CD123 + Represents myeloid DC, CD123 + CD11C - represents conventional DC; CD3 - , TCRγδ - 、CD19 + 、CD20 low 、CD38 + 、CD24 - Plasmablast markers, identified by CD3 - , TCRγδ - 、CD19 + 、CD20 + Marks B cells and further differentiates them based on their expression of CD24 and CD38: CD38 + CD24 - Represents plasmablasts, CD38 + CD24 + Represents transitional B cells, CD38 - CD24 + Represents memory B cells, CD38 - CD24 - represents mature naive B cells; Differentiation of memory B cells based on differential expression of CD27 and CD21: CD27 + CD21 - Represents activated memory B cells, CD27 + CD21 + Represents resting memory B cells, CD27 - CD21 - Stands for tissue-like memory B cells, CD27 - CD21 + represents naive B cells; According to CD21 - CD38 - Differentiate effector B cells, further through IgD + IgM + / - Represents unconverted effector B cells, expressed through IgD - IgM + / - Represents converted effector B cells, through CD21 + CD38 + / - represents the non-effector B cell population; In the non-effector B cell population, further differentiation is made based on the differential expression of IgD and CD27: CD27 - IgD - Represents double negative B cells, CD27 - IgD + Represents transitional B cells and naive B cells, CD27 + IgD + Represents unconverted memory B cells, CD27 + IgD - stands for switched memory B cells; Differentiation of converted memory B cells based on the differential expression of IgA and IgG: IgA - IgG - Represents Ig-negative memory B cells, IgA + IgG - Represents memory B cells expressing IgA, IgA - IgG + Represents memory B cells expressing IgG; or based on IgM and IgG, the converted memory B cells are divided into memory B cells that only express IgM, and IgM + IgG - represent; Among the unconverted memory B cells, they are further differentiated based on the different expression of IgM and CD27: IgM + CD27 + Stands for marginal zone B cells, IgM - CD27 + represents memory B cells that express only IgD; CD3 - , TCRγδ - 、CD19 - 、CD20 - 、CD123 - 、CD14 - 、CD127 + , HLA-DR - Labeling of ILC cells; CD3 + , TCRγδ - 、CD56 - Mark T cells and further distinguish CD4 T cells and CD8 T cells based on CD4 / CD8. Among them, the subtypes of CD4 T cells are distinguished based on CXCR5, CXCR3, CXCR4, and CXCR6: CXCR5 - CXCR4 + stands for helper T cell 2, CXCR3 + CCR6 - stands for helper T cell 1, CXCR3 - CCR6 + Represents helper T cell 17, CXCR5 + CXCR3 + / - stands for follicular helper T cell, CCR4 - CCR6 + represents helper T cell 9; CD4 T cells distinguish memory cell subsets based on CCR7 and CD45RA: CD45RA + CCR7 + Represents naive CD4 T cells, CD45RA - CCR7 + Represents central memory CD4 T cells, CD45RA - CCR7 - Represents effector memory CD4 T cells, CD45RA + CCR7 - represents terminally differentiated effector memory CD4 T cells that re-express CD45RA, and the activation status of each subtype was further tested based on the expression of CD27 and CD28; Regulatory T cells through CD4 + CD25 + CD127 - mark; CD8 T cells distinguish memory cell subsets based on CXCR7 and CD45RA: CD45RA + CCR7 + Represents naive CD8 T cells, CD45RA - CCR7 + Represents central memory CD8 T cells, CD45RA - CCR7 - Represents effector memory CD8 T cells, CD45RA + CCR7 - Represents terminally differentiated effector memory CD8 T cells that re-express CD45RA, and the activation status of each subtype is detected based on the expression of CD27 and CD28.

11. The method for detecting the immune profile of systemic lupus erythematosus based on full-spectrum flow cytometry according to claim 1, characterized in that: Methods for conducting sample testing and collecting particle information for analysis include: The negative sample is loaded, and the voltages of FSC and SSC of the spectral flow cytometer are adjusted to display all target cell populations. After the setting is completed, it is checked whether the fluorescence intensity of the test sample exceeds the maximum detection range under this setting; The single-stained reference samples are loaded sequentially, and particle information is collected. During the process of collecting the particle information, it is observed whether there is positive expression and whether the spectrum that appears is correct; After all the particle information of the single-stained reference samples is collected, the gate position is adjusted in the analysis window of the spectral flow cytometer, the population to be analyzed is selected, the negative gate and the positive gate are moved to appropriate positions in the histogram, and the spectrum of the positive area is checked to ensure that the spectrum of the positive area is correct and the background of the negative area is clean; Draw a rough circle and then load the test sample. After the test, save the analysis data.

Images