CN114264825B - Method and kit for immunophenotyping of B lymphocyte development subpopulation - Google Patents
Method and kit for immunophenotyping of B lymphocyte development subpopulation Download PDFInfo
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to the technical field of immunology, in particular to a method and a kit for immunophenotyping of B lymphocyte development subpopulations. The method comprises the following steps: mixing different fluorescent-labeled antibodies with a sample to be detected, incubating, and detecting by flow cytometry to obtain detection data; the antibody comprises: anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, anti-CD 38 antibodies. The typing method provided by the invention realizes more comprehensive immune typing of the B lymphocyte development subgroup, needs less sample to be detected, is simple to operate, needs short time and has high accuracy, and can be widely applied to the immune typing of the B lymphocyte development subgroup.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method and a kit for immunophenotyping of B lymphocyte development subpopulations.
Background
B lymphocytes, abbreviated as B cells, are differentiated from lymphoid precursor cells in mammalian bone marrow or avian bursa of Fabricius. Mature B cells mainly colonize the lymph nodes in the superficial cortex of the lymph nodes and in the red and white medulla of the spleen. In peripheral blood, B cells account for about 20% of the total lymphocytes. B cells are cells that produce antibodies in vivo and have antigen presenting functions. B cells and antibodies produced thereby are central elements of humoral immunity, and as part of the adaptive immune system, can protect the human body against an almost limitless variety of pathogens. Defects in B cell development, selection and function lead to autoimmune, malignant, immunodeficiency and allergic diseases.
B cells can be divided into different developmental subgroups based on their different cell surface markers. Initial development of human B cells occurs in bone marrow, including the development of progenitor cells into pre-B cells and pre-B cell populations. After leaving the bone marrow, transitional B cells develop further in peripheral blood lymphoid organs and circulate in the blood as primary B cells. The primary B cells encounter antigens and are stimulated in the hair center and the border areas of lymphoid organs via T-dependent and independent pathways to form memory B cells, plasmablasts and long-lived plasma cells.
Serum immunoglobulin levels are widely recognized as the most reliable surrogate markers of B cell function in healthy donors and patients with various immune-related diseases. Whereas B cell development is important to understand the underlying mechanisms of B cell directed therapies and their impact on B cell autoantibody production and other functions. In addition to the treatment of hematological malignancies such as non-hodgkin's lymphoma and chronic lymphocytic leukemia, B-cell directed therapies also play an important role in the treatment of autoimmune diseases. Recent clinical studies in rheumatoid arthritis and systemic lupus erythematosus have shown that depletion of memory cells with rituximab is curative, but memory B cells are not the only target for antibody depletion, and thus these interventions may modulate other B cell populations with therapeutic relevance (Joshua a. Boyce et al., B cell-directed therapies for autoimmune disease and correlates of disease response and relay.j ALLERGY CLIN IMMUNOL,2008, 121:13-21.). Thus, research into B cell subsets is more urgent.
B lymphocytes in a blood sample are closely related to the immune system of an organism, and the total number of B cells and the change of the composition of the B cells are detected by using peripheral blood to be more dynamic. B cells have unique differentiation antigens and peripheral blood CD19 is routinely analyzed by flow cytometry for total B cells, however, no further detection of B cell development is performed. The surface of B cells has various receptors, such as membrane surface immunoglobulins, and most of them are detected by indirect fluorescence immunoassay or enzyme immunohistochemistry. Whether the flow cytometry is used for detecting the total B cells or the indirect fluorescence immunoassay or the enzyme immunohistochemical method is used, the detection surface of the methods for detecting the B cells is narrow, the operation is complex, the sample requirement is large, and the time consumption is relatively long. If more elaborate immunophenotyping of B cell development is desired, simpler, time-saving and less demanding methods and quantitative analysis of sample size are needed.
Disclosure of Invention
In view of the above, the invention provides a method and a kit for immunophenotyping of B lymphocyte development subpopulations, which can realize more comprehensive and fine immunophenotyping and quantitative analysis of B lymphocytes, have high efficiency, save the consumption of a sample to be tested, have short time consumption and are more suitable for the typing and quantitative analysis of B lymphocyte development subpopulations.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for immunophenotyping of B lymphocyte development sub-populations, which comprises the following steps:
mixing the antibodies with different fluorescent markers with a sample to be detected, incubating, detecting by a flow cytometry to obtain detection data, and judging the cell type according to the detection data;
the antibody comprises:
anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, and anti-CD 38 antibodies;
the method for judging the cell type comprises the following steps:
cell surface marker CD19 + CD10 + CD38 + IgD + Representing transitional B cells;
cell surface marker CD19 + CD10 - CD38 - IgD - Representing memory B cells;
cell surface marker CD19 + CD10 - CD38 + IgD - Represents plasmablasts;
cell surface marker CD19 + CD10 - CD38 - IgD + Representing untransformed B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 - IgD + Representing primary B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 + IgD + Representing border zone B cells.
In the present invention, the antibody used in the flow cytometry may be an antibody homologous to the sample to be measured or may be an antibody non-homologous to the sample to be measured, as long as the antibody is capable of generating an antigen-antibody specific binding reaction with a cell surface marker in the sample to be measured.
In the present invention, "+" represents positive, i.e., indicates that the antigen is expressed on the cell surface;
"++" indicates strong positive, i.e., high expression of the antigen on the cell surface;
"-" indicates negative, i.e., indicates that the antigen is not expressed on the cell surface.
In different stages of normal differentiation and maturation and in the activation process of lymphocytes, the cell membrane surfaces of the lymphocytes express surface markers for identification, and the monoclonal antibodies marked by fluorescein are used as molecular probes to carry out flow cytometry detection on the cell surface markers so as to analyze the types, subclasses and functional characteristics of the cells. The invention provides a method for aiming at T cell development subgroup immunophenotyping through a great number of creative researches and experimental verification: cell surface marker CD19 + CD10 + CD38 + IgD + When in use, transitional B cells can be subjected to immune typing; cell surface marker CD19 + CD10 - CD38 - IgD - When the cell is used, the memory B cells can be subjected to immune typing; cell surface marker CD19 + CD10 - CD38 + IgD - When in use, the plasma blast cells can be subjected to immune typing; cell surface marker CD19 + CD10 - CD38 - IgD + When the kit is used, the unconverted B cells can be subjected to immunophenotyping; cell surface marker CD19 + CD10 - CD38 - CD27 - IgD + When the method is used, the initial B cells can be subjected to immunophenotyping; cell surface marker CD19 + CD10 - CD38 - CD27 + IgD + In this case, the border region B cells can be immunophenotyped. According to the invention, the optimal antibody combination of the cell surface markers is designed and obtained according to the cell surface markers corresponding to the cells, and the B lymphocyte development subgroup is subjected to more comprehensive immune typing by flow cytometry. The method for the development and the immune typing of the B lymphocyte subpopulation provided by the invention needs few samples to be detected, and is simple and accurate to operateHigh sex, short time and can be used for the immunophenotyping of B lymphocyte development subgroup.
Preferably, in the method for immunophenotyping B lymphocyte development subpopulations, fluorescence minus a control (FMO control) is further arranged, and the FMO control is arranged in the sense of evaluating interference of other fluorescent dyes on a target channel, so that a threshold value of positive staining can be accurately determined, and positive gates can be accurately arranged.
In the method for immunophenotyping of B lymphocyte development subpopulations provided by the invention, a fluorescence minus one control (FMO control) is adopted as a control group. When detecting Percp-cy5.5-CD38, the FMO control tube is filled with all fluorescent antibodies except for Percp-cy5.5-CD 38. When detecting V450-CD27, the FMO control tube is filled with all fluorescent antibodies except V450-CD 27.
In the embodiment of the invention, the method for immunophenotyping the B lymphocyte development subgroup provided by the invention comprises the following specific steps:
mixing a sample to be detected with erythrocyte lysate, placing the obtained mixture in a water bath at 36.5-37.5 ℃ for 6-8 min, centrifuging to obtain a first product, mixing a fluorescent-labeled anti-CD 19 antibody, an anti-CD 10 antibody, an anti-IgD antibody, an anti-CD 27 antibody and an anti-CD 38 antibody with the first product, incubating for 15-20 min at room temperature (namely 20-25 ℃), washing, performing on-machine detection to obtain detection data, and judging the cell type according to the detection data;
the method for determining the cell type comprises the following steps:
cell surface marker CD19 + CD10 + CD38 + IgD + Representing transitional B cells;
cell surface marker CD19 + CD10 - CD38 - IgD - Representing memory B cells;
cell surface marker CD19 + CD10 - CD38 + IgD - Represents plasmablasts;
cell surface marker CD19 + CD10 - CD38 - IgD + Representing untransformed B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 - IgD + Representing primary B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 + IgD + Representing border zone B cells.
In the method provided by the invention, the fluorescent label in each fluorescent-labeled antibody is not limited by the invention, and a person skilled in the art can select a suitable fluorescent label and control method according to the actual situation. In an embodiment of the invention, the fluorescent labels of the five antibodies include APC, BV510, FITC, V450, or Percp-cy5.5, including but not limited to.
The invention provides an immunophenotyping method of a B lymphocyte development subgroup, which further comprises the step of detecting the number of B lymphocytes in a sample.
In an embodiment of the present invention, the method for detecting the number of B lymphocyte development groups in a sample to be tested according to the present invention comprises the steps of:
detecting the total number of lymphocytes in a sample to be detected;
detecting the percentage of B lymphocyte development sub-population in a sample to be detected to the lymphocytes;
and calculating to obtain the cell number of the B lymphocyte development subgroup in the sample to be detected.
In the method provided by the invention, the number of B lymphocytes is obtained by multiplying the total number of the B lymphocytes by the percentage of the B lymphocytes in the number of the B lymphocytes in the detection sample. In the invention, the absolute numbers of the cells of each B cell subgroup can be obtained by carrying out immunotyping and relative number statistics on each B cell development subgroup in a sample to be tested and multiplying the absolute numbers of the B lymphocytes.
In an embodiment of the present invention, the method provided by the present invention, the step of detecting the percentage of B lymphocytes in the sample, comprises:
mixing the fluorescent-labeled antibody with the sample to be detected, incubating, detecting by a flow cytometry to obtain detection data, analyzing the detection data, and calculating the percentage of B lymphocytes in the sample to be detected;
wherein the antibody used in detecting the percentage of B lymphocytes in the sample to be tested is an anti-CD 19 antibody.
In some embodiments of the invention, the methods of preparation provided herein wherein the step of detecting the percentage of B lymphocytes in the sample comprises fluorescence labeling of the anti-CD 19 antibody with APC.
In the method provided by the invention, the step of detecting the percentage of B lymphocytes in the sample is a conventional lymphocyte immunophenotyping and quantitative analysis method, and the method is not limited by the invention, and a method for determining the percentage of B lymphocytes in the sample to be detected can be selected by a person skilled in the art according to actual conditions.
In the method provided by the invention, the total number of lymphocytes in the sample to be detected is counted by adopting a cytometer or a cell counter. In the method provided by the invention, the method for detecting the total number of lymphocytes in the sample to be detected is a conventional method, and the method is not limited by the invention, and a person skilled in the art can select the method for detecting the total number of lymphocytes in the sample to be detected according to the actual situation.
The invention also provides a method for immunophenotyping of the B lymphocyte development subgroup, which comprises the step of immunophenotyping of the B lymphocyte development subgroup provided by the invention;
the method for immunophenotyping the B lymphocyte development subgroup comprises the following steps:
mixing different fluorescent-labeled antibodies with a sample to be tested, incubating, detecting by flow cytometry to obtain detection data, and judging cell typing according to the detection data;
the antibody comprises:
anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, anti-CD 38 antibodies;
the method for cell typing comprises the following steps:
cell surface marker CD19 + CD10 + CD38 + IgD + Representing transitional B cells;
cell surface marker CD19 + CD10 - CD38 - IgD - Representing memory B cells;
cell surface marker CD19 + CD10 - CD38 + IgD - Represents plasmablasts;
cell surface marker CD19 + CD10 - CD38 - IgD + Representing untransformed B cells;
cell surface marker CD19 + CD10 - CD38 - CD27-IgD + Representing primary B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 + IgD + Representing border region B cells;
the invention also provides a kit for immunophenotyping of a B lymphocyte developmental subpopulation, comprising the following differently fluorescently labeled antibodies:
anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, anti-CD 38 antibodies.
The difference here means that the above five antibodies use different fluorescent labels.
In the kit for B lymphocyte development subgroup immunophenotyping, the fluorescent labeled antibody can be prepared by independently placing a fluorescent marker and the antibody, and coupling the fluorescent marker and the antibody to obtain the fluorescent labeled antibody when the kit is used; the antibody may be directly fluorescent labeled, or may be directly used when in use.
In some embodiments of the invention, the kit provided by the invention for immunophenotyping of a B lymphocyte developmental subpopulation comprises a fluorescent label and an antibody:
the antibody comprises:
anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, anti-CD 38 antibodies.
The fluorescent labels include APC, BV510, FITC, V450 or Percp-cy5.5, including but not limited to the above fluorescent labels.
In some embodiments of the invention, the fluorescent label of the anti-CD 19 antibody is APC in the kit provided herein for immunophenotyping of a subpopulation of B lymphocyte development.
In some embodiments of the invention, the fluorescent label of the anti-CD 10 antibody in the kit for immunophenotyping of a B lymphocyte developmental subpopulation provided by the invention is BV510.
In some embodiments of the invention, the fluorescent label of the anti-IgD antibody in the kit provided by the invention for immunophenotyping of the B lymphocyte developmental subpopulation is FITC.
In some embodiments of the invention, the invention provides a kit for immunophenotyping of a B lymphocyte developmental subpopulation, wherein the fluorescent label of the anti-CD 27 antibody is V450.
In some embodiments of the invention, the fluorescent label of the anti-CD 38 antibody in the kit for immunophenotyping of B lymphocyte developmental subpopulations provided by the invention is Percp-cy5.5.
In the method for immunophenotyping of B lymphocyte development subpopulations provided by the invention, a fluorescence minus one control (FMO control) is adopted as a control group. When detecting Percp-cy5.5-CD38, the FMO control tube is filled with all fluorescent antibodies except for Percp-cy5.5-CD 38. When detecting V450-CD27, the FMO control tube is filled with all fluorescent antibodies except V450-CD 27.
In the kit provided by the invention, the fluorescent markers in the respective fluorescent-labeled antibodies are not limited by the invention, and a person skilled in the art can select a suitable fluorescent marker and a corresponding control according to practical situations.
In the invention, in the flow cytometry detection process, the accuracy of the target cells can be judged through the target cell grouping and the fluorescence intensity of the target cells; the target cells are clearly grouped, and the fluorescence intensity of the target cells is high, so that the representation accuracy is high.
The invention provides a method and a kit for immunophenotyping of B lymphocyte development sub-populations. The invention provides a method for immunophenotyping of B lymphocyte development sub-populations, which comprises the following steps: taking different fluorescenceMixing the labeled antibody with a sample to be detected, incubating, detecting by a flow cytometry to obtain detection data, and analyzing the obtained detection data; the antibody comprises: anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, anti-CD 38 antibodies. The method of analysis includes: cell surface marker CD19 + CD10 + CD38 + IgD + Representing transitional B cells; cell surface marker CD19 + CD10 - CD38 - IgD - Representing memory B cells; cell surface marker CD19 + CD10 - CD38 + IgD - Represents plasmablasts; cell surface marker CD19 + CD10 - CD38 - IgD + Representing untransformed B cells; cell surface marker CD19 + CD10 - CD38 - CD27 - IgD + Representing primary B cells; cell surface marker CD19 + CD10 - CD38 - CD27 + IgD + Representing border zone B cells. Experimental results prove that the optimal antibody combination of the cell surface markers is obtained by the design, and the more comprehensive immunophenotyping and quantitative analysis of the B lymphocyte development subgroup are realized by flow cytometry. In some embodiments of the invention, the method provided by the invention has the advantages of small required sample quantity to be detected, simple operation, obvious grouping and short time consumption. In some embodiments of the invention, the method provided by the invention has good repeatability, obvious grouping and high accuracy, and can be widely used for the immunophenotyping and quantitative analysis of B lymphocyte development sub-populations.
Drawings
FIG. 1 shows the results of B lymphocyte classification in example 1;
FIG. 2 is a typing result of a B lymphocyte development sub-population in example 1;
FIG. 3 shows the results of B lymphocyte classification in example 2;
FIG. 4 is the typing results of the B lymphocyte development subpopulation in example 2;
FIG. 5 shows the results of B lymphocyte classification in example 3;
FIG. 6 is a typing result of a B lymphocyte development sub-population in example 3;
FIG. 7 shows the results of B lymphocyte classification in example 4;
FIG. 8 is a typing result of a B lymphocyte development sub-population in example 4;
FIG. 9 shows the results of B lymphocyte classification in example 5;
FIG. 10 shows the typing results of the B lymphocyte development sub-population in example 5.
Detailed Description
The invention provides a method and a kit for immunophenotyping of B lymphocyte development sub-populations. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
In the invention, the Chinese and English comparison is as follows:
the reagent and the raw materials used in the method and the kit for the immunophenotyping of the B lymphocyte development subgroup provided by the invention can be purchased from the market.
The fluorescent markers FITC, percp-cy5.5, BV510, V450 and APC used in the invention are common fluorescent markers and can be purchased from the market, and the antibodies of the fluorescent markers can be purchased from the market.
The invention is further illustrated by the following examples:
example 1B lymphocyte developmental subpopulation immunophenotyping and quantitative analysis
Experimental materials:
sample to be measured: anticoagulated peripheral blood samples, derived from healthy volunteers, were normal peripheral blood.
The anti-CD 19 antibody, the anti-CD 10 (BV 510) antibody, the anti-CD 38 (Percp-Cy5.5) antibody of the fluorescent-labeled APC were purchased from BioLegend, the anti-IgD (FITC) antibody, and the anti-CD 27 (V450) antibody were purchased from BDbiosciences.
Erythrocyte lysis red fluid (cat RT 122-02) was purchased from Tiangen Biochemical technologies Co.
The experimental method comprises the following steps:
taking 300 μl of anticoagulated peripheral blood sample, taking 200 μl of sample to be measured, and measuring lymphocyte absolute number by hemocytometer to obtain lymphocyte absolute number of 2.58X10 9 and/L.
100. Mu.L was left for detection of B lymphocyte development subpopulations for immunophenotyping and quantitative analysis of B lymphocyte development subpopulations.
B lymphocyte developmental subpopulation immunophenotyping
1. Taking two flow pipes, respectively marking as B-1 and B-2, wherein B-1 is a control group, B-2 is a group to be detected, respectively adding 50 mu L of sample to be detected into the two flow pipes B-1 and B-2, respectively adding 1mL of erythrocyte lysis red liquid into the flow pipes, fully vortex, carrying out water bath at 37 ℃ for 6 min-8 min, centrifuging 500g and 5min, and pouring out supernatant.
2. Adding the following antibodies into the B-1 and B-2 flow tubes according to the table 1, and incubating for 20min at room temperature in a dark place after full vortex;
TABLE 1 class of antibodies added and volume added in each flow tube
3. 1mL of LPBS,500g of each was added, centrifuged for 5min, washed once, suspended by 200. Mu.L of LPBS, and the results were analyzed by flow-on-machine.
Analysis of results:
by the above procedure, the relative numbers (percentages) of each B lymphocyte development subpopulation and the absolute numbers of lymphocytes were obtained. The absolute numbers of the B lymphocyte development subgroups are obtained by the relative numbers (percentage) of the B lymphocyte development subgroups and the absolute numbers of the lymphocyte development subgroups.
B lymphocyte development subpopulation immunophenotyping and quantitative analysis results (analyzed using FlowJo analysis software)
The results of B lymphocyte immunophenotyping are shown in FIG. 1-A, FIG. 2-B and FIG. 2-C, and the relative numbers (percentages) of B cells and various B cell development sub-populations are shown in the figure, and the absolute numbers of the respective B cell sub-populations are calculated according to the absolute numbers of lymphocytes and the relative numbers (percentages) of the respective B cell sub-populations, and the specific experimental results are shown in Table 2.
TABLE 2 cell surface markers for immunophenotyping of individual B cell subsets, relative and absolute numbers of individual B cell subsets
Normal value range reference: elena Blanco et al, age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018, J ALLERGY CLIN IMMUNOL, 141 (6): 2208-2219."-" represents the absolute number of B cells in the border region that was not examined from the literature.
According to FIGS. 1 and 2, the method used in the present case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, unconverted B cells, initial B cells, marginal zone B cells, and quantitatively analyze the B lymphocytes to obtain the relative numbers and absolute numbers of the respective B lymphocyte development subgroups.
In summary, the method provided by the invention can realize the immunotyping of the development subpopulations of the B lymphocyte of the sample to be detected by adopting a small amount of sample to be detected, and count the content of each cell subpopulation, and can accurately and clearly carry out the immunotyping of each B lymphocyte development subpopulation. As can be seen from Table 2, the absolute numbers of the individual cell subsets substantially agree with the normal human reference range. The experimental result of the invention is stable and accurate. Except that the values of plasmablasts and unconverted B cells are not within the reference range, it is possible to relate to normal human sources in the literature, which are from foreign sources.
In summary, the method provided by the invention adopts fewer samples to be detected, so that lymphocyte immunophenotyping of the samples to be detected is realized, and the content of each cell subgroup is counted; the method provided by the invention can accurately carry out immunophenotyping on each cell subgroup.
Example 2B lymphocyte developmental subpopulation immunophenotyping and quantitative analysis
Experimental materials:
sample to be measured: the anticoagulated peripheral blood sample is derived from a venous blood sample (informed consent) of a patient who visits from a hospital and is peripheral blood of a patient who is about to diagnose neutrophil-mediated autoinflammation.
The anti-CD 19 antibody, the anti-CD 10 (BV 510) antibody, the anti-CD 38 (Percp-Cy5.5) antibody of the fluorescent-labeled APC were purchased from BioLegend, the anti-IgD (FITC) antibody, and the anti-CD 27 (V450) antibody were purchased from BDbiosciences.
Erythrocyte lysis red fluid (cat RT 122-02) was purchased from Tiangen Biochemical technologies Co.
The experimental method comprises the following steps:
taking 300 μl of anticoagulated peripheral blood sample, taking 200 μl of sample to be measured, and measuring lymphocyte absolute number by hemocytometer to obtain lymphocyte absolute number of 2.92×10 9 and/L.
100. Mu.L was left for detection of B lymphocyte development subpopulations for immunophenotyping and quantitative analysis of B lymphocyte development subpopulations.
B lymphocyte developmental subpopulation immunophenotyping
In the same manner as in the B lymphocyte classification method described in example 1, 50. Mu.L of a sample to be measured was added to the flow tube, then 1mL of a erythrocyte lysis solution was added to the flow tube, and after sufficient vortexing, the mixture was centrifuged at 37℃in a water bath for 6 to 8 minutes, 500g for 5min and the supernatant was decanted.
Antibodies were then added to the flow tube: APC-labeled anti-CD 19 antibody, BV 510-labeled anti-CD 10 antibody, percp-cy 5.5-labeled anti-CD 38 antibody, FITC-labeled anti-IgD antibody, V450-labeled anti-CD 27 antibody, after sufficient vortexing, incubated at room temperature in the dark for 20min.
B lymphocyte immunophenotyping and quantitative analysis results (analyzed using FlowJo analysis software)
The results of B lymphocyte immunophenotyping are shown in FIGS. 3-A,3-B, and FIGS. 4-A, 4-B and 4-C, wherein the absolute numbers of the B cells and the respective B cell subsets are calculated from the relative numbers (percentages) of the B cells and the respective B cell subsets, and the specific experimental results are shown in Table 3.
TABLE 3 cell surface markers for immunophenotyping of individual B cell subsets, relative and absolute numbers of individual B cell subsets
Normal value range reference: elena Blanco et al, age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood 2018, J ALLERGY CLIN IMMUNOL, 141 (6): 2208-2219; yuan Ding et al Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018, JALLERGY CLIN IMMUNOL, 1-4."-" represents the relative and absolute numbers of B cells in B lymphocytes from the border region that were not examined in the literature.
From fig. 3 and fig. 4, it can be seen that the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, unconverted B cells, initial B cells, and marginal zone B cells, and quantitatively analyze the same to obtain the relative numbers and absolute numbers of the respective B lymphocyte development subgroups. From the results, the relative numbers and absolute values of memory B cells and plasmablasts were lower than normal, while the absolute values of primary B cells and unconverted B cells were higher than normal, suggesting a humoral immune dysfunction in the infant.
In summary, the method provided by the invention can realize the immunotyping of the development subpopulations of the B lymphocyte of the sample to be detected by adopting a small amount of sample to be detected, and count the content of each cell subpopulation, and can accurately and clearly carry out the immunotyping of each B lymphocyte development subpopulation.
Example 3B lymphocyte developmental subpopulation immunophenotyping and quantitative analysis
Experimental materials:
sample to be measured: the anticoagulated peripheral blood sample is derived from a venous blood sample (informed consent) of a patient who visits in our hospital and is peripheral blood of a patient who is about to diagnose liver failure.
The anti-CD 19 antibody, the anti-CD 10 (BV 510) antibody, the anti-CD 38 (Percp-Cy5.5) antibody of the fluorescent-labeled APC were purchased from BioLegend, the anti-IgD (FITC) antibody, and the anti-CD 27 (V450) antibody were purchased from BDbiosciences.
Erythrocyte lysate (cat RT 122-02) was purchased from Tiangen Biochemical technologies Co.
The experimental method comprises the following steps:
taking 300 μl of anticoagulated peripheral blood sample, taking 200 μl of sample to be measured, and measuring lymphocyte absolute number by hemocytometer to obtain lymphocyte absolute number of 5.14X10 9 and/L.
100. Mu.L was left for detection of B lymphocyte development subpopulations for immunophenotyping and quantitative analysis of B lymphocyte development subpopulations.
B lymphocyte developmental subpopulation immunophenotyping
In the same manner as in the B lymphocyte classification method described in example 1, 50. Mu.L of a sample to be measured was added to the flow tube, then 1mL of a erythrocyte lysis solution was added to the flow tube, and after sufficient vortexing, the mixture was centrifuged at 37℃in a water bath for 6 to 8 minutes, 500g for 5min and the supernatant was decanted.
Antibodies were then added to the flow tube: APC-labeled anti-CD 19 antibody, BV 510-labeled anti-CD 10 antibody, percp-cy 5.5-labeled anti-CD 38 antibody, FITC-labeled anti-IgD antibody, V450-labeled anti-CD 27 antibody, after sufficient vortexing, incubated at room temperature in the dark for 20min.
B lymphocyte immunophenotyping and quantitative analysis results (analyzed using FlowJo analysis software)
The results of B lymphocyte immunophenotyping are shown in FIGS. 5-A,5-B, and FIGS. 6-A, 6-B and 6-C, wherein the absolute numbers of the B cells and the respective B cell subsets are calculated from the relative numbers (percentages) of the B cells and the respective B cell subsets, and the specific experimental results are shown in Table 4.
TABLE 4 cell surface markers for immunophenotyping of individual B cell subsets, relative and absolute numbers of individual B cell subsets
Note that: * Normal value range reference: elena Blanco et al, age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018, J ALLERGY CLIN IMMUNOL, 141 (6): 2208-2219.Yuan Ding et al Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018, JALLERGY CLIN IMMUNOL, 1-4."-" represents the relative and absolute numbers of B cells in B lymphocytes from the border region that were not examined in the literature.
From fig. 5 and fig. 6, it can be seen that the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, unconverted B cells, initial B cells, marginal zone B cells, and quantitatively analyze the same to obtain the relative numbers and absolute numbers of the respective B lymphocyte development subgroups. From the results, it was found that the relative numbers and absolute numbers of total and transitional B cells were increased, the absolute values of unconverted B cells were higher than normal, and the relative numbers and absolute numbers of plasmablasts were decreased, suggesting that the infant had humoral immune dysfunction.
In summary, the method provided by the invention can realize the immunotyping of the development subpopulations of the B lymphocyte of the sample to be detected by adopting a small amount of sample to be detected, and count the content of each cell subpopulation, and can accurately and clearly carry out the immunotyping of each B lymphocyte development subpopulation.
Embodiment 4B lymphocyte development subpopulation immunophenotyping and quantitative analysis
Experimental materials:
sample to be measured: the anticoagulation peripheral blood sample is derived from a venous blood sample (informed consent) of the sick child who is treated in the hospital and is the peripheral blood of the sick child who is about to treat biliary tract locking.
The anti-CD 19 antibody, the anti-CD 10 (BV 510) antibody, the anti-CD 38 (Percp-Cy5.5) antibody of the fluorescent-labeled APC were purchased from BioLegend, the anti-IgD (FITC) antibody, and the anti-CD 27 (V450) antibody were purchased from BDbiosciences.
Erythrocyte lysate (cat RT 122-02) was purchased from Tiangen Biochemical technologies Co.
The experimental method comprises the following steps:
taking 300 μl of anticoagulated peripheral blood sample, taking 200 μl of sample to be tested, and measuring lymphocyte absolute number by hemocytometer to obtain lymphocyte absolute number of 7.31X10 9 and/L.
100. Mu.L was left for detection of B lymphocyte development subpopulations for immunophenotyping and quantitative analysis of B lymphocyte development subpopulations.
B lymphocyte developmental subpopulation immunophenotyping
In the same manner as in the B lymphocyte classification method described in example 1, 50. Mu.L of a sample to be measured was added to the flow tube, then 1mL of a erythrocyte lysis solution was added to the flow tube, and after sufficient vortexing, the mixture was centrifuged at 37℃in a water bath for 6 to 8 minutes, 500g for 5min and the supernatant was decanted.
Antibodies were then added to the flow tube: APC-labeled anti-CD 19 antibody, BV 510-labeled anti-CD 10 antibody, percp-cy 5.5-labeled anti-CD 38 antibody, FITC-labeled anti-IgD antibody, V450-labeled anti-CD 27 antibody, after sufficient vortexing, incubated at room temperature in the dark for 20min.
B lymphocyte immunophenotyping and quantitative analysis results (analyzed using FlowJo analysis software)
The results of B lymphocyte immunophenotyping are shown in FIGS. 7-A,7-B, and FIGS. 8-A, 8-B and 8-C, wherein the absolute numbers of the B cells and the respective B cell subsets are calculated from the relative numbers (percentages) of the B cells and the respective B cell subsets, and the specific experimental results are shown in Table 5.
TABLE 5 cell surface markers for immunophenotyping of individual B cell subsets, relative and absolute numbers of individual B cell subsets
* Normal value range reference: elena Blanco et al, age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018, J ALLERGY CLIN IMMUNOL, 141 (6): 2208-2219.Yuan Ding et al Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018, JALLERGY CLINIMMUNOL, 1-4.
"-" represents the relative and absolute numbers of B cells in B lymphocytes from the border region that were not examined in the literature.
From fig. 7 and 8, it can be seen that the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, unconverted B cells, initial B cells, marginal zone B cells, and quantitatively analyze the relative numbers and absolute numbers of the respective B lymphocyte development subgroups. From the results, the absolute numbers of transitional B cells and absolute numbers were decreased, the absolute values of unconverted B cells were higher than normal, and the relative numbers of initial B cells and memory B cells were decreased, but the absolute values were normal, suggesting that the infant had humoral immune dysfunction.
In summary, the method provided by the invention can realize the immunotyping of the development subpopulations of the B lymphocyte of the sample to be detected by adopting a small amount of sample to be detected, and count the content of each cell subpopulation, and can accurately and clearly carry out the immunotyping of each B lymphocyte development subpopulation.
Example 5B lymphocyte developmental subpopulation immunophenotyping and quantitative analysis
Experimental materials:
sample to be measured: anticoagulated peripheral blood samples, derived from healthy volunteers (women, 26 years old), were normal peripheral blood.
The anti-CD 19 antibody, the anti-CD 10 (BV 510) antibody, the anti-CD 38 (Percp-Cy5.5) antibody of the fluorescent-labeled APC were purchased from BioLegend, the anti-IgD (FITC) antibody, and the anti-CD 27 (V450) antibody were purchased from BDbiosciences.
Lymphocyte isolates were purchased from daceae, biotechnology limited.
The experimental method comprises the following steps:
taking 3mL of anticoagulated peripheral blood sample, taking 200 μl of sample to be tested, and measuring lymphocyte absolute number by using a hemocytometer to obtain lymphocyte absolute number of 1.13X10 9 and/L.
The remaining peripheral blood was used to extract Peripheral Blood Mononuclear Cells (PBMC) for detection and quantitative analysis of B lymphocyte development subpopulations.
B lymphocyte developmental subpopulation immunophenotyping
After peripheral blood is diluted by PBS, lymphocyte separation liquid is added into the diluted peripheral blood, centrifugation is carried out for 800g and 20min, and after centrifugation, an intermediate buffy coat is sucked, namely PBMC.
Taking outAntibody was added to the flow tube: APC-labeled anti-CD 19 antibody, BV 510-labeled anti-CD 10 antibody, percp-cy 5.5-labeled anti-CD 38 antibody, FITC-labeled anti-IgD antibody, V450-labeled anti-CD 27 antibody, after sufficient vortexing, incubated at room temperature in the dark for 20min.
B lymphocyte immunophenotyping and quantitative analysis results (analyzed using FlowJo analysis software)
The results of B lymphocyte immunophenotyping are shown in FIGS. 9-A,9-B, and FIGS. 10-A, 10-B and 10-C, wherein the absolute numbers of the B cells and the respective B cell subsets are calculated from the relative numbers (percentages) of the B cells and the respective B cell subsets, and the specific experimental results are shown in Table 6.
TABLE 6 cell surface markers for immunophenotyping of individual B cell subsets, relative and absolute numbers of individual B cell subsets
* Normal value range reference: elena Blanco et al, age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood.2018, J ALLERGY CLIN IMMUNOL, 141 (6): 2208-2219.Yuan Ding et al Reference values for peripheral blood lymphocyte subsets of healthy children in China.2018, JALLERGY CLIN IMMUNOL, 1-4."-" represents the absolute number of B cells in the border region that was not examined from the literature.
From fig. 9 and 10, it can be seen that the method used in this case can accurately divide B lymphocytes into transitional B cells, memory B cells, plasmablasts, unconverted B cells, initial B cells, marginal zone B cells, and quantitatively analyze the relative numbers and absolute numbers of the respective B lymphocyte development subgroups. As can be seen from Table 2, the absolute numbers of the individual cell subsets substantially agree with the normal human reference range. The experimental result of the invention is stable and accurate.
In summary, the method provided by the invention can realize the immunotyping of the development subpopulations of the B lymphocyte of the sample to be detected by adopting a small amount of sample to be detected, and count the content of each cell subpopulation, and can accurately and clearly carry out the immunotyping of each B lymphocyte development subpopulation.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A method of immunophenotyping a developmental subpopulation of B lymphocytes, comprising:
mixing the antibodies with different fluorescent markers with a sample to be detected, incubating, detecting by a flow cytometry to obtain detection data, and judging the cell type according to the detection data;
the antibody comprises:
anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, and anti-CD 38 antibodies;
the method for judging the cell type comprises the following steps:
cell surface marker CD19 + CD10 + CD38 + IgD + Representing transitional B cells;
cell surface marker CD19 + CD10 - CD38 - IgD - Representing memory B cells;
cell surface marker CD19 + CD10 - CD38 + IgD - Represents plasmablasts;
cell surface marker CD19 + CD10 - CD38 - IgD + Representing untransformed B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 - IgD + Representing primary B cells;
cell surface marker CD19 + CD10 - CD38 - CD27 + IgD + Representing border region B cells;
the fluorescent label includes APC, BV510, FITC, V450 or Percp-cy5.5.
2. The method of claim 1, further comprising the step of counting the number of B lymphocytes in the sample to be tested, comprising:
1) Detecting the total number of lymphocytes in the sample to be detected;
2) Detecting the percentage of B lymphocytes in the sample to be detected to the lymphocytes;
3) And obtaining the number of B lymphocytes in the sample to be detected through calculation.
3. The method according to claim 2, wherein said step 2) comprises:
mixing the fluorescent-labeled antibody with the sample to be detected, incubating, detecting by flow cytometry to obtain detection data, and analyzing the detection data;
the antibody is an anti-CD 19 antibody.
4. A kit for immunophenotyping of a subpopulation of B lymphocyte development comprising fluorescently labeled antibodies to:
anti-CD 19 antibodies, anti-CD 10 antibodies, anti-IgD antibodies, anti-CD 27 antibodies, and anti-CD 38 antibodies;
the B lymphocyte developmental subpopulations include transitional B cells, memory B cells, plasmablasts, unconverted B cells, naive B cells, and marginal zone B cells.
5. The kit of claim 4, wherein the fluorescent label comprises APC, BV510, FITC, V450, or Percp-cy5.5.
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