CN104360053B - A kind of method of bone-marrow-derived lymphocyte immunophenotyping and test kit - Google Patents
A kind of method of bone-marrow-derived lymphocyte immunophenotyping and test kit Download PDFInfo
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Abstract
The invention belongs to immunological technique field, disclose method and the test kit of a kind of bone-marrow-derived lymphocyte immunophenotyping.The method of the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, including: take different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain detection data, analyze gained detection data；This antibody includes: the antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27.The inventive method achieves and bone-marrow-derived lymphocyte carries out more comprehensively fine immunophenotyping, required testing sample is few, simple to operate, required time is short, accuracy is high, can be widely applied to lymphocyte subgroup immunophenotyping.
Description
Technical field
The invention belongs to immunological technique field, particularly to method and the test kit of a kind of bone-marrow-derived lymphocyte immunophenotyping.
Background technology
Lymphocyte (lymphocyte) is also referred to as lymph corpuscle, for the one that volume in leukocyte is minimum, diameter 6-8 micron；
Accounting for leukocyte count purpose 20%-30%, round cell core at human body, Cytoplasm is little；Produced by lymphatic organ, be that body is exempted from
The important cells composition of epidemic disease answering, is the class cell line with Immune discrimination function.Lymphocyte is the heterogeneous of complexity
Sexual cell colony, can be divided into different classes of according to its phenotype and functional character, such as T cell, B cell, NK cell etc., these cells
Some subgroups can also be further divided into.Lymphocyte and subgroup thereof cooperate during immunne response, mutually restrict, altogether
With completing the identification to antigenic substance, response and removing, thus maintain body homeostasis.
In early days research is migrated by it according to lymphocyte, surface molecular and the difference of function, generally that lymph is thin
Born of the same parents are divided into T cell, B cell and NKT (NK) cell.Wherein T cell can be divided into cytotoxic T cell (cytotoxic T
Lymphocyte, Tc), helper T cell (helper T lymphocyte, Th), memory T cell (memory T
Lymphocyte, Tm) and regulation/suppression T cell (regulatory/suppressor T lymphocyte)；B cell can be entered
One step is divided into memory B cell (memory B cell), plasma cell (plasma cell), naive B cell
Deng subclass.
Along with the continuous progress of scientific research, scientist is found that more cell surface marker, can be by all kinds of lymphs
Cell carries out finer cell subsets classification.For example, it is possible to T cell is further divided into: TCR α β+Double negative t cells
(double negative T lymphocyte, DNT), gamma delta T cells, auxiliary T cells, auxiliary exhaust T cell, auxiliary
Central memory T cell, secondary effects memory T cell, cytotoxicity T cells, cytotoxicity exhaust T cell, cytotoxicity
Central memory T cell, cytotoxic effect memory T cell.
The kind of blood sample medium-sized lymphocyte has important relationship with the immune system performance of body.By to blood sample
Product medium-sized lymphocyte carries out immunophenotyping and quantitative analysis, it is possible to more accurately evaluate the immunologic function of body, it is also possible to more
Study well the generation of some disease, development and treatment effectiveness evaluation.At present, the lymphocyte being usually used in blood sample is exempted from
The method of epidemic disease typing and quantitative analysis mainly has: immunoenzyme labeling method, common Lymphocyte subset, thinner lymphocyte subgroup are examined
Survey.Immunoenzyme labeling method is just for a certain or specific antigen of a class or antibody generation specific reaction, and detection faces is narrow, operation is multiple
Miscellaneous, big to sample requirements to be detected, waste time and energy.Common Lymphocyte subset, is widely used in lymphocyte and detects, but only
Lymphocyte can be divided into T cell, Tc cell, Th cell, B cell and NK cell, it is impossible to further cell is carried out typing.Relatively
Thin Lymphocyte subtypes test depends on flow cytometry, and lymphocyte is carried out the most detailed by the method using polychrome streaming
Typing, except traditional T cell, Tc cell, Th cell, B cell and NK extracellular, also detection Tm cell and cell quantity.But
It is, if it is desired to if further cell subsets being carried out immunophenotyping and quantitative analysis, in addition it is also necessary to the cell by means of other is exempted from
Epidemic disease classifying method, operation complexity, big to the demand of testing sample, it is not appropriate for actual application.So, in addition it is also necessary to thin to lymph
The method of born of the same parents' immunophenotyping and quantitative analysis is studied, in the hope of utilizing simple method to carry out more smart by lymphocyte as far as possible
Thin ground immunophenotyping and quantitative analysis.
Summary of the invention
In view of this, the goal of the invention of the present invention is to provide method and the test kit of a kind of bone-marrow-derived lymphocyte immunophenotyping.
The method of this bone-marrow-derived lymphocyte immunophenotyping can carry out fine immunophenotyping and quantitative analysis more comprehensively to bone-marrow-derived lymphocyte,
Efficiency is high, and has saved the consumption of testing sample, the immunophenotyping of more suitable bone-marrow-derived lymphocyte and quantitative analysis.
In order to realize the goal of the invention of the present invention, the present invention adopts the following technical scheme that:
A kind of method that the invention provides bone-marrow-derived lymphocyte immunophenotyping, including:
Take different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain inspection
Survey data, analyze described detection data；
This antibody includes:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27；
The method of this analysis includes:
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, used in flow cytometry antibody can be the antibody with testing sample homology or can also
For antibody nonhomologous with testing sample, as long as this antibody can produce antigen-anti-with cell surface marker in testing sample
Body is specific binding to react.
In the present invention, "+" represent the positive, i.e. represent that this antigen has expression at cell surface；
" ++ " represents strong positive, i.e. represents that this antigen is at cell surface high expressed；
"-" represents feminine gender, i.e. represents that this antigen is not expressed at cell surface.
The surface marker of lymphocyte refers to be present in the membrane molecule of lymphocytic cell surface, and they are that lymphocyte identification resists
Former and other immunocytes interact and accept signal stimulus and produce the material base of response, are also to differentiate and separate
The important evidence of lymphocyte.During the differentiation and development of lymphocyte, it is thin that lymphoid stem cells is further differentiated into each
Born of the same parents' subgroup, imparts the specific surface marker of each cell subsets.Some surface marker is that lymphocyte has, some surface
Mark be a certain class or a few quasi-lymphocyte distinctive, so when carrying out lymphocyte immunity typing, can have numerous kinds
Combination.But being not the every kind of combination immunophenotyping that can realize lymphocyte, the present invention is by substantial amounts of creative labor
Dynamic discovery: for B cell, it is a discovery of the invention that cell surface marker is CD19+D27+Time, memory B cell can be carried out immunity
Typing；Cell surface marker is CD19+D27-IgD+Time, naive B cell can be carried out immunophenotyping；Cell surface marker is
CD19+CD24++CD38++Time, transition B cell can be carried out immunophenotyping；Cell surface marker is CD19+CD24-CD38++
Time, plasmablast can be carried out immunophenotyping.The present invention is according to the cell surface marker corresponding to each cell, and design obtains
Obtain the Antibody Combination of the cell surface marker of optimum, by flow cytometry, bone-marrow-derived lymphocyte is carried out fine immunophenotyping
And quantitative analysis, the most perfect fine immunoassay to lymphocyte and quantitative analysis.
Preferably, the present invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, also sets up matched group and Isotype control group.
The meaning arranging matched group is to get rid of the impact of irrelevant variable, increases credibility and the cogency of experimental result；Homotype is set
The meaning of matched group is to distinguish the background signal impact that identical hypotype is caused during antibody staining.
In some embodiments of the invention, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, matched group institute
Antibody specific be: the antibody of anti-CD19.
In the other embodiment of the present invention, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, homotype
Antibody specific used by comparison is: the Isotype antibody of BV450 labelling, the Isotype antibody of BV510 labelling, the homotype of PE labelling resist
Body, the Isotype antibody of Percp-cy5.5 labelling.
In the other embodiment of the present invention, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, homotype
Antibody specific used by comparison is: the Isotype antibody of APC labelling, the Isotype antibody of BV510 labelling, the Isotype antibody of PE labelling,
The Isotype antibody of Percp-cy5.5 labelling.
In some embodiments of the invention, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, anti-CD19's
The fluorescent labeling of antibody is APC or APC-Cy7.
In the other embodiment of the present invention, the present invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, anti-IgD
The fluorescent labeling of antibody be BV510.
In the other embodiment of the present invention, the present invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, AntiCD3 McAb 8
The fluorescent labeling of antibody be Percp-cy5.5.
In the other embodiment of the present invention, the present invention provides in the method for bone-marrow-derived lymphocyte immunophenotyping, anti-CD24
The fluorescent labeling of antibody be PE.
In the other embodiment of the present invention, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, anti-
The fluorescent labeling of the antibody of CD27 is BV450 or APC.
Preferably, the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, particularly as follows:
Take the antibody of fluorescently-labeled anti-CD19, the antibody of fluorescently-labeled anti-IgD, fluorescently-labeled AntiCD3 McAb 8 anti-
Body, the antibody of fluorescently-labeled anti-CD24, the antibody of fluorescently-labeled anti-CD27, mix with detected sample, room temperature (20 DEG C～
25 DEG C) under the conditions of, after hatching 25min～35min, mix with erythrocyte cracked liquid, after splitting erythrocyte, washing, in streaming
Machine testing, obtains detection data；
The method of this analysis includes:
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 other embodiment of the present invention, in the method that the present invention provides, the method for bone-marrow-derived lymphocyte immunophenotyping
In, operation corresponding to matched group and Isotype control group particularly as follows:
Take the antibody of fluorescently-labeled anti-CD19, the Isotype antibody of BV450 labelling, the Isotype antibody of BV510 labelling, PE mark
The Isotype antibody of note, the Isotype antibody of Percp-cy5.5 labelling, mix with detected sample, under the conditions of 20 DEG C～25 DEG C, hatch
After 25min～35min, mix with erythrocyte cracked liquid, after splitting erythrocyte, washing, machine testing in streaming, obtain detection number
According to.
In the other embodiment of the present invention, in the method that the present invention provides, the method for bone-marrow-derived lymphocyte immunophenotyping
In, operation corresponding to matched group and Isotype control group particularly as follows:
Take the antibody of fluorescently-labeled anti-CD19, the Isotype antibody of APC labelling, the Isotype antibody of BV510 labelling, PE labelling
Isotype antibody, the Isotype antibody of Percp-cy5.5 labelling, mix with detected sample, under the conditions of 20 DEG C～25 DEG C, hatch
After 25min～35min, mix with erythrocyte cracked liquid, after splitting erythrocyte, washing, machine testing in streaming, obtain detection number
According to.
In the present invention, in the method that the present invention provides, the fluorescent marker in each fluorescently-labeled antibody is not by this
The restriction of invention, those skilled in the art can select suitable fluorescent marker, and the homotype of correspondence according to practical situation
Comparison.
Preferably, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, also include that adding up B in testing sample drenches
The step of bar cell number.
In some embodiments of the invention, in the method for the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides, detect sample
The step of middle bone-marrow-derived lymphocyte number includes:
The sum of detection testing sample medium-sized lymphocyte；
In detection testing sample, bone-marrow-derived lymphocyte accounts for the percentage ratio of described lymphocyte；
By calculating, obtain the number of bone-marrow-derived lymphocyte in testing sample.
In the method that the present invention provides, in detection sample in bone-marrow-derived lymphocyte number, total lymphocyte count is multiplied by wherein B and drenches
The percentage ratio of bar cell, obtains the number of bone-marrow-derived lymphocyte.In the present invention by each B cell subgroup of testing sample is carried out
Immunophenotyping and relative number statistics, in conjunction with the absolute number of bone-marrow-derived lymphocyte, the cell that can obtain each B cell subgroup is absolute
Number.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step, including:
Take different fluorescently-labeled antibody, mix with new described testing sample, after hatching, examine through flow cytometry
Survey, obtain detection data, analyze described detection data；
In detection testing sample, in the percentage ratio of bone-marrow-derived lymphocyte, antibody used includes:
The antibody of anti-CD45, the antibody of AntiCD3 McAb, the antibody of anti-CD16, the antibody of anti-CD56, the antibody of anti-CD19；
The antibody of anti-CD19 used in the percentage ratio of bone-marrow-derived lymphocyte in detection testing sample divides with bone-marrow-derived lymphocyte immunity
The antibody of anti-CD19 described in type, independently of one another with fluorescent labeling.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, antibody used by matched group 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 the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the antibody fluorescence of anti-CD45 is labeled as Percp.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the fluorescent labeling of the antibody of AntiCD3 McAb is FITC.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the fluorescent labeling of the antibody of anti-CD16 is PE.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the fluorescent labeling of the antibody of anti-CD56 is PE.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the fluorescent labeling of the antibody of anti-CD19 is APC.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the fluorescent labeling of the antibody of anti-CD4 is APC.
In some embodiments of the invention, in the method that the present invention provides, the percentage ratio of bone-marrow-derived lymphocyte in detection sample
Step in, the fluorescent labeling of the antibody of anti-CD8 is PE.
In the present invention, in the method that the present invention provides, in detection sample, the step of the percentage ratio of bone-marrow-derived lymphocyte is conventional
Lymphocyte immunity typing and quantitative analysis method, the method is not limited by the present invention, and those skilled in the art can root
Select to measure the method for the percentage ratio of bone-marrow-derived lymphocyte in testing sample according to practical situation.
In the other embodiment of the present invention, in the method that the present invention provides, detect testing sample medium-sized lymphocyte
Sum method for use blood-counter system count.In the present invention, in the method that the present invention provides, detect to be measured
The method of the sum of sample medium-sized lymphocyte is conventional method, and the method is not limited by the present invention, those skilled in the art
The method that can select the sum of detection testing sample medium-sized lymphocyte according to practical situation.
A kind of method that present invention also offers lymphocyte immunity typing, it includes the bone-marrow-derived lymphocyte that the present invention provides
The step of immunophenotyping；
The method of this bone-marrow-derived lymphocyte immunophenotyping includes:
Take different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain inspection
Survey data, analyze described detection data；
This antibody includes:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27；
The method of described analysis includes:
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 test kit for bone-marrow-derived lymphocyte immunophenotyping, including the most different fluorescence marks
The antibody of note:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27.
In the present invention, the present invention provide in the test kit of bone-marrow-derived lymphocyte immunophenotyping, fluorescently-labeled anti-
Body, can be that fluorescent marker is individually placed with antibody, in use both couplings is obtained fluorescently-labeled antibody；Can also
It is fluorescently-labeled antibody, in use, directly uses.
In some embodiments of the invention, the test kit for bone-marrow-derived lymphocyte immunophenotyping that the present invention provides includes
Fluorescent marker and antibody；
This antibody includes:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27.
In some embodiments of the invention, the present invention provide in the test kit of bone-marrow-derived lymphocyte immunophenotyping, anti-
The fluorescent labeling of the antibody of CD19 is APC or APC-Cy7.
In the other embodiment of the present invention, the test kit for bone-marrow-derived lymphocyte immunophenotyping that the present invention provides
In, the fluorescent labeling of the antibody of anti-IgD is BV510.
In the other embodiment of the present invention, the test kit for bone-marrow-derived lymphocyte immunophenotyping that the present invention provides
In, the fluorescent labeling of the antibody of AntiCD3 McAb 8 is Percp-cy5.5.
In the other embodiment of the present invention, the test kit for bone-marrow-derived lymphocyte immunophenotyping that the present invention provides
In, the fluorescent labeling of the antibody of anti-CD24 is PE.
In the other embodiment of the present invention, the test kit for bone-marrow-derived lymphocyte immunophenotyping that the present invention provides
In, the fluorescent labeling of the antibody of anti-CD27 is BV450 or APC.
In the other embodiment of the present invention, in the test kit of the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides
Antibody, also includes isotype control Ab, particularly as follows: the Isotype antibody of BV450 labelling, the Isotype antibody of BV510 labelling, PE labelling
Isotype antibody, the Isotype antibody of Percp-cy5.5 labelling.
In the other embodiment of the present invention, in the test kit of the bone-marrow-derived lymphocyte immunophenotyping that the present invention provides
Isotype control Ab in antibody, particularly as follows: the Isotype antibody of APC labelling, the Isotype antibody of BV510 labelling, PE labelling is same
Type antibody, the Isotype antibody of Percp-cy5.5 labelling.
In the present invention, in the test kit that the present invention provides, the fluorescent marker in each fluorescently-labeled antibody is not subject to
The restriction of the present invention, those skilled in the art can select suitable fluorescent marker according to practical situation, and correspondence is same
Type compares.
Present invention also offers a kind of test kit for lymphocyte immunity typing, it includes the most different fluorescence marks
The antibody of note:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27.
In some embodiments of the invention, the present invention provide in the test kit of lymphocyte immunity typing, anti-
The fluorescent labeling of the antibody of CD19 is APC or APC-Cy7.
In the other embodiment of the present invention, the test kit for lymphocyte immunity typing that the present invention provides
In, the fluorescent labeling of the antibody of anti-IgD is BV510.
In the other embodiment of the present invention, the test kit for lymphocyte immunity typing that the present invention provides
In, the fluorescent labeling of the antibody of AntiCD3 McAb 8 is Percp-cy5.5.
In the other embodiment of the present invention, the test kit for lymphocyte immunity typing that the present invention provides
In, the fluorescent labeling of the antibody of anti-CD24 is PE.
In the other embodiment of the present invention, the test kit for lymphocyte immunity typing that the present invention provides
In, the fluorescent labeling of the antibody of anti-CD27 is BV450 or APC.
In the present invention, during Flow cytometry, hived off the clearest, target cell fluorescence by target cell
Intensity etc. represent the height of accuracy；Target cell hive off clear, target cell fluorescence intensity is the highest, by same specimen
Carry out being repeated several times experiment, no difference of science of statistics, then it represents that accuracy is high.
The invention provides method and the test kit of a kind of bone-marrow-derived lymphocyte immunophenotyping.The bone-marrow-derived lymphocyte that the present invention provides
The method of immunophenotyping, including: take different fluorescently-labeled antibody, mix with detected sample, after hatching, through fluidic cell
Art detects, and obtains detection data, analyzes gained detection data；This antibody includes: the antibody of anti-CD19, the antibody of anti-IgD, AntiCD3 McAb 8
Antibody, the antibody of anti-CD24 and the antibody of anti-CD27；The method of described analysis includes: 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++Generation
Table transition B cell；Cell surface marker CD19+CD24-CD38++Represent plasmablast.Experimental result confirms, present invention design obtains
Obtain the Antibody Combination of the cell surface marker of optimum, pass through flow cytometry, it is achieved that carry out more comprehensive to bone-marrow-derived lymphocyte
Immunophenotyping and quantitative analysis.In the other embodiment of the present invention, the method that the present invention provides, required testing sample
Less, simple to operate, required time is short.In the other embodiment of the present invention, the method that the present invention provides is reproducible, accurate
Really property is high, can be widely applied to bone-marrow-derived lymphocyte subgroup immunophenotyping and quantitative analysis.
Accompanying drawing explanation
Fig. 1 shows embodiment 1 medium-sized lymphocyte classification results；
Fig. 2 shows T lymphocyte genotyping result in embodiment 1；Wherein, Fig. 2-A, Fig. 2-B show TCR α β+Double negative t cells
Cell typing result；Fig. 2-C shows the cell typing result of gamma delta T cells；Fig. 2-D shows the cell typing knot of helper T cell subclass
Really；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-sized lymphocyte classification results；
Fig. 5 shows T lymphocyte genotyping result in embodiment 2；Wherein, Fig. 5-A, Fig. 5-B show TCR α β+Double negative t cells
Cell typing result；Fig. 5-C shows the cell typing result of gamma delta T cells；Fig. 5-D shows the cell typing knot of helper T cell subclass
Really；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-sized lymphocyte classification results；
Fig. 8 shows T lymphocyte genotyping result in embodiment 3；Wherein, Fig. 8-A, Fig. 8-B show TCR α β+Double negative t cells
Cell typing result；Fig. 8-C shows the cell typing result of gamma delta T cells；Fig. 8-D shows the cell typing knot of helper T cell subclass
Really；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；
Figure 10 shows when in comparative example, CD19, CD38, CD24, IgM are combined, the streaming figure of gained.
Detailed description of the invention
The invention discloses method and the test kit of a kind of bone-marrow-derived lymphocyte immunophenotyping.Those skilled in the art can join
Examine present disclosure, implement the method, it is achieved its application, it is accordingly required in particular to it is noted that all similar replacements and change are to ability
Being apparent from for field technique personnel, they are considered as being included in the present invention.The method of the present invention and application are
Being described by preferred embodiment, related personnel substantially can be to this in without departing from present invention, spirit and scope
Literary composition is preparation method and application be modified or suitably change and combine, and realizes and applies the technology of the present invention.
Reagent used in the method for a kind of bone-marrow-derived lymphocyte immunophenotyping that the present invention provides and test kit and raw material are equal
Can be buied by market.
Fluorescent labeling Percp used in the present invention, FITC, PE, APC, BV421, Percp-cy5.5, BV510, PE-
Cy7, BV450 are common fluorescent labeling and can be buied by market, and each fluorescently-labeled antibody can also be buied by market.
In order to make those skilled in the art better understood when technical scheme, below in conjunction with enforcement
Example, is expanded on further the present invention:
The fine immunophenotyping of embodiment 1 lymphocyte and quantitative analysis
Experiment material:
Testing sample: anticoagulation cirumferential blood sample, derives from healthy volunteer, for the peripheral blood of normal person.
BD Biosciences Lymphocyte subset test kit (cat 340503), buys in BD biosciences, its
Middle mixed antibody 1 includes: the anti-CD45 antibody of fluorescent labeling Percp, the antibody of AntiCD3 McAb (FITC), the antibody of anti-CD4 (APC),
The antibody of anti-CD8 (PE)；Mixed antibody 2 includes: the anti-CD45 antibody of fluorescent labeling Percp, the antibody of anti-CD19 (APC), anti-
The antibody of 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 liquid4Cl 8.29g、KHCO31g,EDTA 0.37g。
Experimental technique:
Take 500 μ L anticoagulation cirumferential blood samples, take 200 μ L testing samples, record lymphocyte by blood-counter system exhausted
Logarithm, the absolute number obtaining lymphocyte is 4.22 × 109Individual/L.
Remaining 300 μ L are used for detecting each subgroup of lymphocyte, carry out lymphocyte immunity typing and quantitative analysis.
Lymphocyte subset
1, take two streaming pipes, be respectively labeled as L-1, L-2, by BD Biosciences Lymphocyte subset test kit
In (cat 340503), two kinds of mixed antibodies are added separately in the streaming pipe of correspondence, and wherein mixed antibody 1 joins No. L-1 stream
In formula pipe；Mixed antibody 2 is separately added in L-2 streaming pipe.
2, in L-1, L-2 streaming pipe, each addition 50 μ L testing samples, abundant vortex, room temperature (25 DEG C) lucifuge is hatched
30min；
3, with 1 × BD erythrocyte cracked liquid splitting erythrocyte in test kit, 5min；
4, add (3500rpm/min, 2min) after 1mL PBS washed once, add machine in 200 μ L PBS streamings, and divide
Analysis result.
The fine immunophenotyping of T lymphocyte
1, taking two streaming pipes, be respectively labeled as T-1, T-2, wherein T-1 is matched group and Isotype control group, and T-2 is for treating
Detection group；In each streaming pipe, following antibody is added according to table 1:
The classification of the antibody added in each streaming pipe of table 1 and the volume of addition
2, in T-1, T-2 streaming pipe, each addition 50 μ L testing samples, after abundant vortex, room temperature (25 DEG C) lucifuge is incubated
Educate 30min；
3, in each streaming pipe, following antibody is added according to table 2:
The classification of the antibody added in each streaming pipe of table 2 and the volume of addition
4, after using the erythrocyte cracked liquid splitting erythrocyte of autogamy, 37 DEG C of water-bath 10min；
5, after each addition 1mL PBS washes once, machine analysis result in streaming.
The fine immunophenotyping of bone-marrow-derived lymphocyte
1, taking two streaming pipes, respectively numbered B-1, B-2, wherein, B-1 is matched group and Isotype control group, and B-2 is for treating
Detection group；In each streaming pipe, following antibody is added according to table 3:
The classification of the antibody added in each streaming pipe of table 3 and the volume of addition
2, in B-1, B-2 streaming pipe, each 50 μ L testing samples adding mixing, after mixing, room temperature (25 DEG C) lucifuge is incubated
Educate 30min；
3, by the erythrocyte cracked liquid splitting erythrocyte of autogamy；
4, after addition 1mL PBS washes once, machine analysis result in streaming.
Interpretation of result:
By above three steps, obtain each lymphocyte subgroup relative number (percent) and lymphocyte absolute number.Each lymph
Cell subsets relative number (percent) × lymphocyte absolute number, i.e. obtains each lymphocyte subgroup absolute number.Here each pouring
Bar cell subsets relative number refers to: for during Lymphocyte subset being the percentage that respectively cell such as T, B, NK accounts for lymphocyte
Ratio；It is each cell subsets percentage ratio of accounting for T cell during immunophenotyping fine for T cell；During immunophenotyping fine for B cell
The percentage ratio of B cell is accounted for for each cell subsets.
Lymphocyte immunity typing and quantitative analysis results (using BD auto Analysis to analyze)
Lymphocyte genotyping result is shown in Fig. 1, it can be seen that T cell, Tc cell, Th cell, B cell, the phase of NK cell
Logarithm (percent)；Absolute number according to lymphocyte and the relative number (percent) of each cell subsets, calculate and obtain each
The absolute number of cell subsets, specific experiment the results are shown in Table 4.
Each cell surface marker used by cell subsets immunophenotyping of table 4, the relative number of each cell subsets and definitely
Number
The fine immunophenotyping of T cell and quantitative analysis results (using BD Diva software analysis)
T lymphocyte genotyping result is shown in Fig. 2-A, 2-B, 2-C, 2-D and 2-E, it can be seen that all kinds of T cell subgroup
Relative number (percent), this relative number is the percent relative to T cell number；Absolute number according to lymphocyte and each
The relative number (percent) of cell subsets, calculates the absolute number obtaining each cell subsets.
1、TCRαβ+Double negative t cells (TCR α β+DNT cell)
Understood by Fig. 2-A, Fig. 2-B, TCR α β+DNT (that is, CD3+TCRαβ+CD4-CD8-) it is target cell.Thin by streaming
Born of the same parents' instrument can record target cell and account for the 1.82% of T cell, and the lymphocyte absolute number that can be recorded by blood-counter system calculates,
TCRαβ+The absolute number of double negative t cells is 50/μ L.
2, gamma delta T cells
From Fig. 2-C, CD3+TCRγδ+It is target cell.Target cell can be recorded by flow cytometer and account for T cell
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 T cell subclass
Can obtain from Fig. 2-D, following four cell subsets: 1. assist T cells (i.e. CD4+ CD3+CD4+
CD45RA+CD27+), relative number 34.36%, absolute number 936/μ L；2. auxiliary exhausts T cell (i.e. Q4-2 in figure, CD3+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, following 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 exhausts T cell (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 fine immunophenotyping of B cell and quantitative analysis results (using BD Diva software analysis)
Bone-marrow-derived lymphocyte genotyping result is shown in Fig. 3-A and Fig. 3-B, it can be seen that the relative number (percentage of all kinds of B cell subgroup
Number), this relative number is the percent relative to B cell number；Absolute number according to lymphocyte and the phase of each cell subsets
Logarithm (percent), calculates the absolute number obtaining each cell subsets.
From the figure 3, it may be seen that the cell number of following four cell subsets and this subgroup 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 the literature, the term of reference of the relative number of each cell subsets in the lymphocyte of normal person is shown in Table 5 to root
Shown in.
Each cell subsets relative number in the lymphocyte of table 5 normal person
Wherein, ※ represents these data and does not has general term of reference, is only the experimental result of current research report；
According to Fig. 1, lymphocyte successfully can be divided into T lymphocyte, B lymph thin by the method used by the present embodiment
Born of the same parents and NK cell, and carry out quantitative analysis, it is thus achieved that the relative number of each cell and absolute number.From table 5, result understands, respectively
The relative number of individual cell subsets falls completely within the term of reference of normal person, consistent with the practical situation of detected sample, explanation
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, the method used by the present embodiment can be exactly by T
Lymphocyte is divided into TCR α β+Double negative t cells, gamma delta T cells, auxiliary T cells, auxiliary exhaust T cell, sectional center note
Recall T cell, secondary effects memory T cell, cytotoxicity T cells, cytotoxicity exhaustion T cell, cytotoxicity center note
Recall T cell, cytotoxic effect memory T cell, and carry out quantitative analysis, it is thus achieved that the relative number of each cell subsets is with exhausted
Logarithm.From table 5, result understands, and the relative number of each cell subsets is basically identical with the term of reference of normal person, with to be detected
The practical situation of sample is consistent, illustrates that experimental result of the present invention is stable, accurate.
According to Fig. 3, bone-marrow-derived lymphocyte can be divided into memory B cell, initial by method used by the present embodiment exactly
B cell, transition B cell, plasmablast, and carry out quantitative analysis, it is thus achieved that the relative number of each cell subsets and absolute number.
From table 5, result understands, and the relative number of each cell subsets is basically identical with the term of reference of normal person, with detected sample
Practical situation is consistent, illustrates that experimental result of the present invention is stable, accurate.
In sum, the method that the present invention provides uses less testing sample to be achieved that the lymph to testing sample is thin
Born of the same parents' immunophenotyping, and counted the content of each cell subsets；And the present invention provide method can be accurately by each cell
Subgroup carries out immunophenotyping.
The fine immunophenotyping of embodiment 2 lymphocyte and quantitative analysis
Experiment material:
Testing sample: anticoagulation cirumferential blood sample, derives from infant venous blood sample to our hospital and (has signed informed consent
Book), for intending examining the peripheral blood of systemic lupus erythematosus (sle) (SLE) infant.
BD Biosciences Lymphocyte subset test kit (cat 340503), buys in BD biosciences, its
Middle mixed antibody 1 includes: the anti-CD45 antibody of fluorescent labeling Percp, the antibody of AntiCD3 McAb (FITC), the antibody of anti-CD4 (APC),
The antibody of anti-CD8 (PE)；Mixed antibody 2 includes: the anti-CD45 antibody of fluorescent labeling Percp, the antibody of anti-CD19 (APC), anti-
The antibody of 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 liquid4Cl 8.29g、KHCO31g,EDTA 0.37g。
Experimental technique:
Take 500 μ L anticoagulation cirumferential blood samples, take 200 μ L testing samples, record lymphocyte by blood-counter system exhausted
Logarithm, the absolute number obtaining lymphocyte is 0.68 × 109Individual/L.
Remaining 300 μ L are used for detecting each subgroup of lymphocyte, carry out lymphocyte immunity typing and quantitative analysis.
Lymphocyte subset
Identical with the Lymphocyte subset method described in embodiment 1, temperature when wherein hatching is room temperature (20 DEG C), incubates
The time of educating is 35min.
The fine immunophenotyping of T lymphocyte
Identical with the T Lymphocyte subset method described in embodiment 1, wherein add PE labelling anti-tcr α β antibody,
After the anti-tcr γ anti-δ of BV421 labelling, the temperature hatched is room temperature (20 DEG C), and incubation time is 35min；Add residue antibody
After, the temperature hatched 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 fine immunophenotyping of bone-marrow-derived lymphocyte
1, taking two streaming pipes, respectively numbered B-1, B-2, wherein, B-1 is matched group and Isotype control group, and B-2 is for treating
Detection group；In each streaming pipe, following antibody is added according to table 6:
The classification of the antibody added in each streaming pipe of table 6 and the volume of addition
2, in B-1, B-2 streaming pipe, each 50 μ L testing samples adding mixing, after mixing, room temperature (20 DEG C) lucifuge is incubated
Educate 30min；
3, by the erythrocyte cracked liquid splitting erythrocyte of autogamy；
4, after addition 1mL PBS washes once, machine analysis result in streaming.
Interpretation of result:
By above three steps, obtain each lymphocyte subgroup relative number (percent) and lymphocyte absolute number.Concrete point
The method that analysis method provides with embodiment 1 is identical.
Lymphocyte immunity typing and quantitative analysis results (using BD auto Analysis to analyze)
Lymphocyte genotyping result Fig. 4, it can be seen that T cell, Tc cell, Th cell, B cell, NK cell is relative
(percent)；Absolute number according to lymphocyte and the relative number (percent) of each cell subsets, calculate and obtain each cell
The absolute number of subgroup, specific experiment the results are shown in Table 7.
Each cell surface marker used by cell subsets immunophenotyping of table 7, the relative number of each cell subsets and definitely
Number
The fine immunophenotyping of T cell and quantitative analysis results (using BD Diva software analysis)
T Lymphocyte subset result is shown in Fig. 5-A, 5-B, 5-C, 5-D and Fig. 5-E, it can be seen that all kinds of T cell subgroup
Relative number (percent), this relative number is the percent relative to T cell number；Absolute number according to lymphocyte and each
The relative number (percent) of individual cell subsets, calculates the absolute number obtaining each cell subsets.
1、TCRαβ+Double negative t cells (TCR α β+DNT cell)
Understood by Fig. 5-A, Fig. 5-B, TCR α β+DNT (i.e. CD3+TCRαβ+CD4-CD8-) it is target cell.Thin by streaming
Born of the same parents' instrument can record target cell and account for the 3.04% of T cell, and the lymphocyte absolute number that can be recorded by blood-counter system calculates,
TCRαβ+The absolute number of double negative t cells is 14/μ L.
2, gamma delta T cells
From Fig. 5-C, CD3+TCRγδ+It is target cell.Target cell can be recorded by flow cytometer and account for T cell
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 T cell subclass
Can obtain from Fig. 5-D, following four cell subsets: 1. assist T cells (i.e. CD4+ CD3+CD4+
CD45RA+CD27+), relative number 16.87%, absolute number 77/μ L；2. auxiliary exhausts T cell (i.e. Q4-2 in figure, CD3+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, following 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 exhausts T cell (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 fine immunophenotyping of B cell and quantitative analysis results (using BD Diva software analysis)
Bone-marrow-derived lymphocyte genotyping result is shown in Fig. 6-A and Fig. 6-B, it can be seen that the relative number (percentage of all kinds of B cell subgroup
Number), this relative number is the percent relative to B cell number；Absolute number according to lymphocyte and the phase of each cell subsets
Logarithm (percent), calculates the absolute number obtaining each cell subsets.
It will be appreciated from fig. 6 that the cell number of following four cell subsets and this subgroup 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. slurry is female thin
Born of the same parents (CD19+CD24-CD38++), relative number 13.8%, absolute number 29/μ L.
According to the literature, the term of reference of the relative number of each cell subsets in the lymphocyte of normal person is shown in Table 5 to root
Shown in.
According to Fig. 4, lymphocyte successfully can be divided into T lymphocyte, B lymph thin by the method used by the present embodiment
Born of the same parents and NK cell, and carry out quantitative analysis, it is thus achieved that the relative number of each cell and absolute number.As can be known from the results, test sample is treated
NK cell (CD16 in product+CD56+) content is significantly lower than normal reference range lower limit (7%-40%), and B cell (CD19+)
Higher than the normal reference range upper limit (5-18%), remaining Non Apparent Abnormality, point out B cell abnormal activation or increasing in this testing sample
Grow.
According to Fig. 5, T lymphocyte can be divided into TCR α β by method used by the present embodiment exactly+Double negative t
Cell, gamma delta T cells, auxiliary T cells, auxiliary exhaust that T cell, sectional center memory T cell, secondary effects memory T are thin
Born of the same parents, cytotoxicity T cells, cytotoxicity exhaust the memory of T cell, cytotoxicity Central memory T cell, cytotoxic effect
T cell, and carry out quantitative analysis, it is thus achieved that the relative number of each cell subsets and absolute number.As can be known from the results, each cell
The relative number of subgroup is basically identical with the term of reference of normal person, Non Apparent Abnormality.
According to Fig. 6, bone-marrow-derived lymphocyte can be divided into memory B cell, initial by method used by the present embodiment exactly
B cell, transition B cell, plasmablast, and carry out quantitative analysis, it is thus achieved that the relative number of each cell subsets and absolute number.
As can be known from the results, in testing sample, transition B cell (Transitional B cell) only accounts for the 0.05% of B cell, does not almost have
Have, point out its humoral immunization transition activation or imbalance.
In sum, the method that the present invention provides uses less testing sample to be achieved that the lymph to testing sample is thin
Born of the same parents' immunophenotyping, and counted the content of each cell subsets；And the present invention provide method can be accurately by each cell
Subgroup carries out immunophenotyping.
The fine immunophenotyping of embodiment 3 lymphocyte and quantitative analysis
Experiment material:
Testing sample: anticoagulation cirumferential blood sample, derives from infant venous blood sample to our hospital and (has signed informed consent
Book), for intending examining the peripheral blood of high IgM syndrome in children.
BD Biosciences Lymphocyte subset test kit (cat 340503), buys in BD biosciences, its
Middle mixed antibody 1 includes: the anti-CD45 antibody of fluorescent labeling Percp, the antibody of AntiCD3 McAb (FITC), the antibody of anti-CD4 (APC),
The antibody of anti-CD8 (PE)；Mixed antibody 2 includes: the anti-CD45 antibody of fluorescent labeling Percp, the antibody of anti-CD19 (APC), anti-
The antibody of 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 liquid4Cl 8.29g、KHCO31g,EDTA 0.37g。
Experimental technique:
Take 500 μ L anticoagulation cirumferential blood samples, take 200 μ L testing samples, record lymphocyte by blood-counter system exhausted
Logarithm, the absolute number obtaining lymphocyte is 0.89 × 109Individual/L.
Remaining 300 μ L are used for detecting each subgroup of lymphocyte, carry out lymphocyte immunity typing and quantitative analysis.
Lymphocyte subset
Identical with the Lymphocyte subset method described in embodiment 1, temperature when wherein hatching is room temperature (22 DEG C), incubates
The time of educating is 30min.
The fine immunophenotyping of T lymphocyte
Identical with the T Lymphocyte subset method described in embodiment 1, wherein add PE labelling anti-tcr α β antibody,
After the anti-tcr γ anti-δ of BV421 labelling, the temperature hatched is room temperature (22 DEG C), and incubation time is 25min；Add residue antibody
After, the temperature hatched 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 fine immunophenotyping of bone-marrow-derived lymphocyte
Identical with the bone-marrow-derived lymphocyte sorting technique described in embodiment 1, temperature when wherein hatching is room temperature (22
DEG C), incubation time is 25min.
Interpretation of result:
By above three steps, obtain each lymphocyte subgroup relative number (percent) and lymphocyte absolute number.Concrete point
The method that analysis method provides with embodiment 1 is identical.
Lymphocyte immunity typing and quantitative analysis results (using BD auto Analysis to analyze)
Lymphocyte genotyping result is shown in Fig. 7, it can be seen that T cell, Tc cell, Th cell, B cell, the phase of NK cell
To (percent)；Absolute number according to lymphocyte and the relative number (percent) of each cell subsets, each is thin to calculate acquisition
The absolute number of born of the same parents' subgroup, specific experiment the results are shown in Table 8.
Each cell surface marker used by cell subsets immunophenotyping of table 8, the relative number of each cell subsets and definitely
Number
The fine immunophenotyping of T cell and quantitative analysis results (using BD Diva software analysis)
T Lymphocyte subset result is shown in Fig. 8-A, 8-B, 8-C, 8-D and Fig. 8-E, it can be seen that all kinds of T cell subgroup
Relative number (percent), this relative number is the percent relative to T cell number；Absolute number according to lymphocyte and each
The relative number (percent) of individual cell subsets, calculates the absolute number obtaining each cell subsets.
1、TCRαβ+Double negative t cells (TCR α β+DNT cell)
Understood by Fig. 8-A, Fig. 8-B, TCR α β+DNT (i.e. CD3+TCRαβ+CD4-CD8-) it is target cell.Thin by streaming
Born of the same parents' instrument can record target cell and account for the 1.24% of T cell, and the lymphocyte absolute number that can be recorded by blood-counter system calculates,
TCRαβ+The absolute number of double negative t cells is 8/μ L.
2, gamma delta T cells
From Fig. 8-C, CD3+TCRγδ+It is target cell.Target cell can be recorded by flow cytometer and account for T cell
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 T cell subclass
Can obtain from Fig. 8-D, following four cell subsets: 1. assist T cells (i.e. CD4+ CD3+CD4+
CD45RA+CD27+), relative number 23.36%, absolute number 146/μ L；2. auxiliary exhausts T cell (i.e. Q4-2 in figure, CD3+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, following 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 exhausts T cell (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 fine immunophenotyping of B cell and quantitative analysis results (using BD Diva software analysis)
Bone-marrow-derived lymphocyte genotyping result is shown in Fig. 9-A and Fig. 9-B, it can be seen that the relative number (percentage of all kinds of B cell subgroup
Number), this relative number is the percent relative to B cell number；Absolute number according to lymphocyte and the phase of each cell subsets
Logarithm (percent), calculates the absolute number obtaining each cell subsets.
As shown in Figure 9, following four cell subsets and the cell number of this subgroup 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%, absolutely
Logarithm 216/μ L；3. transition B cell (CD19+CD24++CD38++), relative number 28.3%, absolute number 64/μ L；4. slurry is female thin
Born of the same parents (CD19+CD24-CD38++), relative number 0.9%, absolute number 2/μ L.
According to the literature, the term of reference of the relative number of each cell subsets in the lymphocyte of normal person is shown in Table 5 to root
Shown in.
According to Fig. 7, lymphocyte successfully can be divided into T lymphocyte, B lymph thin by the method used by the present embodiment
Born of the same parents and NK cell, and carry out quantitative analysis, it is thus achieved that the relative number of each cell and absolute number.As can be known from the results, test sample is treated
NK cell (CD16 in product+CD56+) it is 2.22%, hence it is evident that less than normal reference range lower limit (7-40%), and B cell (CD19+) it is 25.42%, higher than the normal reference range upper limit (5-18%), remaining Non Apparent Abnormality, prompting B cell abnormal activation or increasing
Grow.
According to Fig. 8, T lymphocyte can be divided into TCR α β by method used by the present embodiment exactly+Double negative t
Cell, gamma delta T cells, auxiliary T cells, auxiliary exhaust that T cell, sectional center memory T cell, secondary effects memory T are thin
Born of the same parents, cytotoxicity T cells, cytotoxicity exhaust the memory of T cell, cytotoxicity Central memory T cell, cytotoxic effect
T cell, and carry out quantitative analysis, it is thus achieved that the relative number of each cell subsets and absolute number.As can be known from the results, testing sample
Middle cytotoxicity exhausts T cell (CD8+TEMRA) content accounts for the 36.5% of T cell, is significantly higher than normal child of the same age, prompting
Infant there may be long-term chronic virus and infects；In testing sample, memory T cell (all kinds of centers/effect memory T) is just below
Often term of reference, points out this infant to there may be cellular immune abnormality；In testing sample, the content of gamma delta T cells accounts for T cell
18.1%, far above normal reference range (less than 5%), prompting infant virus infection, T cell abnormal activation or increment.
According to Fig. 9, bone-marrow-derived lymphocyte can be divided into memory B cell, initial by method used by the present embodiment exactly
B cell, transition B cell, plasmablast, and carry out quantitative analysis, it is thus achieved that the relative number of each cell subsets and absolute number.
As can be known from the results, naive B cellToo much, and memory B cell (memory B) only accounts for the 0.5% of B cell, the lowest
In normal limits, point out infant humoral immune function obstacle.
In sum, the method that the present invention provides uses less testing sample to be achieved that the lymph to testing sample is thin
Born of the same parents' immunophenotyping, and counted the content of each cell subsets；And the present invention provide method can be accurately by each cell
Subgroup carries out immunophenotyping.
Bone-marrow-derived lymphocyte surface markers immunophenotyping fine on bone-marrow-derived lymphocyte that comparative example is different and the impact of quantitative analysis
During B cell fine immunophenotyping method is set up, it is also desirable to (that is, surface resists to select suitable surface marker
Former combination) and the most easy stroke of door strategy.As a example by transition B cell (transitional B cell), there is multiple mistake at present
Cross specific surface antigen's combination of B cell, such as CD19+CD38++CD24++IgM++/CD19+CD38++CD24++(+represent the positive,
++ represent strong positive).
Combined with CD19, CD38, CD24, IgM, with the antibody of anti-CD19, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24,
The antibody of anti-IgM, as detection antibody, carries out lymphocyte immunity typing to testing sample.During data analysis, the first step exists
CD19+Cell in iris out CD24++IgM++Cell P1, obtain Figure 10-A；Second step, irises out CD24 with P1 for door++CD38++'s
Target cell (Transitional B cell), obtains Figure 10-B.As can be known from the results, owing to IgM and CD24 is continuous expression,
The boundary of P1 cell mass is unclear, it is impossible to accurately define the expression positive/strong positive.
Below it is only the preferred embodiment of the present invention, it is noted that it is right that above-mentioned preferred implementation is not construed as
The restriction of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, it is also possible to make some improvements and modifications, these change
Enter and retouch and also should be regarded as protection scope of the present invention.
Claims (9)
1. the method for a bone-marrow-derived lymphocyte immunophenotyping, it is characterised in that including:
Take different fluorescently-labeled antibody, mix with detected sample, after hatching, through Flow cytometry, obtain detection number
According to, analyze described detection data；
Described antibody includes:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27；
The method of described analysis includes:
Cell surface marker CD19+CD27+Represent memory B cell；
Cell surface marker CD19+CD27-IgD+Represent naive B cell；
Cell surface marker CD19+CD24++CD38++Represent transition B cell；
Cell surface marker CD19+CD24-CD38++Represent plasmablast.
Method the most according to claim 1, it is characterised in that also include adding up bone-marrow-derived lymphocyte number in described testing sample
Purpose step, including:
Detect the sum of described testing sample medium-sized lymphocyte；
Detect bone-marrow-derived lymphocyte in described testing sample and account for the percentage ratio of described lymphocyte；
By calculating, obtain the number of bone-marrow-derived lymphocyte in described testing sample.
3. the method for a lymphocyte immunity typing, it is characterised in that it includes that B lymph as claimed in claim 1 or 2 is thin
The step of born of the same parents' immunophenotyping.
4. the test kit for the method for bone-marrow-derived lymphocyte immunophenotyping as claimed in claim 1, it is characterised in that bag
Include the most different fluorescently-labeled antibody:
The antibody of anti-CD19, the antibody of anti-IgD, the antibody of AntiCD3 McAb 8, the antibody of anti-CD24 and the antibody of anti-CD27.
Test kit the most according to claim 4, it is characterised in that the fluorescent labeling of the antibody of described anti-CD19 be APC or
APC-Cy7。
6. according to the test kit described in claim 4 or 5, it is characterised in that the fluorescent labeling of the antibody of described anti-IgD is
BV510。
7. according to the test kit described in claim 4 or 5, it is characterised in that the fluorescent labeling of the antibody of described AntiCD3 McAb 8 is
Percp-cy5.5。
8. according to the test kit described in claim 4 or 5, it is characterised in that the fluorescent labeling of the antibody of described anti-CD24 is PE.
9. according to the test kit described in claim 4 or 5, it is characterised in that the fluorescent labeling of the antibody of described anti-CD27 is
BV450 or APC.
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| CN105758783B (en) * | 2016-05-05 | 2019-07-26 | 苏州大学 | A method of utilizing Flow cytometry popularity road trypsin like proteases 4 |
| CN106520693B (en) * | 2016-10-20 | 2019-11-22 | 清华大学 | Memory B cell precursor and application thereof |
| CN106771205B (en) * | 2017-01-18 | 2017-11-21 | 浙江博真生物科技有限公司 | Ten color antibody compositions and its application in leukemia-lymphoma parting |
| CN108226016A (en) * | 2018-01-12 | 2018-06-29 | 浙江普罗亭健康科技有限公司 | The mass spectrum flow cytometer detection kit of the accurate parting of tumor vaccine cells subgroup |
| CN108375675A (en) * | 2018-01-29 | 2018-08-07 | 李小峰 | Lymphocyte subpopulation cell concentration detection kit and its detection method |
| CN110716051B (en) * | 2018-07-13 | 2022-11-29 | 迈健医药科技无锡有限公司 | Immune cell full-dimensional analysis method |
| 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 |
| CN114264825B (en) * | 2021-12-22 | 2023-08-15 | 重庆医科大学附属儿童医院 | Method and kit for immunophenotyping of B lymphocyte development subpopulation |
| AU2022436042A1 (en) * | 2022-02-22 | 2023-09-07 | Cas Lamvac (Guangzhou) Biomedical Technology Co., Ltd. | Method for detecting a phenotype and a function of a cd141+ dendritic cell subset and kit for use |
| CN115792200B (en) * | 2022-11-18 | 2023-09-15 | 无锡市人民医院 | Antibody combination and kit for absolute count of peripheral blood B cell subpopulations after B cell removal treatment and application of antibody combination and kit |
| CN117169517B (en) * | 2023-11-03 | 2024-01-19 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD28 antibody residues in T lymphocyte preparation |
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