CN104360053A - B lymphocyte immunophenotyping method and kit - Google Patents

B lymphocyte immunophenotyping method and kit Download PDF

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CN104360053A
CN104360053A CN201410485865.1A CN201410485865A CN104360053A CN 104360053 A CN104360053 A CN 104360053A CN 201410485865 A CN201410485865 A CN 201410485865A CN 104360053 A CN104360053 A CN 104360053A
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antibody
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lymphocyte
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CN104360053B (en
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赵晓东
周丽娜
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Childrens Hospital of Chongqing Medical University
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    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/5047—Cells of the immune system
    • G01N33/5052—Cells of the immune system involving B-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

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Abstract

The invention belongs to the field of immunological technique and discloses a B lymphocyte immunophenotyping method and a kit. The B lymphocyte immunophenotyping method provided by the invention comprises the following steps: taking different fluorescently-labeled antibodies and mixing the antibodies with a sample to be tested, carrying out incubation, carrying out flow cytometry detection to obtain test data, and analyzing the test data. The antibodies contain an anti-CD19 antibody, an anti-IgD antibody, an anti-CD38 antibody, an anti-CD24 antibody and an anti-CD27 antibody. By the above method, more comprehensive fine B lymphocyte immunophenotyping is realized. Few samples to be tested are required. The method is simple to operate. Required time is short. And the method has high accuracy and can widely be applied in lymphocyte subpopulation immunophenotyping.

Description

A kind of method of bone-marrow-derived lymphocyte immunophenotyping and kit
Technical field
The invention belongs to immunological technique field, particularly a kind of method of bone-marrow-derived lymphocyte immunophenotyping and kit.
Background technology
Lymphocyte (lymphocyte) also claim lymph corpuscle, is the one that volume in leucocyte is minimum, diameter 6-8 micron; Account for leukocyte count object 20%-30% at human body, round cell core, tenuigenin is little; Being produced by lymphoid organ, is the important cells composition of immune response function, is the clone that a class has Immune discrimination function.Lymphocyte is complicated heterogeneous population body, and can be divided into different classes of according to its phenotype and functional character, as T cell, B cell, NK cell etc., these cells can also be further divided into some subgroups.Lymphocyte and subgroup thereof mutually cooperate, mutually restrict in immune response process, jointly complete the identification to antigenic substance, response and removing, thus maintain body homeostasis.
Early stage research is moved by it according to lymphocyte, the difference of surface molecular and function, substantially lymphocyte is divided into T cell, B cell and natural killer (NK) cell.Wherein T cell can be divided into cytotoxic T cell (cytotoxic T lymphocyte, Tc), helper cell (helper T lymphocyte, Th), memory T cell (memory T lymphocyte, Tm) and adjustment/suppressor T cell (regulatory/suppressor T lymphocyte); B cell can be further divided into memory B cell (memory B cell), thick liquid cell (plasma cell), naive B cell deng subclass.
Along with the continuous progress of scientific research, scientist has found more cell surface marker, each quasi-lymphocyte can be carried out meticulousr cell subsets classification.Such as, can be by T cell Further Division: TCR α β +double negative t cells (double negative T lymphocyte, DNT), gamma delta T cells, auxiliary T cells, auxiliary deplete T cells, sectional center memory T cell, secondary effects memory T cell, cytotoxicity T cells, cytotoxicity deplete T cells, cytotoxicity Central memory T cell, cytotoxic effect memory T cell.
The kind of blood sample medium size lymphocyte and the immune system performance of body have important relationship.By carrying out immunophenotyping and quantitative test to blood sample medium size lymphocyte, the immunologic function of body can be evaluated more exactly, also can study the generation of some disease, development and treatment effectiveness evaluation better.At present, the method being usually used in lymphocyte immunity somatotype in blood sample and quantitative test mainly contains: immunoenzyme labeling method, common Lymphocyte subset, thinner Lymphocyte subtypes test.Immunoenzyme labeling method is just for a certain or specific antigen of a class or antibody generation idiosyncrasy, and detection faces is narrow, complicated operation, large to sample requirements to be detected, wastes time and energy.Common Lymphocyte subset, is widely used in lymphocyte and detects, but lymphocyte can only be divided into T cell, Tc cell, Th cell, B cell and NK cell, can not carry out somatotype further to cell.Thinner Lymphocyte subtypes test depends on flow cytometry, adopts the method for polychrome streaming, carries out comparatively detailed somatotype to lymphocyte, except traditional T cell, Tc cell, Th cell, B cell and NK extracellular, also detects Tm cell and cell quantity.But, if think to carry out immunophenotyping and quantitative test to cell subsets further, also need the cellular immunity classifying method by means of other, complicated operation, large to the demand of testing sample, and be not suitable for practical application.So, also needing to study the method for lymphocyte immunity somatotype and quantitative test, in the hope of utilizing simple method as far as possible, lymphocyte being carried out immunophenotyping and quantitative test more subtly.
Summary of the invention
In view of this, goal of the invention of the present invention is the method and the kit that provide a kind of bone-marrow-derived lymphocyte immunophenotyping.The method of this bone-marrow-derived lymphocyte immunophenotyping can carry out meticulous immunophenotyping and quantitative test more comprehensively to bone-marrow-derived lymphocyte, and efficiency is high, and has saved the consumption of testing sample, the immunophenotyping of more suitable bone-marrow-derived lymphocyte and quantitative test.
In order to realize goal of the invention of the present invention, the present invention adopts following technical scheme:
The invention provides a kind of method of bone-marrow-derived lymphocyte immunophenotyping, comprising:
Get different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain detection data, analyze described detection data;
This antibody comprises:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27;
The method of this analysis comprises:
Cell surface marker CD19 +d27 +represent memory B cell;
Cell surface marker CD19 +d27 -igD +represent naive B cell;
Cell surface marker CD19 +cD24 ++cD38 ++represent transition B cell;
Cell surface marker CD19 +cD24 -cD38 ++represent plasmablast.
In the present invention, antibody used in flow cytometry can be with the antibody of testing sample homology or can be also antibody nonhomologous with testing sample, reacts as long as this antibody can produce Ag-Ab specific binding with cell surface marker in testing sample.
In the present invention, "+" represents the positive, namely represents that this antigen has expression at cell surface;
" ++ " represents strong positive, namely represents that this antigen is at cell surface high expressed;
"-" represents feminine gender, namely represents that this antigen is not expressed at cell surface.
Lymphocytic surface marker refers to the membrane molecule being present in lymphocytic cell surface, they are lymphocyte identification antigen, interact and acknowledge(ment) signal stimulates and produces the material base of replying with other immunocytes, are also discriminatings and are separated lymphocytic important evidence.In lymphocytic differentiation and development process, lymphoid stem cells is divided into each cell subsets further, imparts the specific surface marker of each cell subsets.Some surface marker is that lymphocyte has, and some surface marker is that a certain class or a few quasi-lymphocyte are distinctive, so when carrying out lymphocyte immunity somatotype, can have many multiple combinations.But be not that often kind of combination can both realize lymphocytic immunophenotyping, the present invention is found by a large amount of creative works: for B cell, the present invention finds, cell surface marker is CD19 +d27 +time, immunophenotyping can be carried out to memory B cell; Cell surface marker is CD19 +d27 -igD +time, immunophenotyping can be carried out to naive B cell; Cell surface marker is CD19 +cD24 ++cD38 ++time, immunophenotyping can be carried out to transition B cell; Cell surface marker is CD19 +cD24 -cD38 ++time, immunophenotyping can be carried out to plasmablast.The present invention is according to the cell surface marker corresponding to each cell, design obtains the Antibody Combination of optimum cell surface marker, pass through flow cytometry, meticulous immunophenotyping and quantitative test have been carried out to bone-marrow-derived lymphocyte, perfect in further lymphocytic meticulous immunoassay and quantitative test.
Preferably, the invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, control group and Isotype control group are also set.The meaning arranging control group is the impact getting rid of irrelevant variable, increases confidence level and the cogency of experimental result; The meaning arranging Isotype control group is to distinguish the background signal impact that identical hypotype in antibody staining process causes.
In some embodiments of the invention, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, control group antibody specific used is: the antibody of anti-CD19.
In other embodiment of the present invention, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, Isotype control antibody specific used is: the Isotype antibody of BV450 mark, the Isotype antibody of BV510 mark, the Isotype antibody of PE mark, the Isotype antibody of Percp-cy5.5 mark.
In other embodiment of the present invention, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, Isotype control antibody specific used is: the Isotype antibody of APC mark, the Isotype antibody of BV510 mark, the Isotype antibody of PE mark, the Isotype antibody of Percp-cy5.5 mark.
In some embodiments of the invention, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of anti-CD19 is APC or APC-Cy7.
In other embodiment of the present invention, the invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, the fluorescence labeling of the antibody of anti-IgD is BV510.
In other embodiment of the present invention, the invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, the fluorescence labeling of the antibody of AntiCD3 McAb 8 is Percp-cy5.5.
In other embodiment of the present invention, the invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, the fluorescence labeling of the antibody of anti-CD24 is PE.
In other embodiment of the present invention, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of anti-CD27 is BV450 or APC.
Preferably, the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, is specially:
Get antibody, the antibody of fluorescently-labeled AntiCD3 McAb 8, the antibody of fluorescently-labeled anti-CD24, the antibody of fluorescently-labeled anti-CD27 of the antibody of fluorescently-labeled anti-CD19, fluorescently-labeled anti-IgD, mix with detected sample, under room temperature (20 DEG C ~ 25 DEG C) condition, after hatching 25min ~ 35min, mix with erythrocyte cracked liquid, after splitting erythrocyte, washing, machine testing in streaming, obtains detection data;
The method of this analysis comprises:
Cell surface marker CD19 +d27 +represent memory B cell;
Cell surface marker CD19 +d27 -igD +represent naive B cell;
Cell surface marker CD19 +cD24 ++cD38 ++represent transition B cell;
Cell surface marker CD19 +cD24 -cD38 ++represent plasmablast.
In other embodiment of the present invention, in method provided by the invention, in the method for bone-marrow-derived lymphocyte immunophenotyping, control group and the operation corresponding to Isotype control group are specially:
Get the antibody of fluorescently-labeled anti-CD19, the Isotype antibody of BV450 mark, the Isotype antibody of BV510 mark, the Isotype antibody of PE mark, the Isotype antibody of Percp-cy5.5 mark, mix with detected sample, under 20 DEG C ~ 25 DEG C conditions, after hatching 25min ~ 35min, mix with erythrocyte cracked liquid, after splitting erythrocyte, washing, machine testing in streaming, obtains detection data.
In other embodiment of the present invention, in method provided by the invention, in the method for bone-marrow-derived lymphocyte immunophenotyping, control group and the operation corresponding to Isotype control group are specially:
Get the antibody of fluorescently-labeled anti-CD19, the Isotype antibody of APC mark, the Isotype antibody of BV510 mark, the Isotype antibody of PE mark, the Isotype antibody of Percp-cy5.5 mark, mix with detected sample, under 20 DEG C ~ 25 DEG C conditions, after hatching 25min ~ 35min, mix with erythrocyte cracked liquid, after splitting erythrocyte, washing, machine testing in streaming, obtains detection data.
In the present invention, in method provided by the invention, the fluorescent marker in each fluorescently-labeled antibody is not by restriction of the present invention, and those skilled in the art can select suitable fluorescent marker according to actual conditions, and the Isotype control of correspondence.
Preferably, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, also comprise the step of bone-marrow-derived lymphocyte number in statistics testing sample.
In some embodiments of the invention, in the method for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the step detecting bone-marrow-derived lymphocyte number in sample comprises:
Detect the sum of testing sample medium size lymphocyte;
Detect bone-marrow-derived lymphocyte in testing sample and account for described lymphocytic number percent;
By calculating, obtain the number of bone-marrow-derived lymphocyte in testing sample.
In method provided by the invention, detect in sample in bone-marrow-derived lymphocyte number, TLC is multiplied by the number percent of wherein bone-marrow-derived lymphocyte, obtains the number of bone-marrow-derived lymphocyte.In the present invention by carrying out immunophenotyping and relative number statistics to each B cell subgroup of testing sample, then in conjunction with the absolute number of bone-marrow-derived lymphocyte, the cell absolute number of each B cell subgroup can be obtained.
In some embodiments of the invention, in method provided by the invention, detect the step of the number percent of bone-marrow-derived lymphocyte in sample, comprising:
Get different fluorescently-labeled antibody, mix with new described testing sample, after hatching, through Flow cytometry, obtain detection data, analyze described detection data;
Detect antibody used in the number percent of bone-marrow-derived lymphocyte in testing sample to comprise:
The antibody of the antibody of the antibody of anti-CD45, the antibody of AntiCD3 McAb, anti-CD16, the antibody of anti-CD56, anti-CD19;
Detect the antibody of anti-CD19 described in the antibody of anti-CD19 used in the number percent of bone-marrow-derived lymphocyte in testing sample and bone-marrow-derived lymphocyte immunophenotyping, independently of one another with fluorescence labeling.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, control group antibody used is: fluorescently-labeled anti-CD45 antibody, fluorescently-labeled anti-cd 3 antibodies, fluorescently-labeled CD4 antibody, fluorescently-labeled CD8 antibody.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the antibody fluorescence of anti-CD45 is labeled as Percp.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the fluorescence labeling of the antibody of AntiCD3 McAb is FITC.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the fluorescence labeling of the antibody of anti-CD16 is PE.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the fluorescence labeling of the antibody of anti-CD56 is PE.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the fluorescence labeling of the antibody of anti-CD19 is APC.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the fluorescence labeling of the antibody of anti-CD4 is APC.
In some embodiments of the invention, in method provided by the invention, detect in the step of the number percent of bone-marrow-derived lymphocyte in sample, the fluorescence labeling of the antibody of anti-CD8 is PE.
In the present invention, in method provided by the invention, the step detecting the number percent of bone-marrow-derived lymphocyte in sample is conventional lymphocyte immunity somatotype and quantitative analysis method, the method is not by restriction of the present invention, and those skilled in the art can select the method for the number percent measuring bone-marrow-derived lymphocyte in testing sample according to actual conditions.
In other embodiment of the present invention, in method provided by the invention, the method detecting the sum of testing sample medium size lymphocyte counts for adopting blood-counter system.In the present invention, in method provided by the invention, the method detecting the sum of testing sample medium size lymphocyte is conventional method, and the method is not by restriction of the present invention, and those skilled in the art can select the method for the sum detecting testing sample medium size lymphocyte according to actual conditions.
Present invention also offers a kind of method of lymphocyte immunity somatotype, it comprises the step of bone-marrow-derived lymphocyte immunophenotyping provided by the invention;
The method of this bone-marrow-derived lymphocyte immunophenotyping comprises:
Get different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain detection data, analyze described detection data;
This antibody comprises:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27;
The method of described analysis comprises:
Cell surface marker CD19 +d27 +represent memory B cell;
Cell surface marker CD19 +d27 -igD +represent naive B cell;
Cell surface marker CD19 +cD24 ++cD38 ++represent transition B cell;
Cell surface marker CD19 +cD24 -cD38 ++represent plasmablast.
Present invention also offers a kind of kit for bone-marrow-derived lymphocyte immunophenotyping, comprise fluorescently-labeled antibody different as follows:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27.
In the present invention, in the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, fluorescently-labeled antibody, can be that fluorescent marker and antibody are placed separately, in use both couplings be obtained fluorescently-labeled antibody; Also can be fluorescently-labeled antibody, in use, directly use.
In some embodiments of the invention, the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention comprises fluorescent marker and antibody;
This antibody comprises:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27.
In some embodiments of the invention, in the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of anti-CD19 is APC or APC-Cy7.
In other embodiment of the present invention, in the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of anti-IgD is BV510.
In other embodiment of the present invention, in the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of AntiCD3 McAb 8 is Percp-cy5.5.
In other embodiment of the present invention, in the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of anti-CD24 is PE.
In other embodiment of the present invention, in the kit for bone-marrow-derived lymphocyte immunophenotyping provided by the invention, the fluorescence labeling of the antibody of anti-CD27 is BV450 or APC.
In other embodiment of the present invention, antibody in the kit of bone-marrow-derived lymphocyte immunophenotyping provided by the invention, also comprise isotype control Ab, be specially: the Isotype antibody of BV450 mark, the Isotype antibody of BV510 mark, the Isotype antibody of PE mark, the Isotype antibody of Percp-cy5.5 mark.
In other embodiment of the present invention, isotype control Ab in antibody in the kit of bone-marrow-derived lymphocyte immunophenotyping provided by the invention, is specially: the Isotype antibody of APC mark, the Isotype antibody of BV510 mark, the Isotype antibody of PE mark, the Isotype antibody of Percp-cy5.5 mark.
In the present invention, in kit provided by the invention, the fluorescent marker in each fluorescently-labeled antibody is not by restriction of the present invention, and those skilled in the art can select suitable fluorescent marker according to actual conditions, and the Isotype control of correspondence.
Present invention also offers a kind of kit for lymphocyte immunity somatotype, it comprises fluorescently-labeled antibody different as follows:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27.
In some embodiments of the invention, in the kit for lymphocyte immunity somatotype provided by the invention, the fluorescence labeling of the antibody of anti-CD19 is APC or APC-Cy7.
In other embodiment of the present invention, in the kit for lymphocyte immunity somatotype provided by the invention, the fluorescence labeling of the antibody of anti-IgD is BV510.
In other embodiment of the present invention, in the kit for lymphocyte immunity somatotype provided by the invention, the fluorescence labeling of the antibody of AntiCD3 McAb 8 is Percp-cy5.5.
In other embodiment of the present invention, in the kit for lymphocyte immunity somatotype provided by the invention, the fluorescence labeling of the antibody of anti-CD24 is PE.
In other embodiment of the present invention, in the kit for lymphocyte immunity somatotype provided by the invention, the fluorescence labeling of the antibody of anti-CD27 is BV450 or APC.
In the present invention, in Flow cytometry process, represent the height of accuracy by target cell whether clear, target cell fluorescence intensity etc. of hiving off; Target cell hive off clear, target cell fluorescence intensity is accurately high, by same sample is repeatedly repeated experiment, no difference of science of statistics, then represent accuracy high.
The invention provides a kind of method and kit of bone-marrow-derived lymphocyte immunophenotyping.The method of bone-marrow-derived lymphocyte immunophenotyping provided by the invention, comprising: get different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtains detection data, analyzes gained and detects data; This antibody comprises: the antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27; The method of described analysis comprises: cell surface marker CD19 +d27 +represent memory B cell; Cell surface marker CD19 +d27 -igD +represent naive B cell; Cell surface marker CD19 +cD24 ++cD38 ++represent transition B cell; Cell surface marker CD19 +cD24 -cD38 ++represent plasmablast.Experimental result confirms, the present invention's design obtains the Antibody Combination of optimum cell surface marker, by flow cytometry, achieves and carries out more comprehensively immunophenotyping and quantitative test to bone-marrow-derived lymphocyte.In other embodiment of the present invention, method provided by the invention, required testing sample is few, simple to operate, required time is short.In other embodiment of the present invention, method provided by the invention is reproducible, accuracy is high, can be widely used in bone-marrow-derived lymphocyte subgroup immunophenotyping and quantitative test.
Accompanying drawing explanation
Fig. 1 shows embodiment 1 medium size lymphocyte classification results;
Fig. 2 shows T lymphocyte genotyping result in embodiment 1; Wherein, Fig. 2-A, Fig. 2-B show TCR α β +the cell typing result of double negative t cells; Fig. 2-C shows the cell typing result of gamma delta T cells; Fig. 2-D shows the cell typing result of helper cell subclass; Fig. 2-E shows the cell typing result of cytotoxic T cell subclass;
Fig. 3 shows bone-marrow-derived lymphocyte genotyping result in embodiment 1;
Fig. 4 shows embodiment 2 medium size lymphocyte classification results;
Fig. 5 shows T lymphocyte genotyping result in embodiment 2; Wherein, Fig. 5-A, Fig. 5-B show TCR α β +the cell typing result of double negative t cells; Fig. 5-C shows the cell typing result of gamma delta T cells; Fig. 5-D shows the cell typing result of helper cell subclass; Fig. 5-E shows the cell typing result of cytotoxic T cell subclass;
Fig. 6 shows bone-marrow-derived lymphocyte genotyping result in embodiment 2;
Fig. 7 shows embodiment 3 medium size lymphocyte classification results;
Fig. 8 shows T lymphocyte genotyping result in embodiment 3; Wherein, Fig. 8-A, Fig. 8-B show TCR α β +the cell typing result of double negative t cells; Fig. 8-C shows the cell typing result of gamma delta T cells; Fig. 8-D shows the cell typing result of helper cell subclass; Fig. 8-E shows the cell typing result of cytotoxic T cell subclass;
Fig. 9 shows bone-marrow-derived lymphocyte genotyping result in embodiment 3;
When Figure 10 shows that in comparative example, CD19, CD38, CD24, IgM are combined, the streaming figure of gained.
Embodiment
The invention discloses a kind of method and kit of bone-marrow-derived lymphocyte immunophenotyping.Those skilled in the art can with reference to present disclosure, implements the method, realizes its application, special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, and they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope this paper preparation method and application are changed or suitably change with combination, realize and apply the technology of the present invention.
Reagent used in the method for a kind of bone-marrow-derived lymphocyte immunophenotyping provided by the invention and kit and raw material all can be buied by market.
Fluorescence labeling Percp, FITC, PE, APC, BV421, Percp-cy5.5, BV510, PE-Cy7, BV450 of using in the present invention are common fluorescence labeling and can be buied by market, and each fluorescently-labeled antibody also can be buied by market.
In order to enable those skilled in the art understand technical scheme of the present invention better, below in conjunction with embodiment, set forth the present invention further:
The meticulous immunophenotyping of embodiment 1 lymphocyte and quantitative test
Experiment material:
Testing sample: anticoagulation cirumferential blood sample, derives from healthy volunteer is the peripheral blood of normal person.
BD Biosciences Lymphocyte subset kit (cat 340503), buy in BD biosciences, wherein mixed antibody 1 comprises: the antibody of the anti-CD45 antibody of fluorescence labeling Percp, the antibody of AntiCD3 McAb (FITC), the antibody of anti-CD4 (APC), anti-CD8 (PE); Mixed antibody 2 comprises: the antibody of the anti-CD45 antibody of fluorescence labeling Percp, the antibody of anti-CD19 (APC), anti-CD56 and CD16 (PE), the antibody of AntiCD3 McAb (FITC).The erythrocyte cracked liquid that BD clinical reagent box carries.
The erythrocyte cracked liquid of autogamy: containing NH in every 1L erythrocyte cracked liquid 4cl 8.29g, KHCO 31g, EDTA 0.37g.
Experimental technique:
Get 500 μ L anticoagulation cirumferential blood samples, get 200 μ L testing samples, record lymphocyte absolute number by blood-counter system, obtaining lymphocytic absolute number is 4.22 × 10 9individual/L.
Remaining 300 μ L, for detecting each subgroup of lymphocyte, carry out lymphocyte immunity somatotype and quantitative test.
Lymphocyte subset
1, two streaming pipes are got, be labeled as L-1, L-2 respectively, joined respectively in corresponding streaming pipe by two kinds of mixed antibodies in BD Biosciences Lymphocyte subset kit (cat 340503), wherein mixed antibody 1 joins in L-1 streaming pipe; Mixed antibody 2 adds respectively in L-2 streaming pipe.
2, in L-1, L-2 streaming pipe, respectively add 50 μ L testing samples, abundant vortex, room temperature (25 DEG C) lucifuge hatches 30min;
3, by 1 × BD erythrocyte cracked liquid splitting erythrocyte in kit, 5min;
4, add 1mL PBS and wash (3500rpm/min, 2min) once, add machine in 200 μ L PBS streamings, and analysis result.
The meticulous immunophenotyping of T lymphocyte
1, get two streaming pipes, be labeled as T-1, T-2 respectively, wherein T-1 is control group and Isotype control group, and T-2 is to be detected group; In each streaming pipe, following antibody is added according to table 1:
Classification and the volume added of the antibody added in each streaming pipe of table 1
2, in T-1, T-2 streaming pipe, respectively add 50 μ L testing samples, after abundant vortex, room temperature (25 DEG C) lucifuge hatches 30min;
3, in each streaming pipe, following antibody is added according to table 2:
Classification and the volume added of the antibody added in each streaming pipe of table 2
4, with after the erythrocyte cracked liquid splitting erythrocyte of autogamy, 37 DEG C of water-bath 10min;
5, respectively add 1mL PBS to wash once, machine in streaming analysis result.
The meticulous immunophenotyping of bone-marrow-derived lymphocyte
1, get two streaming pipes, be numbered B-1, B-2 respectively, wherein, B-1 is control group and Isotype control group, and B-2 is to be detected group; In each streaming pipe, following antibody is added according to table 3:
Classification and the volume added of the antibody added in each streaming pipe of table 3
2, in B-1, B-2 streaming pipe, respectively add 50 μ L testing samples of mixing, after mixing, room temperature (25 DEG C) lucifuge hatches 30min;
3, by the erythrocyte cracked liquid splitting erythrocyte of autogamy;
4, add 1mL PBS to wash once, machine in streaming analysis result.
Interpretation of result:
By above three steps, obtain each lymphocyte subgroup relative number (percentage) and lymphocyte absolute number.Each lymphocyte subgroup relative number (percentage) × lymphocyte absolute number, namely obtains each lymphocyte subgroup absolute number.Here each lymphocyte subgroup relative number refers to: account for lymphocytic number percent for during Lymphocyte subset for being respectively the cells such as T, B, NK; Be the number percent that each cell subsets accounts for T cell during immunophenotyping meticulous for T cell; Be the number percent that each cell subsets accounts for B cell during immunophenotyping meticulous for B cell.
Lymphocyte immunity somatotype and quantitative analysis results (adopting the analysis of BD auto Analysis)
Lymphocyte genotyping result is shown in Fig. 1, as we know from the figure, and the relative number (percentage) of T cell, Tc cell, Th cell, B cell, NK cell; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets, specific experiment the results are shown in Table 4.
The relative number of each cell subsets immunophenotyping of table 4 cell surface marker used, each cell subsets and absolute number
The meticulous immunophenotyping of T cell and quantitative analysis results (adopting BD Diva software analysis)
T lymphocyte genotyping result is shown in Fig. 2-A, 2-B, 2-C, 2-D and 2-E, as we know from the figure, the relative number (percentage) of all kinds of T cell subgroup, this relative number is the percentage relative to T cell number; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets.
1, TCR α β +double negative t cells (TCR α β +dNT cell)
Known by Fig. 2-A, Fig. 2-B, TCR α β +dNT (that is, CD3 +tCR α β +cD4 -cD8 -) be target cell.Can record that target cell accounts for T cell by flow cytometer 1.82%, the lymphocyte absolute number that can be recorded by blood-counter system calculates, TCR α β +the absolute number of double negative t cells is 50/μ L.
2, gamma delta T cells
From Fig. 2-C, CD3 +tCR γ δ +be target cell.Can record that target cell accounts for T cell by flow cytometer 3.4%, the lymphocyte absolute number that can be recorded by blood-counter system calculates, and the absolute number of gamma delta T cells is 93/μ L.
3, helper cell subclass
Can obtain from Fig. 2-D, below four cell subsets: 1. auxiliary T cells (i.e. CD4 + cD3 +cD4 +cD45RA +cD27 +), relative number 34.36%, absolute number 936/μ L; 2. auxiliary deplete T cells (i.e. Q4-2, CD3 in figure +cD4 +cD45RA +cD27-) relative number 0.12%, absolute number 3/μ L; 3. sectional center memory T cell (i.e. CD4 +cM, CD3 +cD4 +cD45RA -cD27 +) relative number 6.34%, absolute number 173/μ L; 4. secondary effects memory T cell (i.e. CD4 +eM, CD3 +cD4 +cD45RA -cD27 -) relative number 0.37%, absolute number 10/μ L.
4, cytotoxic T cell subclass
Can be obtained by Fig. 2-E, below four cell subsets: 1. cytotoxicity T cells (i.e. CD8 + cD3 +cD8 +cD45RA +cD27 +), relative number 17.68%, absolute number 482/μ L; 2. cytotoxicity deplete T cells (i.e. CD8 +tEMRA, CD3 +cD8 +cD45RA +cD27 -) relative number 2.69%, absolute number 73/μ L; 3. cytotoxicity Central memory T cell (i.e. CD8 +cM, CD8 +cD45RA -cD27 +) relative number 1.09%, absolute number 30/μ L; 4. cytotoxic effect memory T cell (i.e. CD8 +eM, CD3 +cD8 +cD45RA -cD27 -) relative number 0.28%, absolute number 8/μ L.
The meticulous immunophenotyping of B cell and quantitative analysis results (adopting BD Diva software analysis)
Bone-marrow-derived lymphocyte genotyping result is shown in Fig. 3-A and Fig. 3-B, as we know from the figure, the relative number (percentage) of all kinds of B cell subgroup, this relative number is the percentage relative to B cell number; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets.
As shown in Figure 3, the cell number of four cell subsets and this subgroup below in testing sample: 1. memory B cell (CD19 +d27 +), relative number 12.0%, absolute number 127/μ L; 2. naive B cell (CD19 +d27 -igD +), relative number 69.5%, absolute number 605/μ L; 3. transition B cell (CD19 +cD24 ++cD38 ++), relative number 9.0%, absolute number 95/μ L; 4. plasmablast (CD19 +cD24 -cD38 ++), relative number 3.4%, absolute number 37/μ L.
According to bibliographical information, the term of reference of the relative number of each cell subsets in the lymphocyte of normal person is shown in Table 5.
Each cell subsets relative number in the lymphocyte of table 5 normal person
Wherein, ※ represents the not general term of reference of these data, is only the experimental result of research report at present;
According to Fig. 1, lymphocyte successfully can be divided into T lymphocyte, bone-marrow-derived lymphocyte and NK cell by the present embodiment method used, and carries out quantitative test, obtains relative number and the absolute number of each cell.From table 5, result is known, and the relative number of each cell subsets falls into the term of reference of normal person completely, consistent with the actual conditions of detected sample, illustrates that experimental result of the present invention is stable, accurate.
Can obtain according to Fig. 2-A, Fig. 2-B, Fig. 2-C, Fig. 2-D, Fig. 2-E, T lymphocyte can be divided into TCR α β by the present embodiment method used exactly +double negative t cells, gamma delta T cells, auxiliary T cells, auxiliary deplete T cells, sectional center memory T cell, secondary effects memory T cell, cytotoxicity T cells, cytotoxicity deplete T cells, cytotoxicity Central memory T cell, cytotoxic effect memory T cell, and carry out quantitative test, obtain relative number and the absolute number of each cell subsets.From table 5, result is known, the relative number of each cell subsets and the term of reference of normal person basically identical, consistent with the actual conditions of detected sample, illustrate that experimental result of the present invention is stable, accurately.
According to Fig. 3, bone-marrow-derived lymphocyte can be divided into memory B cell, naive B cell, transition B cell, plasmablast by the present embodiment method used exactly, and carries out quantitative test, obtains relative number and the absolute number of each cell subsets.From table 5, result is known, the relative number of each cell subsets and the term of reference of normal person basically identical, consistent with the actual conditions of detected sample, illustrate that experimental result of the present invention is stable, accurately.
In sum, method provided by the invention adopts less testing sample just to achieve lymphocyte immunity somatotype to testing sample, and has counted the content of each cell subsets; And each cell subsets accurately can be carried out immunophenotyping by method provided by the invention.
The meticulous immunophenotyping of embodiment 2 lymphocyte and quantitative test
Experiment material:
Testing sample: anticoagulation cirumferential blood sample, derives from infant venous blood sample (signing Informed Consent Form) to our hospital, for intending the peripheral blood of examining systemic loupus erythematosus (SLE) infant.
BD Biosciences Lymphocyte subset kit (cat 340503), buy in BD biosciences, wherein mixed antibody 1 comprises: the antibody of the anti-CD45 antibody of fluorescence labeling Percp, the antibody of AntiCD3 McAb (FITC), the antibody of anti-CD4 (APC), anti-CD8 (PE); Mixed antibody 2 comprises: the antibody of the anti-CD45 antibody of fluorescence labeling Percp, the antibody of anti-CD19 (APC), anti-CD56 and CD16 (PE), the antibody of AntiCD3 McAb (FITC).The erythrocyte cracked liquid that BD clinical reagent box carries.
The erythrocyte cracked liquid of autogamy: containing NH in every 1L erythrocyte cracked liquid 4cl 8.29g, KHCO 31g, EDTA 0.37g.
Experimental technique:
Get 500 μ L anticoagulation cirumferential blood samples, get 200 μ L testing samples, record lymphocyte absolute number by blood-counter system, obtaining lymphocytic absolute number is 0.68 × 10 9individual/L.
Remaining 300 μ L, for detecting each subgroup of lymphocyte, carry out lymphocyte immunity somatotype and quantitative test.
Lymphocyte subset
Identical with the Lymphocyte subset method recorded in embodiment 1, temperature when wherein hatching is room temperature (20 DEG C), and incubation time is 35min.
The meticulous immunophenotyping of T lymphocyte
Identical with the T Lymphocyte subset method recorded in embodiment 1, after wherein adding the anti-tcr γ anti-δ of the anti-tcr α β antibody of PE mark, BV421 mark, the temperature of hatching is room temperature (20 DEG C), and incubation time is 35min; After adding residue antibody, the temperature of hatching is room temperature (20 DEG C), and incubation time is 35min; After the erythrocyte cracked liquid splitting erythrocyte of autogamy, 36.5 DEG C of water-bath 11min.
The meticulous immunophenotyping of bone-marrow-derived lymphocyte
1, get two streaming pipes, be numbered B-1, B-2 respectively, wherein, B-1 is control group and Isotype control group, and B-2 is to be detected group; In each streaming pipe, following antibody is added according to table 6:
Classification and the volume added of the antibody added in each streaming pipe of table 6
2, in B-1, B-2 streaming pipe, respectively add 50 μ L testing samples of mixing, after mixing, room temperature (20 DEG C) lucifuge hatches 30min;
3, by the erythrocyte cracked liquid splitting erythrocyte of autogamy;
4, add 1mL PBS to wash once, machine in streaming analysis result.
Interpretation of result:
By above three steps, obtain each lymphocyte subgroup relative number (percentage) and lymphocyte absolute number.Specific analytical method is identical with the method that embodiment 1 provides.
Lymphocyte immunity somatotype and quantitative analysis results (adopting the analysis of BD auto Analysis)
Lymphocyte genotyping result Fig. 4, as we know from the figure, relative (percentage) of T cell, Tc cell, Th cell, B cell, NK cell; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets, specific experiment the results are shown in Table 7.
The relative number of each cell subsets immunophenotyping of table 7 cell surface marker used, each cell subsets and absolute number
The meticulous immunophenotyping of T cell and quantitative analysis results (adopting BD Diva software analysis)
T Lymphocyte subset the results are shown in Figure 5-A, 5-B, 5-C, 5-D and Fig. 5-E, as we know from the figure, the relative number (percentage) of all kinds of T cell subgroup, this relative number is the percentage relative to T cell number; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets.
1, TCR α β +double negative t cells (TCR α β +dNT cell)
Known by Fig. 5-A, Fig. 5-B, TCR α β +dNT (i.e. CD3 +tCR α β +cD4 -cD8 -) be target cell.Can record that target cell accounts for T cell by flow cytometer 3.04%, the lymphocyte absolute number that can be recorded by blood-counter system calculates, TCR α β +the absolute number of double negative t cells is 14/μ L.
2, gamma delta T cells
From Fig. 5-C, CD3 +tCR γ δ +be target cell.Can record that target cell accounts for T cell by flow cytometer 1.32%, the lymphocyte absolute number that can be recorded by blood-counter system calculates, and the absolute number of gamma delta T cells is 6/μ L.
3, helper cell subclass
Can obtain from Fig. 5-D, below four cell subsets: 1. auxiliary T cells (i.e. CD4 + cD3 +cD4 +cD45RA +cD27 +), relative number 16.87%, absolute number 77/μ L; 2. auxiliary deplete T cells (i.e. Q4-2, CD3 in figure +cD4 +cD45RA +cD27 -) relative number 0.09%, absolute number 0.3/μ L; 3. sectional center memory T cell (i.e. CD4 +cM, CD3 +cD4 +cD45RA -cD27 +) relative number 22.16%, absolute number 101/μ L; 4. secondary effects memory T cell (i.e. CD4 +eM, CD3 +cD4 +cD45RA -cD27 -) relative number 6.43%, absolute number 29/μ L.
4, cytotoxic T cell subclass
Can be obtained by Fig. 5-E, below four cell subsets: 1. cytotoxicity T cells (i.e. CD8 + cD3 +cD8 +cD45RA +cD27 +), relative number 12.33%, absolute number 56/μ L; 2. cytotoxicity deplete T cells (i.e. CD8 +tEMRA, CD3 +cD8 +cD45RA +cD27 -) relative number 1.97%, absolute number 9/μ L; 3. cytotoxicity Central memory T cell (i.e. CD8 +cM, CD8 +cD45RA -cD27 +) relative number 3.78%, absolute number 17/μ L; 4. cytotoxic effect memory T cell (i.e. CD8 +eM, CD3 +cD8 +cD45RA -cD27 -) relative number 2.24%, absolute number 10/μ L.
The meticulous immunophenotyping of B cell and quantitative analysis results (adopting BD Diva software analysis)
Bone-marrow-derived lymphocyte genotyping result is shown in Fig. 6-A and Fig. 6-B, as we know from the figure, the relative number (percentage) of all kinds of B cell subgroup, this relative number is the percentage relative to B cell number; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets.
As shown in Figure 6, the cell number of four cell subsets and this subgroup below in testing sample: 1. memory B cell (CD19 +d27 +), relative number 28.0%, absolute number 59/μ L; 2. naive B cell (CD19 +d27 -igD +), relative number 48.5%, absolute number 102/μ L; 3. transition B cell (CD19 +cD24 ++cD38 ++), relative number 0.5%, absolute number 1/μ L; 4. plasmablast (CD19 +cD24 -cD38 ++), relative number 13.8%, absolute number 29/μ L.
According to bibliographical information, the term of reference of the relative number of each cell subsets in the lymphocyte of normal person is shown in Table 5.
According to Fig. 4, lymphocyte successfully can be divided into T lymphocyte, bone-marrow-derived lymphocyte and NK cell by the present embodiment method used, and carries out quantitative test, obtains relative number and the absolute number of each cell.As can be known from the results, the NK cell (CD16 in testing sample +cD56 +) content is starkly lower than normal reference range lower limit (7%-40%), and B cell (CD19 +) higher than the normal reference range upper limit (5-18%), all the other Non Apparent Abnormalities, point out B cell abnormal activation or propagation in this testing sample.
According to Fig. 5, T lymphocyte can be divided into TCR α β by the present embodiment method used exactly +double negative t cells, gamma delta T cells, auxiliary T cells, auxiliary deplete T cells, sectional center memory T cell, secondary effects memory T cell, cytotoxicity T cells, cytotoxicity deplete T cells, cytotoxicity Central memory T cell, cytotoxic effect memory T cell, and carry out quantitative test, obtain relative number and the absolute number of each cell subsets.As can be known from the results, the relative number of each cell subsets and the term of reference of normal person basically identical, Non Apparent Abnormality.
According to Fig. 6, bone-marrow-derived lymphocyte can be divided into memory B cell, naive B cell, transition B cell, plasmablast by the present embodiment method used exactly, and carries out quantitative test, obtains relative number and the absolute number of each cell subsets.As can be known from the results, in testing sample, transition B cell (TransitionalB cell) only accounts for 0.05% of B cell, does not almost have, and points out its humoral immunity transition to activate or imbalance.
In sum, method provided by the invention adopts less testing sample just to achieve lymphocyte immunity somatotype to testing sample, and has counted the content of each cell subsets; And each cell subsets accurately can be carried out immunophenotyping by method provided by the invention.
The meticulous immunophenotyping of embodiment 3 lymphocyte and quantitative test
Experiment material:
Testing sample: anticoagulation cirumferential blood sample, derives from infant venous blood sample (signing Informed Consent Form) to our hospital, for intending the peripheral blood of examining high IgM syndrome in children.
BD Biosciences Lymphocyte subset kit (cat 340503), buy in BD biosciences, wherein mixed antibody 1 comprises: the antibody of the anti-CD45 antibody of fluorescence labeling Percp, the antibody of AntiCD3 McAb (FITC), the antibody of anti-CD4 (APC), anti-CD8 (PE); Mixed antibody 2 comprises: the antibody of the anti-CD45 antibody of fluorescence labeling Percp, the antibody of anti-CD19 (APC), anti-CD56 and CD16 (PE), the antibody of AntiCD3 McAb (FITC).The erythrocyte cracked liquid that BD clinical reagent box carries.
The erythrocyte cracked liquid of autogamy: containing NH in every 1L erythrocyte cracked liquid 4cl 8.29g, KHCO 31g, EDTA 0.37g.
Experimental technique:
Get 500 μ L anticoagulation cirumferential blood samples, get 200 μ L testing samples, record lymphocyte absolute number by blood-counter system, obtaining lymphocytic absolute number is 0.89 × 10 9individual/L.
Remaining 300 μ L, for detecting each subgroup of lymphocyte, carry out lymphocyte immunity somatotype and quantitative test.
Lymphocyte subset
Identical with the Lymphocyte subset method recorded in embodiment 1, temperature when wherein hatching is room temperature (22 DEG C), and incubation time is 30min.
The meticulous immunophenotyping of T lymphocyte
Identical with the T Lymphocyte subset method recorded in embodiment 1, after wherein adding the anti-tcr γ anti-δ of the anti-tcr α β antibody of PE mark, BV421 mark, the temperature of hatching is room temperature (22 DEG C), and incubation time is 25min; After adding residue antibody, the temperature of hatching is room temperature (22 DEG C), and incubation time is 25min; After the erythrocyte cracked liquid splitting erythrocyte of autogamy, 37.5 DEG C of water-bath 9min.
The meticulous immunophenotyping of bone-marrow-derived lymphocyte
Identical with the bone-marrow-derived lymphocyte sorting technique recorded in embodiment 1, temperature when wherein hatching is room temperature (22 DEG C), and incubation time is 25min.
Interpretation of result:
By above three steps, obtain each lymphocyte subgroup relative number (percentage) and lymphocyte absolute number.Specific analytical method is identical with the method that embodiment 1 provides.
Lymphocyte immunity somatotype and quantitative analysis results (adopting the analysis of BD auto Analysis)
Lymphocyte genotyping result is shown in Fig. 7, as we know from the figure, and relative (percentage) of T cell, Tc cell, Th cell, B cell, NK cell; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets, specific experiment the results are shown in Table 8.
The relative number of each cell subsets immunophenotyping of table 8 cell surface marker used, each cell subsets and absolute number
The meticulous immunophenotyping of T cell and quantitative analysis results (adopting BD Diva software analysis)
T Lymphocyte subset the results are shown in Figure 8-A, 8-B, 8-C, 8-D and Fig. 8-E, as we know from the figure, the relative number (percentage) of all kinds of T cell subgroup, this relative number is the percentage relative to T cell number; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets.
1, TCR α β +double negative t cells (TCR α β +dNT cell)
Known by Fig. 8-A, Fig. 8-B, TCR α β +dNT (i.e. CD3 +tCR α β +cD4 -cD8 -) be target cell.Can record that target cell accounts for T cell by flow cytometer 1.24%, the lymphocyte absolute number that can be recorded by blood-counter system calculates, TCR α β +the absolute number of double negative t cells is 8/μ L.
2, gamma delta T cells
From Fig. 8-C, CD3 +tCR γ δ +be target cell.Can record that target cell accounts for T cell by flow cytometer 18.1%, the lymphocyte absolute number that can be recorded by blood-counter system calculates, and the absolute number of gamma delta T cells is 113/μ L.
3, helper cell subclass
Can obtain from Fig. 8-D, below four cell subsets: 1. auxiliary T cells (i.e. CD4 + cD3 +cD4 +cD45RA +cD27 +), relative number 23.36%, absolute number 146/μ L; 2. auxiliary deplete T cells (i.e. Q4-2, CD3 in figure +cD4 +cD45RA +cD27 -) relative number 0.17%, absolute number 1/μ L; 3. sectional center memory T cell (i.e. CD4 +cM, CD3 +cD4 +cD45RA -cD27 +) relative number 9.47%, absolute number 59/μ L; 4. secondary effects memory T cell (i.e. CD4 +eM, CD3 +cD4 +cD45RA -cD27 -) relative number 0.71%, absolute number 4/μ L.
4, cytotoxic T cell subclass
Can be obtained by Fig. 8-E, below four cell subsets: 1. cytotoxicity T cells (i.e. CD8 + cD3 +cD8 +cD45RA +cD27 +), relative number 15.66%, absolute number 98/μ L; 2. cytotoxicity deplete T cells (i.e. CD8 +tEMRA, CD3 +cD8 +cD45RA +cD27 -) relative number 10.15%, absolute number 63/μ L; 3. cytotoxicity Central memory T cell (i.e. CD8 +cM, CD8 +cD45RA -cD27 +) relative number 1.20%, absolute number 7/μ L; 4. cytotoxic effect memory T cell (i.e. CD8 +eM, CD3 +cD8 +cD45RA -cD27 -) relative number 0.81%, absolute number 5/μ L.
The meticulous immunophenotyping of B cell and quantitative analysis results (adopting BD Diva software analysis)
Bone-marrow-derived lymphocyte genotyping result is shown in Fig. 9-A and Fig. 9-B, as we know from the figure, the relative number (percentage) of all kinds of B cell subgroup, this relative number is the percentage relative to B cell number; According to the relative number (percentage) of lymphocytic absolute number and each cell subsets, calculate the absolute number obtaining each cell subsets.
As shown in Figure 9, the cell number of four cell subsets and this subgroup below in testing sample: 1. memory B cell (CD19 +d27 +), relative number 0.5%, absolute number 1/μ L; 2. naive B cell (CD19 +d27 -igD +), relative number 95.3%, absolute number 216/μ L; 3. transition B cell (CD19 +cD24 ++cD38 ++), relative number 28.3%, absolute number 64/μ L; 4. plasmablast (CD19 +cD24 -cD38 ++), relative number 0.9%, absolute number 2/μ L.
According to bibliographical information, the term of reference of the relative number of each cell subsets in the lymphocyte of normal person is shown in Table 5.
According to Fig. 7, lymphocyte successfully can be divided into T lymphocyte, bone-marrow-derived lymphocyte and NK cell by the present embodiment method used, and carries out quantitative test, obtains relative number and the absolute number of each cell.As can be known from the results, the NK cell (CD16 in testing sample +cD56 +) be 2.22%, be starkly lower than normal reference range lower limit (7-40%), and B cell (CD19 +) be 25.42%, higher than the normal reference range upper limit (5-18%), all the other Non Apparent Abnormalities, prompting B cell abnormal activation or propagation.
According to Fig. 8, T lymphocyte can be divided into TCR α β by the present embodiment method used exactly +double negative t cells, gamma delta T cells, auxiliary T cells, auxiliary deplete T cells, sectional center memory T cell, secondary effects memory T cell, cytotoxicity T cells, cytotoxicity deplete T cells, cytotoxicity Central memory T cell, cytotoxic effect memory T cell, and carry out quantitative test, obtain relative number and the absolute number of each cell subsets.As can be known from the results, cytotoxicity deplete T cells (CD8 in testing sample +tEMRA) content accounts for 36.5% of T cell, is significantly higher than normal children of the same age, and prompting infant may exist long-term chronic virus infections; In testing sample, memory T cell (all kinds of center/effect memory T) is all lower than normal reference range, points out this infant there is cellular immune abnormality; In testing sample, the content of gamma delta T cells accounts for 18.1% of T cell, far above normal reference range (lower than 5%), and prompting infant virus infections, T cell abnormal activation or increment.
According to Fig. 9, bone-marrow-derived lymphocyte can be divided into memory B cell, naive B cell, transition B cell, plasmablast by the present embodiment method used exactly, and carries out quantitative test, obtains relative number and the absolute number of each cell subsets.As can be known from the results, naive B cell too much, and memory B cell (memory B) only accounts for 0.5% of B cell, far below normal limits, and prompting infant humoral immune function obstacle.
In sum, method provided by the invention adopts less testing sample just to achieve lymphocyte immunity somatotype to testing sample, and has counted the content of each cell subsets; And each cell subsets accurately can be carried out immunophenotyping by method provided by the invention.
The different bone-marrow-derived lymphocyte surface indicia of comparative example is on the impact of the meticulous immunophenotyping of bone-marrow-derived lymphocyte and quantitative test
In B cell meticulous immunophenotyping method establishment process, also need select suitable surface marker (that is, surface antigen combination) and accurately draw door strategy easily.For transition B cell (transitional B cell), the specific surface antigen of multiple transition B cell is had to combine, as CD19 at present +cD38 ++cD24 ++igM ++/ CD19 +cD38 ++cD24 ++(+represent is positive, ++ represent strong positive).
Combined with CD19, CD38, CD24, IgM, using the antibody of the antibody of the antibody of the antibody of anti-CD19, AntiCD3 McAb 8, anti-CD24, anti-IgM as detection antibody, lymphocyte immunity somatotype is carried out to testing sample.During data analysis, the first step is at CD19 +cell in iris out CD24 ++igM ++cell P1, obtain Figure 10-A; Second step is that door irises out CD24 with P1 ++cD38 ++target cell (Transitional Bcell), obtain Figure 10-B.As can be known from the results, because IgM and CD24 is continuous expression, the boundary of P1 cell mass is unclear, accurately cannot define the expression positive/strong positive.
Below be only the preferred embodiment of the present invention, it should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. a method for bone-marrow-derived lymphocyte immunophenotyping, is characterized in that, comprising:
Get different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain detection data, analyze described detection data;
Described antibody comprises:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27;
The method of described analysis comprises:
Cell surface marker CD19 +d27 +represent memory B cell;
Cell surface marker CD19 +d27 -igD +represent naive B cell;
Cell surface marker CD19 +cD24 ++cD38 ++represent transition B cell;
Cell surface marker CD19 +cD24 -cD38 ++represent plasmablast.
2. method according to claim 1, is characterized in that, also comprises the step of bone-marrow-derived lymphocyte number in the described testing sample of statistics, comprising:
Detect the sum of described testing sample medium size lymphocyte;
Detect bone-marrow-derived lymphocyte in described testing sample and account for described lymphocytic number percent;
By calculating, obtain the number of bone-marrow-derived lymphocyte in described testing sample.
3. a method for lymphocyte immunity somatotype, is characterized in that, it comprises the step of bone-marrow-derived lymphocyte immunophenotyping as claimed in claim 1 or 2.
4. for a kit for bone-marrow-derived lymphocyte immunophenotyping, it is characterized in that, comprise fluorescently-labeled antibody different as follows:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27.
5. kit according to claim 4, is characterized in that, the fluorescence labeling of the antibody of described anti-CD19 is APC or APC-Cy7.
6. the kit according to claim 4 or 5, is characterized in that, the fluorescence labeling of the antibody of described anti-IgD is BV510.
7. the kit according to any one of claim 4 to 6, is characterized in that, the fluorescence labeling of the antibody of described AntiCD3 McAb 8 is Percp-cy5.5.
8. the kit according to any one of claim 4 to 7, is characterized in that, the fluorescence labeling of the antibody of described anti-CD24 is PE.
9. the kit according to any one of claim 4 to 8, is characterized in that, the fluorescence labeling of the antibody of described anti-CD27 is BV450 or APC.
10. for a kit for lymphocyte immunity somatotype, it is characterized in that, it comprises fluorescently-labeled antibody different as follows:
The antibody of the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and anti-CD27.
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CN110687292A (en) * 2019-11-11 2020-01-14 北京大学人民医院(北京大学第二临床医学院) Application of double negative B cell and regulatory B10 cell in preparation of autoimmune disease diagnostic reagent
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WO2023115703A1 (en) * 2021-12-22 2023-06-29 重庆医科大学附属儿童医院 Method and kit for immunophenotyping of b lymphocyte development subgroups
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CN115792200A (en) * 2022-11-18 2023-03-14 无锡市人民医院 Antibody combination and kit for absolute counting of peripheral blood B cell subsets after B cell clearance treatment and application of antibody combination and kit
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