CN109813903A - A method for the diagnosis of Mycobacterium tuberculosis infection based on the detection of tuberculosis-specific T cell immunophenotype by flow cytometry - Google Patents
A method for the diagnosis of Mycobacterium tuberculosis infection based on the detection of tuberculosis-specific T cell immunophenotype by flow cytometry Download PDFInfo
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Abstract
本发明涉及基于流式细胞术检测结核特异性T细胞免疫表型用于诊断结核分枝杆菌感染的方法，包括首先对细胞的培养和刺激，接着进行多色免疫表型标记，再用流式细胞术检测CD4+T淋巴细胞经结核特异性抗原ESAT‑6和CFP‑10刺激后，其细胞表型CD25+CD134+的表达增加。本发明的方法有着操作简单的优点，且还增加了诊断的灵敏度和缩短了检测时间，适合在结核病的检测中推广。
The invention relates to a method for diagnosing Mycobacterium tuberculosis infection based on the detection of tuberculosis-specific T cell immunophenotype based on flow cytometry. Cytometry detection of CD4 + T lymphocytes stimulated by tuberculosis-specific antigens ESAT-6 and CFP-10 increased the expression of their cell phenotype CD25 + CD134 + . The method of the invention has the advantages of simple operation, increases the sensitivity of diagnosis and shortens the detection time, and is suitable for promotion in the detection of tuberculosis.
Description
Technical field
The present invention relates to medical sciences, in particular to are based on Flow cytometry tuberculosis Specific T cell immunity
The method that phenotype is used for diagnosis of tuberculosis mycobacterial infections.
Background technique
Tuberculosis is one kind and machine caused by mycobacterium tuberculosis (mycobacterium tuberculosis, MTB)
Closely related chronic infectious disease is immunized in body cell, seriously threatens human health.In recent years, continuous with MTB persister
Increase and send out and BCG vaccine prevention declines, tuberculosis is in rebound significantly trend in the whole world, and popularity is increasingly serious.In
State is severely afflicated area lungy, and Tuberculosis number accounts for the 11.36% of the whole world, occupies the whole world second.After MTB infects human body,
According to the Immunity of individual, three kinds of final results can occur: the first final result is that individual immunity power is vigorous, directly by infection
MTB is killed；Second is that immunity of organisms is not enough to kill whole MTB, but can inhibit a large amount of proliferation of MTB, is made up to flat
Weighing apparatus state, so as to cause latent tuberculosis infects；The third, which is that the immunity of body is too low, causes MTB to be largely proliferated, and initiation is faced
Bed disease.It is estimated that having nearly 1/3 population infection tubercle bacillus in the world, wherein only 5%~10% or so people can send out
Disease, remaining 90%~95% crowd are in latent infection.However, can lead to LTBI transformation in body's immunity disorder
For active tuberculosis.Therefore, efficiently timely tuberculosis early diagnosis is tuberculosis normalization to tubercular especially LTBI patient
The starting point for the treatment of.
Laboratory diagnostic technique lungy is always the bottleneck of tuberculosis prevention and control.Existing diagnostic means include that aetology is examined
It is disconnected as MTB is separately cultured, the methods of smear for microscopic examination and Molecular Detection diagnosis.But the above method has respective limitation, as MTB is separated
Time-consuming (generally requiring 6~8 weeks) for culture, and sensitivity is not high, and there are bio-safety risks, only opens in tuberculosis fixed hospital
Exhibition.Smear for microscopic examination susceptibility is low (only 20%~30% positive rate), and cannot distinguish between mycobacterium tuberculosis and non-tuberculosis point
Branch bacillus, often delay treatment.Molecular diagnosis is complicated for operation, and the biological characteristics of tubercle bacillus, the sensitivity of this method and
Specificity is not able to satisfy clinical demand.The above method requires to obtain sample such as phlegm, hydrothorax, ascites, brain from infection site
Spinal fluid etc., but it is negative for sputum smear negative or etiological examination, and clinical strong suspicion tuberculosis；Disease can not especially be obtained
Stove sample such as bone tuberculosis, intestinal tuberculosis etc. is badly in need of a kind of new laboratory inspection method to diagnosis lungy and exclusion diagnosis, is exempted from
Epidemiology diagnosis is come into being.In recent years, scientist has found that Mycobacterium tuberculosis has and BCG vaccine by comparing genomics
No gene region with other mycobacteriums, and it is located at two albumen Early insulin secretion antigens -6 (ESAT-6) and the training in this region
Supporting base filtration albumen -10 (CFP-10) has stronger Th epitope.Herein on basis, in conjunction with T cell immunoassay technology
Progress establishes a kind of novel tuberculosis Specific T cell immunity external detection method --- based on elispot assay
Tuberculosis infection T lymphocyte spot test (T-SPOT.TB) and T lymphocyte γ-IFN body based on Enzyme-linked Immunosorbent Assay technology
Outer release test (IGRA).
The principle of T-SPOT.TB and IGRA is almost the same, is body to the cell immune response occurred after MTB infection.
It is former using the tuberculosis antigen ESAT-6 and CFP-10 of specificity as stimulation, it is stimulated by the antigen presenting cell in whole blood special
Property Th cell generate IFN-γ, in the lymphocyte quantity (T- of 37 DEG C of generation IFN-γ of detection after in vitro culture 16-24 hour
SPOT.TB IFN-γ content (IGRA.TB)) or in culture solution judges in whole blood with the presence or absence of tuberculosis specific T-cells and its work
Power.Due to, as stimulation original, not influenced by BCG vaccine and non-tuberculous mycobacteria, to tuberculosis sense using the peculiar antigen of tulase
The detection sensitivity and specificity of dye are higher.
The two methods of value of T-SPOT.TB and IGRA in diagnosis and treatment tuberculosis is without significant difference, but in technological layer, T-
SPOT.TB and IGRA.TB suffers from common defect: being 1. the not CD4 using total lymphocyte as research object+T lymph it is thin
Born of the same parents, and CD4+T lymphocyte is that the principal immune of the anti-MTB of the mankind adjusts cell, the specificity of testing result and sensitivity meeting
It is affected.2. being the variation of T-SPOT.TB detection intracellular cytokine, IGRA.TB testing inspection is to discharge into the cell
Cell factor out, detection process is more complex, and influence factor is more.3. be for T-SPOT.TB, in detection process to point
From PBMC count requirement it is accurate, otherwise influence result accuracy；When as a result judging, with the naked eye there is subjectivity, otherwise needs to use
Instrument readings, and have overlapping phenomenon when blurring；And for IGRA.TB test, though not having to accurate counting PBMC number, use
The IFN-γ of ELISA detection culture upper liquid, detection is both needed to do standard curve every time, is not suitable for single specimen examination, and calculate
It is more complex.
Summary of the invention
In order to overcome the above-mentioned deficiency in the presence of the prior art, the present invention provides be based on Flow cytometry tuberculosis
The method that Specific T cell immunity phenotype is used for diagnosis of tuberculosis mycobacterial infections.Detection method of the invention is more clinical than at present
Widely applied T-SPOT.TB and IGRA operation is simpler, and cost can be some more low, and the diagnosis spirit to mycobacterium tuberculosis infection
Sensitivity and specificity are higher.
The present invention the technical solution to solve the technical problem is that: exempted from based on Flow cytometry tuberculosis specific T-cells
The method that epidemic disease phenotype is used for diagnosis of tuberculosis mycobacterial infections, comprising the following steps:
1) cell culture:
If three pipes；First pipe is negative control pipe: being added in 1640 cell culture mediums containing 9~11% calf serums
CD28 and CD49d adds 0.4~0.6mL liver after so that CD28 and CD49d concentration is respectively 0.9~0.12ug/mL
The anticoagulant peripheral blood of element；Second pipe is positive control pipe: being added in 1640 cell culture mediums containing 9~11% calf serums
PHA, CD28 and CD49d, making 9~11ug/mL of PHA concentration and CD28, CD49d concentration in culture medium is respectively 0.9~0.12ug/
After mL, the peripheral blood of 0.4~0.6mL anticoagulant heparin is added；Third pipe is measurement pipe: containing 9~11% calf serums
ESAT-6, CFP-10, CD28, CD49d are added in 1640 cell culture mediums, makes ESAT-6 the and CFP-10 concentration in culture medium respectively be
After 4.5~5.5ug/mL, CD28 and CD49d concentration are respectively 0.9~1ug/mL, the periphery of 0.4~0.6mL anticoagulant heparin is added
Blood；
Negative control pipe, positive control pipe and measurement pipe be put into 36~38 DEG C, culture 20 in 4~6%CO2 incubator~
24 hours；
2) polychrome immunophenotypic marker and Flow cytometry: 45~55uL is respectively taken to distinguish from 3 pipe whole bloods of culture
It is added in three streaming pipes, then, CD3-APC, CD4-PC5, CD25-PerCP, CD134-FITC is added in every streaming pipe
Each 4~6uL, room temperature are protected from light 15~20min；Add NH4Cl 400~450ul of hemolysin, slight oscillatory mix；Adjust fluidic cell
The voltage of forward direction and side scattered light takes CD4 according to CD4-CD3 gating in instrument+CD3+Cell analysis CD25+CD134+Group's cell
Percentage, measurement result are the CD25 for measuring pipe+CD134+Group's cell percentage-negative control pipe CD25+CD134+Group is thin
Born of the same parents' percentage.
It is of the invention to be used to examine based on Flow cytometry tuberculosis Specific T cell immunity phenotype as prioritization scheme
The method of disconnected mycobacterium tuberculosis infection, comprising the following steps:
1) cell culture:
If three pipes；First pipe is negative control pipe: CD28 is added in 1640 cell culture mediums containing 10% calf serum
And CD49d adds the peripheral blood of 0.5mL anticoagulant heparin after so that CD28 and CD49d concentration is respectively 1ug/mL；The
Two pipes are positive control pipe: PHA, CD28 and CD49d being added in 1640 cell culture mediums containing 10% calf serum, makes to cultivate
PHA concentration is 10ug/mL and after CD28, CD49d concentration is respectively 1ug/mL in base, adds the peripheral blood of 0.5mL anticoagulant heparin；
Third pipe be measurement pipe: containing 10% calf serum 1640 cell culture mediums in be added ESAT-6, CFP-10, CD28,
CD49d makes in culture medium ESAT-6 and CFP-10 concentration respectively be 5ug/mL, after CD28 and CD49d concentration is respectively 1ug/mL, then plus
Enter the peripheral blood of 0.5mL anticoagulant heparin；
Negative control pipe, positive control pipe and measurement pipe are put into 37 DEG C, cultivated 24 hours in 5%CO2 incubator；
2) polychrome immunophenotypic marker and Flow cytometry: 45~55uL is respectively taken to distinguish from 3 pipe whole bloods of culture
It is added in three streaming pipes, then, CD3-APC, CD4-PC5, CD25-PerCP, CD134-FITC is added in every streaming pipe
Each 5uL, room temperature are protected from light 15min；Add NH4Cl hemolysin 400ul, slight oscillatory mix；Adjust forward direction and side in flow cytometer
CD4 is taken according to CD4-CD3 gating to the voltage of scattering light+CD3+Cell analysis CD25+CD134+Group's cell percentage, measurement
It as a result is the CD25 of measurement pipe+CD134+Group's cell percentage-negative control pipe CD25+CD134+Group's cell percentage.
As optimization, in the step 1), in three set pipes, the use of 1640 cell culture mediums containing 10% calf serum
Amount is 0.5mL.
Compared with prior art, of the invention to be used for based on Flow cytometry tuberculosis Specific T cell immunity phenotype
The method of diagnosis of tuberculosis mycobacterial infections establishes multi-color marking art, CD4 using polychrome flow cytometry as detection platform+T lymph
Cell is after tuberculosis specific antigen ESAT-6 and CFP-10 stimulation, cell phenotype CD25+CD134+Expression increase, have
Significant progress, specific as follows:
1) CD4 sensitive to tubercle bacillus specific antigen protein (CFP-10 and ESAT-6) stimulation is directly detected+T lymph
Cell (CD4+T lymphocyte is that the principal immune of mankind's Killing Mycobacterium Tuberculosis infection adjusts cell), improve the spy of detection
It is anisotropic；
2) directly detection CD4 after tubercle bacillus specific antigen protein (CFP-10 and ESAT-6) stimulation+T lymphocyte
Surface immunophenotype CD25+CD134+, compared to the method and IGRA.TB examination of T-SPOT.TB detection intracellular cytokine variation
The method that detection releases cell factor into the cell is tested, operation is simpler, and sensibility is higher, and testing cost is lower.
Detailed description of the invention
Fig. 1 is streaming gating scheme of the invention.
Specific embodiment
Below by specific embodiment, and in conjunction with attached drawing, technical solution of the present invention is further elaborated with, but simultaneously
Not as the foundation limited the present invention.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Embodiment
The side of diagnosis of tuberculosis mycobacterial infections is used for based on Flow cytometry tuberculosis Specific T cell immunity phenotype
Method, comprising the following steps:
1) cell culture:
If three pipes；First pipe is negative control pipe: CD28 is added in 1640 cell culture mediums containing 10% calf serum
And CD49d adds the peripheral blood of 0.5mL anticoagulant heparin after so that CD28 and CD49d concentration is respectively 1ug/mL；The
Two pipes are positive control pipe: PHA, CD28 and CD49d being added in 1640 cell culture mediums containing 10% calf serum, makes to cultivate
PHA concentration is 10ug/mL and after CD28, CD49d concentration is respectively 1ug/mL in base, adds the peripheral blood of 0.5mL anticoagulant heparin；
Third pipe be measurement pipe: containing 10% calf serum 1640 cell culture mediums in be added ESAT-6, CFP-10, CD28,
CD49d makes in culture medium ESAT-6 and CFP-10 concentration respectively be 5ug/mL, after CD28 and CD49d concentration is respectively 1ug/mL, then plus
Enter the peripheral blood of 0.5mL anticoagulant heparin；In three set pipes, the dosage of 1640 cell culture mediums containing 10% calf serum is
0.5mL；
Negative control pipe, positive control pipe and measurement pipe are put into 37 DEG C, cultivated 24 hours in 5%CO2 incubator；
2) polychrome immunophenotypic marker and Flow cytometry (streaming gating scheme such as Fig. 1): from 3 pipe whole bloods of culture
In respectively take 50uL to be added separately in three streaming pipes, then, in every streaming pipe be added CD3-APC, CD4-PC5, CD25-
Each 5uL of PerCP, CD134-FITC, room temperature are protected from light 15min；Add NH4Cl hemolysin 400ul, slight oscillatory mix；Adjust streaming
The voltage of forward direction and side scattered light takes CD4 according to CD4-CD3 gating in cell instrument+CD3+Cell analysis CD25+CD134+Group
Cell percentage, measurement result are the CD25 for measuring pipe+CD134+Group's cell percentage-negative control pipe CD25+CD134+
Group's cell percentage.
The periphery whole blood of 113 tuberculosis groups, 95 non-tuberculosis illness groups and 23 Healthy People groups is used of the invention
Method detection: statisticalling analyze through ROC curve, and the area under ROC curve is 86.8%, cutoff value 1.250, and sensitivity is
0.824, specificity is 0.836, positive predictive value 0.716, negative predictive value 0.868.
The patient for collecting 113 doubtful tuberculosis carries out Clinical double-blind experiment, wherein final clinical definite is trouble lungy
Person has 45, remaining 68 patient is non-tuberculosis people.It is verified with method of the invention: sensitivity 0.800, specifically
Property is 0.838, positive prediction 0.766, negative predictive value 0.864, diagnostic accordance rate 0.823.
The diagnostic sensitivity and specificity that method of the invention infects mycobacterium tuberculosis are higher.45 tuberculosis patients
Detected with 68 non-tuberculosis disease patients with this method: sensitivity 91.1%, specificity are 88.2%；It is detected with TB.IGRA, spirit
Sensitivity is 87.5%, and specificity is 82.3%.Method of the invention has more highly sensitive and special compared to the method for TB.IGRA
The opposite sex increases the accuracy in clinical diagnosis.
Note: detection sensitivity indicates are as follows: (tuberculosis patient detects number positive/tuberculosis patient sum) × 100%；Detection
Specificity indicates are as follows: (1- healthy volunteer detects number positive/healthy volunteer's sum) × 100%.
The above-mentioned generality to invention involved in the application is described and be should not be construed as to the description of its specific embodiment
It is the limitation constituted to the inventive technique scheme.Those skilled in the art according to disclosure herein, can without prejudice to
Under the premise of related invention constituent element, the public technology feature in above-mentioned general description or/and embodiment is carried out
Increase, reduce or combine, forms the other technical solutions belonged within the application protection scope.
Claims (3)
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111504886A (en) * | 2020-05-06 | 2020-08-07 | 西安交通大学 | Application of a group of molecules in preparation of auxiliary diagnosis reagent or kit for new coronary pneumonia |
| CN111504886B (en) * | 2020-05-06 | 2021-09-03 | 西安交通大学 | Application of a group of molecules in preparation of auxiliary diagnosis reagent or kit for new coronary pneumonia |
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