CN110487706A - A kind of detection method of human peripheral lymphocyte - Google Patents
A kind of detection method of human peripheral lymphocyte Download PDFInfo
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- CN110487706A CN110487706A CN201910671950.XA CN201910671950A CN110487706A CN 110487706 A CN110487706 A CN 110487706A CN 201910671950 A CN201910671950 A CN 201910671950A CN 110487706 A CN110487706 A CN 110487706A
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- human peripheral
- fluorescence probe
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kind of detection methods of human peripheral lymphocyte.The present invention takes people's anticoagulation cirumferential blood, it is separately added into anti-human CD3, CD4, CD8, CD45 monoclonal antibody, and anti-human CD3, CD56, CD16, CD45 monoclonal antibody, then the hemolysin working solution comprising fluorescence probe is added, to obtain human peripheral blood T lymphocyte subgroup and NK lymphocyte subgroup percentage and absolute counting value respectively, the detection to human peripheral lymphocyte is realized.The present invention has the advantages that easy to operate, the sample stable holding time is long, and accuracy is high, and multi objective exports.
Description
Technical field
The invention belongs to Cell Measurement Technique fields, more particularly to a kind of detection method of human peripheral lymphocyte.
Background technique
Flow cytometry (Flow Cytometry, FCM) is the high science and technology that the seventies grow up, it, which collects, calculates
Machine technology, laser technology, hydrodynamics, cytochemistry, cellular immunology have analysis and sorting cell function in one
Energy.Testing principle is fluorescent marker cell to be tested and single cell suspension is made, according to liquid stream inner cell after reception laser irradiation
Scattering light signal and fluorescence signal analysis cell physicochemical characteristic, such as cell size, the letter of tested cell internal particle
Breath etc..It not only can measure the character of cell size, internal particle, also detectable cell surface and cytoplasmic antigen, intracellular
DNA, rna content etc. can analyze group's cell on individual cell level, test and analyze a large amount of cells in a short time,
And data are collected, store and handle, carry out multi-parameter quantitative analysis；Can a certain cell subset of category sorting, separating purity >
95%.It is widely applied in subjects such as hematology, immunology, oncology, materia medica, molecular biology.
T lymphocyte and NK cell are two kinds of monoids of lymphocyte, can embody the immune function of matrix cells.
Chinese patent CN107063982A discloses a kind of side of Flow cytometry chicken lymphocyte subpopulation
Method.But the method for above-mentioned patent disclosure is only capable of exporting the percent positive of each subgroup, it is as a result more single, and the simple positive
Percentage is only comparative counting, and if lymphocyte whole cell number all reduces, i.e., absolute number reduces, but opposite each group
Percentage may remain unchanged.In addition, the detection method of above-mentioned patent is longer to the extraction operation time of lymphocyte, it is unfavorable for
The absolute counting of lymphocyte, at the same will lead to sample cell damage and apoptotic process it is long, detection sensitivity is relatively low, influences to examine
Survey result.
Summary of the invention
Based on defect of the existing technology, the purpose of the present invention is to provide a kind of detections of human peripheral lymphocyte
Method.The present invention has the advantages that easy to operate, the sample stable holding time is long, and accuracy is high, and multi objective exports.
To achieve the above object, the present invention takes following technical scheme:
A kind of detection method of human peripheral lymphocyte, comprising the following steps:
(1) T lymphocyte subgroup detects:
It takes people's anticoagulation cirumferential blood that streaming bottom of the tube is added, anti-human CD3, CD4, CD8, CD45 monoclonal antibody is added, be vortexed
It mixes, is protected from light incubation at room temperature；Then the hemolysin working solution comprising fluorescence probe is further added, is vortexed and mixes, at room temperature
It is protected from light incubation；It is finally detected using flow cytometer, obtains human peripheral blood T lymphocyte subgroup percentage and absolute counting
Value；
(2) NK lymph subsets counts:
It takes people's anticoagulation cirumferential blood that streaming bottom of the tube is added, anti-human CD3, CD56, CD16, CD45 monoclonal antibody, whirlpool is added
Rotation mixes, and is protected from light incubation at room temperature；Then the hemolysin working solution comprising fluorescence probe is further added, is vortexed and mixes, room temperature
Under be protected from light incubation；Finally detected using flow cytometer, obtain human peripheral NK lymphocyte subgroup percentage and absolutely
Count value.
The present invention directly marks human peripheral using the monoclonal antibody reagent of Cell membrane antigens specific antibody,
In: leucocyte is total to antigen molecule CD45, for distinguishing different lymphocyte populations；Antibody CD3, CD4, CD8 specific marker T leaching
Bar cell and its subgroup molecule；Antibody CD16 and CD56 specific marker NK lymphocyte and its subgroup molecule.
Further, in the above-mentioned methods, the fluorescence probe of the step (1) is mitochondria fluorescence probe；Preferably, institute
Stating fluorescence probe is mitochondria green fluorescence probe.
Further, in the above-mentioned methods, the fluorescence probe of the step (2) is lysosome fluorescence probe；Preferably, institute
Stating fluorescence probe is lysosome green fluorescence probe.
Mitochondria fluorescence probe and lysosome fluorescence probe combination lymph is respectively adopted in step (1) of the present invention and step (2)
Cell completes organelle calibration.Wherein, mitochondria fluorescence probe is mainly combined according to mitochondrial membrane potential variation, lyase
Body fluorescence probe is mainly combined according to lysosome pH value variation.
Further, in the above-mentioned methods, the monoclonal antibody and the volume ratio of fluorescence probe are 5:1~10:1.
Monoclonal antibody is mainly protein component, and fluorescence probe is high-molecular compound, will appear interference between the two
Reaction, the present invention can effectively reduce disturbing reaction by the appropriate titer of optimization antibody and probe, guarantee antigen and antibody, visit
The accurate combination of needle and target.
Further, in the above-mentioned methods, the hemolysin working solution include ammonium chloride, saleratus, EDETATE SODIUM and
Fetal calf serum (FBS).
Further, in the above-mentioned methods, every liter of hemolysin working solution includes 75~85g of ammonium chloride, saleratus
15~25g, 300~400mg of EDETATE SODIUM, fetal calf serum (FBS) 15~30ml.
The hemolysin Working solution prescription that the present invention uses is a kind of mild erythrocyte cracked liquid, can crack human peripheral
Sample makes sample only retain leucocyte, guarantees the integrality of leucocyte membrane surface molecule structure, and maintains the good work of cell
Property, it is ensured that the sample after dyeing maintains the stability in 48 hours.
Further, in the above-mentioned methods, the volumetric concentration of fluorescence probe is 2%~5% in the hemolysin working solution.
Further, in the above-mentioned methods, the volume ratio of people's anticoagulation cirumferential blood and hemolysin working solution is 1:10~1:5.
Further, in the above-mentioned methods, the incubation time that is protected from light is 15-60 minutes.
The present invention has following technical characterstic:
1) what the present invention innovated combines lymphocyte therein using organelle-specificity fluorescence probe, completes organelle mark
Fixed, the signal strength or weakness after fluorescence probe combination cell device being capable of the stable cell mass of Accurate Analysis mass.
2) present invention detects T lymphocyte and NK lymphocyte simultaneously.T lymphocyte is as main in lymphocyte populations
Expression can be improved the sensitivity to lymphocyte detection；NK lymphocyte subgroup is as inherent immunity cell, to where cell
Content environment it is extremely sensitive, by analyzing the variation of its subgroup, the susceptibility of cell detection can be improved.
3) detecting step of the present invention is simple, easy to operate, and detecting step is only two steps, and sample is in conjunction with reagent, red blood cell
Cracking, can go up machine testing, reduce manual operation, reduce operational risk, at the same after preparing sample the stabilization holding time
It is longer
4) present invention accuracy with higher and sensitivity pass through the percent positive and absolute meter of lymphocyte subgroup
Two indices are counted to realize the detection to human peripheral lymphocyte, can be applied to assessment cellular damage situation in the future, or even use
In judging risk.
Detailed description of the invention
Fig. 1 is CD45/SSC scatter plot, for enclosing lymphocyte populations
Fig. 2 is to read CD45+Lymph, and Mito/CD3 scatter plot identifies Mito+CD3+ (i.e. Q3-2)
Fig. 3 is to read Mito+CD3+, and CD4/CD8 scatter plot identifies CD3+CD8+, CD3+CD4+.
Fig. 4 is CD45/SSC scatter plot, for enclosing lymphocyte populations
Fig. 5 is to read CD45+Lymph, and Lyso/CD3 scatter plot identifies Lyso+CD3- (i.e. Q2-1)
Fig. 6 is to read Lyso+CD3-, and CD16/CD56 scatter plot identifies CD3-CD56+CD16+, CD3-
CD56dimCD16-, CD3-CD56brightCD16-.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be to the embodiment of the present invention
Technical solution carries out clear, complete description.Obviously, described embodiment is a part of the embodiments of the present invention, rather than
Whole embodiments.Based on described the embodiment of the present invention, those of ordinary skill in the art are without creative work
Under the premise of every other embodiment obtained, belong to protection scope of the present invention.
Unless otherwise defined, technical term or scientific term used in the disclosure are should be in fields of the present invention
The ordinary meaning for thering is the personage of general technical ability to be understood.
Reagent source used in the specific embodiment of the invention is as shown in table 1.
1 reagent name of table, producer and article No.
Embodiment 1: the T lymphocyte subgroup detection of human peripheral mitochondria expression
The detection reagent of the T lymphocyte subgroup of human peripheral mitochondria expression of the invention include: hemolysin (10 ×),
Mitochondria fluorescent dye, CD3APC, CD4PE-Cy7, CD8PE, CD45APC-Cy7 monoclonal antibody.
(1) the T lymphocyte subgroup reagent of human peripheral mitochondria expression prepares
1. hemolysin
10 × hemolysin is diluted to 1 × working solution (1 × hemolysin) with deionized water, is added according to 50 μ L human peripherals
The amount calculation amount of 0.5mL working solution is prepared.
10 × hemolysin of 1L is prepared to need using 82.9g ammonium chloride, 10g saleratus, 372mg EDETATE SODIUM, 20mL
Fetal calf serum (Fetal calf serum, FBS).
2. mitochondria fluorescent dye and monoclonal antibody
Mitochondria fluorescent dye prepares (1 person-portion dosage): 1 μ L mitochondria fluorescent dye is added to 499 μ L hemolysin works
Make in liquid, low speed vortex mixes, and 2~8 DEG C spare.
CD3APC MAb concentration is 0.05 μ g/test (5 μ L), and CD4PE-Cy7 MAb concentration is 0.1 μ
G/test (5 μ L), CD8PE MAb concentration are 0.02 μ g/test (5 μ L), and CD45APC-Cy7 MAb concentration is
0.01μg/test(5μL)。
(2) the T lymphocyte subgroup of flow cytomery human peripheral mitochondria expression is utilized
1. pipetting 50 μ L anticoagulation cirumferential bloods with suitable micropipettor, it is slowly added into streaming bottom of the tube, as tube wall has blood
Liquid contamination, needs to clean using cotton swab, incomplete to avoid subsequent erythrocyte splitting, and testing result fragment is caused to increase phenomenon；
2. pipetting each antibody reagent using suitable micropipettor is added the sample into step 1, middling speed, which is vortexed, to be mixed,
It is protected from light incubation 15 minutes at room temperature；
3. pipette 0.5mL hemolysin working solution (fluorescent dye containing mitochondria) using suitable micropipettor, slowly plus
Enter in the streaming pipe in step 2, middling speed, which is vortexed, to be mixed, and is protected from light incubation 60 minutes at room temperature；
Machine testing (detection being completed in 6 hours, if do not detected immediately, sample is please placed on 2~8 DEG C and is protected from light down) on 4..
5. streaming graph data interpretation of result, testing result are as shown in Figure 1-Figure 3.Wherein, Fig. 1 is with CD45/SSC scatterplot
Human peripheral leucocytes are divided into three groups of lymphocyte populations, monocyte group and granulocyte group by figure, CD45；Fig. 2 is to read
It is CD45+Lymph groups, is shown as mitochondrial probe expression and T lymphocyte number (CD3) scatter plot, is superimposed in the figure
With the blank tube for the mitochondrial probe that is unstained；Fig. 3 be read be Mito+/CD3+ group (i.e. Q3-2), display CD4/CD8 scatterplot
Scheme, identified respectively helper-inducer T lymphocyte group (CD3+CD4+) in figure, inhibits malicious T lymphocyte group (CD3+CD8+), make
With the volumetric method absolute counting method of ACEA NovoCyte flow cytometer, the absolute counting and positive percentage of three groups is obtained
Than as shown in table 1.
The absolute counting and percent positive of table tri- groups of 1 Q3-2, CD3+CD4+ and CD3+CD8+
| Gate | Count | Abs.Count | %Q3-2 |
| Q3-2 | 9,919 | 1,475 | 100.00% |
| CD3+CD8+ | 4,036 | 599 | 40.69% |
| CD3+CD4+ | 4,701 | 699 | 47.39% |
Embodiment two: the NK Lymphocyte subtypes test of human peripheral lysosome expression
The NK Lymphocyte subtypes test reagent of human peripheral lysosome expression of the invention include: hemolysin (10 ×),
Lysosome fluorescent dye, CD3APC, CD56PE, CD16-PE/Cy7, CD45-APC-Cy7 monoclonal antibody.
(1) the NK lymphocyte subgroup reagent of human peripheral lysosome expression prepares
1. hemolysin
10 × hemolysin is diluted to 1 × working solution (1 × hemolysin) with deionized water, is added according to 100 μ L human peripherals
The amount calculation amount for entering 2mL working solution is prepared.
10 × hemolysin of 1L is prepared to need using 82.9g ammonium chloride, 10g saleratus, 372mgEDTA disodium.
2. lysosome fluorescent dye and monoclonal antibody
Lysosome fluorescent dye prepares (1 person-portion dosage): 1 μ L lysosome fluorescent dye is added to 499 μ L hemolysin works
Make in liquid, low speed vortex mixes, and 2~8 DEG C spare.
CD3APC MAb concentration is 0.05 μ g/test (5 μ L), and CD16PE-Cy7 MAb concentration is 0.05
μ g/test (5 μ L), CD56PE MAb concentration are 0.1 μ g/test (5 μ L), CD45APC-Cy7 MAb concentration
For 0.01 μ g/test (5 μ L).
(2) the NK lymphocyte subgroup of human peripheral lysosome expression
1. pipetting 50 μ L anticoagulation cirumferential bloods with suitable micropipettor, it is slowly added into streaming bottom of the tube, as tube wall has blood
Liquid contamination, needs to clean using cotton swab, incomplete to avoid subsequent erythrocyte splitting, and testing result fragment is caused to increase phenomenon；
2. pipetting each antibody reagent using suitable micropipettor is added the sample into step 1, middling speed, which is vortexed, to be mixed,
It is protected from light incubation 15 minutes at room temperature；
3. being added in step 2 using the hemolysin (fluorescent dye containing lysosome) that suitable micropipettor pipettes 0.5mL
Streaming pipe in, middling speed be vortexed mix, be protected from light at room temperature incubation 60 minutes；
Machine testing (detection being completed in 6 hours, if do not detected immediately, sample is please placed on 2~8 DEG C and is protected from light down) on 4..
5. streaming graph data interpretation of result, testing result are as shown in Figure 4-Figure 6.Wherein, Fig. 4 is dissipated with CD45/SSC
Human peripheral leucocytes are divided into three groups of lymphocyte populations, monocyte group and granulocyte group by point diagram, CD45；Fig. 5 is to read
Be CD45+Lymph groups, be shown as lysosome probe expression and T lymphocyte number (CD3) scatter plot, be superimposed in the figure
With the blank tube for the lysosome probe that is unstained；Fig. 6 be read be Lyso+/CD3- group (i.e. Q2-1), display CD16/CD56
Scatter plot identifies NK1 lymphocyte populations (CD3-CD56+CD16+) in figure, NK2 lymphocyte populations (CD3- respectively
CD56dimCD16-) and NK3 lymphocyte populations (CD3-CD56brightCD16-), ACEA NovoCyte flow cytometer is used
Volumetric method absolute counting method, obtain the absolute counting and percent positive of four groups, as shown in table 2.
The absolute counting and percent positive of table tetra- groups of 2 Q2-1, NK1, NK2 and NK3
| Gate | Count | Abs.Count | %Q2-1 |
| Q2-1 | 3,414 | 596 | 100.00% |
| NK1 | 1,841 | 321 | 53.93% |
| NK2 | 57 | 9.9 | 1.67% |
| NK3 | 8 | 1.39 | 0.23% |
To sum up, group can be divided by the quality of streaming map analysis organelle from the embodiment of the present invention 1 and embodiment 2, according to
The stable cell mass of mass is precisely analyzed according to the signal strength or weakness after fluorescence probe combination cell device.Stablize cell mass by analyzing
T lymphocyte subgroup and NK lymphocyte subgroup, the percentage and absolute counting value of two kinds of subgroups are read, to realize outside people
The detection of all blood lymphocytes.Method of the invention effectively raises the accuracy and sensitivity of lymphocyte detection.
The method of the present invention that the above embodiments are only used to help understand and its core concept.It should be pointed out that for
For those skilled in the art, without departing from the principle of the present invention, if can also be carried out to the present invention
Dry improvement and modification, these improvement and modification are also fallen into the claims in the present invention protection scope.
Claims (9)
1. a kind of detection method of human peripheral lymphocyte, which comprises the following steps:
(1) T lymphocyte subgroup detects:
It takes people's anticoagulation cirumferential blood that streaming bottom of the tube is added, anti-human CD3, CD4, CD8, CD45 monoclonal antibody is added, be vortexed and mix,
It is protected from light incubation at room temperature；Then the hemolysin working solution comprising fluorescence probe is further added, is vortexed and mixes, be protected from light incubate at room temperature
It educates；It is finally detected using flow cytometer, obtains human peripheral blood T lymphocyte subgroup percentage and absolute counting value；
(2) NK Lymphocyte subtypes test:
It takes people's anticoagulation cirumferential blood that streaming bottom of the tube is added, anti-human CD3, CD56, CD16, CD45 monoclonal antibody is added, be vortexed mixed
It is even, it is protected from light incubation at room temperature；Then the hemolysin working solution comprising fluorescence probe is further added, is vortexed and mixes, keep away at room temperature
Light is incubated for；It is finally detected using flow cytometer, obtains human peripheral NK lymphocyte subgroup percentage and absolute counting
Value.
2. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that the step (1)
Fluorescence probe is mitochondria fluorescence probe；Preferably, the fluorescence probe is mitochondria green fluorescence probe.
3. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that the step (2)
Fluorescence probe is lysosome fluorescence probe；Preferably, the fluorescence probe is lysosome green fluorescence probe.
4. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that the monoclonal antibody
Volume ratio with fluorescence probe is 5:1~10:1.
5. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that the hemolysin work
Liquid includes ammonium chloride, saleratus, EDETATE SODIUM and fetal calf serum (FBS).
6. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that every liter of hemolysin
Working solution include 75~85g of ammonium chloride, 15~25g of saleratus, 300~400mg of EDETATE SODIUM, fetal calf serum (FBS) 15~
30ml。
7. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that the hemolysin work
The volumetric concentration of fluorescence probe is 2%~5% in liquid.
8. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that people's anticoagulation cirumferential blood with
The volume ratio of hemolysin working solution is 1:10~1:5.
9. a kind of detection method of human peripheral lymphocyte according to claim 1, which is characterized in that described when being protected from light incubation
Between be 15-60 minutes.
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| PCT/CN2020/099875 WO2021012925A1 (en) | 2019-07-24 | 2020-07-02 | Method for measuring human peripheral blood lymphocytes |
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| CN112098646B (en) * | 2020-11-23 | 2021-02-05 | 泛肽生物科技(浙江)有限公司 | Kit for quantitatively detecting lymphocyte subpopulation and detection method thereof |
| CN113218847A (en) * | 2021-04-12 | 2021-08-06 | 武汉凯普瑞生物技术有限公司 | Hemolytic agent for flow cytometry analysis and preparation method and application method thereof |
| CN114578048A (en) * | 2021-12-22 | 2022-06-03 | 重庆医科大学附属儿童医院 | T lymphocyte development subgroup immunophenotyping method and kit |
| CN114578048B (en) * | 2021-12-22 | 2023-08-08 | 重庆医科大学附属儿童医院 | T lymphocyte development subgroup immunophenotyping method and kit |
| CN115141801A (en) * | 2022-08-16 | 2022-10-04 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method and kit for analyzing different lymphocyte subpopulations based on mitochondrial metabolic activity and application of method and kit |
| CN115141801B (en) * | 2022-08-16 | 2024-01-26 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method for analyzing different lymphocyte subpopulations based on mitochondrial metabolic activity, kit and application thereof |
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