CN107478563A - A kind of method based on the double cell factors of Flow cytometry tuberculosis specificity - Google Patents
A kind of method based on the double cell factors of Flow cytometry tuberculosis specificity Download PDFInfo
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Abstract
The present invention relates to medical science, discloses a kind of method based on the double cell factors of Flow cytometry tuberculosis specificity, including：1）It is prepared by PMNC；2）Tuberculosis specific lymphocyte is induced to produce the foundation of IFN γ and TNF α cell culture system；3）Flow cytometry CD4+T lymphocyte intracellular IFN γ and TNF α cell factors.The present invention establishes multi-color marking art using polychrome flow cytometry as detection platform, while detects CD4+T lymphocytes release TNF α and IFN γ technology（TB.FACS）, with the respective advantage of complementary double cytokine release diagnosis of tuberculosis, at the same play flow cytometry can quantify, specificity analysis cell population of interest, it is quick and can single Samples detection the characteristics of.
Description
Technical field
The present invention relates to medical science, more particularly to one kind is based on the double cells of Flow cytometry tuberculosis specificity
The method of the factor.
Background technology
Tuberculosis is a kind of and machine caused by mycobacterium tuberculosis (mycobacterium tuberculosis, MTB)
Closely related chronic infectious disease is immunized in body cell, and serious threat human health.In recent years, it is continuous with MTB persisters
Increase and send out, and BCG vaccine prevention declines, tuberculosis is in rebound significantly trend in the whole world, and popularity is increasingly serious.According to
The World Health Organization estimates that there are about 2,000,000,000 mycobacterium tuberculosis in the whole world（MT）Infected patient, there are about 860 Wan Xinfa tuberculosis every year
Example, 1,600,000 people die from tuberculosis.Therefore, the especially latent early diagnosis of tubercular promptly and accurately of tubercular is tuberculosis
The starting point of normalization treatment, efficient tuberculosis early diagnosis means just seem most important.
And laboratory diagnostic technique lungy is always the bottleneck of tuberculosis prevention and control.Existing diagnostic means include MTB points
The methods of from culture, smear for microscopic examination and Molecular Detection.But the above method has respective limitation, as MTB is separately cultured, time-consuming（One
As need 6 ~ 8 weeks）, smear for microscopic examination susceptibility is low（Only 20%~30% positive rate）, often delay treatment.And molecule diagnoses
Complex operation and due to increasing antibody-resistant bacterium, raises the false negative rate of this method.The nineties in last century scientists
By comparative genomics be found that Mycobacterium tuberculosis possess and BCG vaccine, other mycobacteriums without gene region, and
Two albumen Early insulin secretion antigens -6 positioned at this region（ESAT-6）Albumen -10 is filtered with culture medium（CFP-10）With stronger
Th epitopes.Herein on basis, the progress that foreign scholar combines T cell immunoassay technology establishes a kind of new tuberculosis
Specific T cell immunity external detection method --- based on elispot assay（ELISpot）Tuberculosis infection T lymphs it is thin
Born of the same parents' spot test（T-SPOT.TB）With the T lymphocyte γ-IFN extracorporeal releasing tests based on Enzyme-linked Immunosorbent Assay technology
（IGRA）.T-SPOT.TB and IGRA principle is basically identical, is the cell immune response occurred after body is infected MTB.
It is former by the use of specific tuberculosis antigen ESAT-6 and CFP-10 as stimulating, stimulated by the antigen presenting cell in whole blood special
Property Th cells produce IFN-γ, after 37 DEG C of in vitro culture 16-24 hours detection produce the lymphocyte quantity of IFN-γ（T-
SPOT.TB）Or IFN-γ content in nutrient solution（IGRA.TB）, judge to whether there is tuberculosis specific T-cells and its work in whole blood
Power.Due to, as original is stimulated, not influenceed using the peculiar antigen of tulase by BCG vaccine and non-tuberculous mycobacteria, to tuberculosis sense
The detection sensitivity and specificity of dye are higher.
Value of the bis- kinds of methods of T-SPOT.TB and IGRA in diagnosis and treatment tuberculosis is without significant difference, but in technological layer,
The defects of T-SPOT.TB and IGRA.TB common：1. it is the not CD4+ T lymphocytes using total lymphocyte as research object,
And CD4+T lymphocytes are the anti-MTB of mankind principal immune regulation cells, the specificity of its testing result and sensitivity can be by
Influence.2. the cell factor for being T-SPOT.TB and IGRA.TB testing inspections is single IFN-γ, and CD4+T lymphocytes
The cell factors such as TNF secretion-α, IL-2 are gone back under mycobacterium tuberculosis specific antigen protein ESAT-6 and CFP-10 stimulation,
T lymphocyte reactions specific to tuberculosis can not be delicately detected by mark of single IFN-γ.3. it is to T-
It is accurate to the PBMC count requirements of separation in detection process for SPOT.TB, otherwise influence result accuracy；When as a result judging,
Subjectivity with the naked eye be present, otherwise need to use instrument readings, and have overlapping phenomenon during blurring；And for IGRA.TB experiments, though
Without accurate counting PBMC numbers, but the IFN-γ for cultivating upper liquid is detected with ELISA, detection every time is both needed to do standard curve, no
Single specimen examination is applicable, and is calculated more complicated.4. it is from clinical value aspect, although T-SPOT.TB and IGRA suit the medicine to the illness
The diagnosis for the Tuberculosis that shape is not true to type, sick body is negative has larger clinical meaning, but the IFN-γ secreted can not still reflect
It is not active tuberculosis and latent tuberculosis.
The content of the invention
In order to solve the above-mentioned technical problem, it is double thin based on Flow cytometry tuberculosis specificity the invention provides one kind
The method of intracellular cytokine.The present invention establishes multi-color marking art using polychrome flow cytometry as detection platform, while detects CD4+T leaching
The technology of bar cell release TNF-α and IFN-γ（TB.FACS）, with the respective excellent of complementary double cytokine release diagnosis of tuberculosis
Gesture, at the same play flow cytometry can quantify, specificity analysis cell population of interest, it is quick and can single Samples detection the characteristics of.
The present invention concrete technical scheme be：A kind of side based on the double cell factors of Flow cytometry tuberculosis specificity
Method, comprise the following steps：
1）It is prepared by PMNC：Selected object peripheral blood 4-6mL is gathered with vacuum anticoagulant heparin pipe, with lymphocyte
Separating liquid presses 1:1.8-2.2 ratio is added in centrifuge tube, and wherein lymphocyte separation medium first adds centrifugation bottom of the tube, and blood adds
On lymphocyte separation medium upper strata, centrifugal treating；Peripheral blood mononuclear cells layer is drawn to new centrifuge tube, adds RPMI1640 to cultivate
Base 9-11mL wash centrifugal treating, abandoning supernatant, again plus RPMI1640 culture mediums 9-11mL piping and druming mix, wash again from
Heart processing, abandoning supernatant, adds the RPMI1640 culture mediums containing hyclone, and mononuclearcell number is adjusted to 1.8-2.2
×106/mL。
2）Tuberculosis specific lymphocyte is induced to produce the foundation of the cell culture system of IFN-γ and TNF-α：It will be resuspended
It is divided into 3 pipes in the PMNC of the culture medium containing hyclone, often pipe 0.4-0.6mL.
Wherein, the first pipe negative control pipe：Add CD28 and CD49d each 0.8-1.2ug/mL, coban 9-11ug/
mL；Second pipe positive control pipe：Add PMA9-11ug/mL, each 0.8-1.2ug/ of ION 0.8-1.2ug/mL, CD28 and CD49d
ML, coban 9-11ug/mL；3rd pipe measure pipe：Add ESAT-6 and CFP-10 each 4.5-5.5ug/mL, CD28 and
CD49d each 0.8-1.2ug/mL, coban 9-11ug/mL.
35-38 DEG C is put into, 4-6%CO216~18h is cultivated in incubator.
3）Flow cytometry CD4+T lymphocyte intracellular IFN-γs and TNF-α cell factor：By the cell after culture
Centrifugal treating, abandoning supernatant, add CD3-PerCP 4.5-5.5uL, CD8-APC 2-3uL, lucifuge 12-18min.Add rupture of membranes agent
I liquid 18-22uL, lucifuge 12-18min；Centrifugal treating, abandoning supernatant, add the liquid 18-22uL of rupture of membranes agent II, jog 4-6min, add
IFN-γ-FITC and TNF-α-PE each 4.5-5.5uL, lucifuge 25-35min；Adjust forward direction and lateral scattering in flow cytometer
The voltage of light, irises out lymphocyte populations, and SS and CD3 scatter diagram is built as door, irises out CD3+Cell mass, using this group as
Door, SS and CD8 scatter diagram is built, irises out CD8- cell masses, the scatter diagram of IFN-γ and TNF-α is built as door, is drawn
The percentage of two cytokine-positive groups, determining the value of pipe, to subtract the value of negative control pipe be testing result.
Preferably, step 1）In, the parameter of centrifugal treating is 2200rpm first, centrifuges 17min；At second of centrifugation
The parameter of reason is 1600 rpm, 7min；The parameter of third time centrifugal treating is 1300 rpm, 7min.
Preferably, step 1）In, hyclone content is 9- in the RPMI1640 culture mediums containing hyclone
11wt%。
Preferably, step 3）In, the parameter of first time centrifugal treating is 2500rpm, 5min；Second centrifugal treating
Parameter is 2000rpm, 5min.
It is compared with the prior art, the beneficial effects of the invention are as follows：The present invention is built using polychrome flow cytometry as detection platform
Vertical multi-color marking art, while detect the technology of CD4+T lymphocytes release TNF-α and IFN-γ（TB.FACS）, with complementary double thin
Intracellular cytokine discharge diagnosis of tuberculosis respective advantage, while play flow cytometry can quantify, specificity analysis cell population of interest,
It is quick and can single Samples detection the characteristics of.
Brief description of the drawings
Fig. 1 is the gating scheme schematic diagram of TB.FACS methods of the present invention.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment
1. establish TB.FACS technologies
Step 1：It is prepared by PMNC：Selected object peripheral blood 5mL is gathered with vacuum anticoagulant heparin pipe, it is thin with lymph
Born of the same parents' separating liquid presses 1：2 ratio is added in centrifuge tube, and lymphocyte separation medium first adds centrifugation bottom of the tube, and blood is added in thereon
Layer.2200rpm is centrifuged, centrifuges 17min.Peripheral blood mononuclear cells layer is drawn to new centrifuge tube, adds RPMI1640 culture mediums
10mL washing centrifugations, 1600 rpm, 7min.Abandoning supernatant, again plus RPMI1640 culture mediums 10mL piping and druming mixes, and washes again
Wash centrifugation, 1300 rpm, 7min.Abandoning supernatant, add the RPMI1640 culture mediums containing 10% hyclone, and single core is thin
Born of the same parents' number is adjusted to 2 × 106/mL。
Step 2：Tuberculosis specific lymphocyte is induced to produce the foundation of the cell culture system of IFN-γ and TNF-α：Will
It is resuspended in the PMNC containing 10% hyclone culture medium and is divided into 3 pipes, often pipe 0.5mL：First pipe negative control
Pipe：Add CD28 and CD49d each 1ug/mL, coban 10ug/mL.Second pipe positive control pipe：Add PMA10ug/mL,
ION1ug/mL, CD28 and CD49d each 1ug/mL, coban 10ug/mL.3rd pipe measure pipe：Add ESAT-6 and CFP-10
Each 5ug/mL, CD28 and CD49d each 1ug/mL, coban 10ug/mL.37 DEG C are put into, 5%CO2Culture 17 is small in incubator
When.
Step 3：Flow cytometry CD4+T lymphocyte intracellular IFN-γs and TNF-α cell factor：After culture
Cell 2500rpm is centrifuged, 5min, abandoning supernatant.Add CD3-PerCP 5uL, CD8-APC 2.5uL, lucifuge 15min.Add brokenly
Film I liquid 20uL, lucifuge 15min.3 centrifugation 2000rpm, 5min, abandoning supernatant, add rupture of membranes agent II liquid 20uL, jog 5min,
Add IFN-γ-FITC and TNF-α-PE each 5uL, lucifuge 30min.Adjust forward direction in flow cytometer（FS）With it is lateral（SS）Scattering
The voltage of light, irises out lymphocyte populations, and SS and CD3 scatter diagram is built as door, irises out CD3+Cell mass, using this group as
Door, SS and CD8 scatter diagram is built, irises out CD8- cell masses, the scatter diagram of IFN-γ and TNF-α is built as door, is drawn
The percentage of two cytokine-positive groups.（The gating scheme of detailed TB.FACS methods as shown in Figure 1）The value of measure pipe subtracts feminine gender
The value of control tube is testing result.
2. make receiver operating curves（ROC curve）：Compare TB.FACS and T-SPOT.TB and IGRA diagnosis of tuberculosis
Performance, including respective specificity, sensitivity, positive predictive value, negative predictive value and diagnosis efficiency.
3. producing IFN-γ and TNF-α cell factor feature according to tuberculosis specific C D4+T lymphocytes intracellular, establish thin
Intracellular cytokine combines.
Conclusion
ROC curve is analyzed, display：
CD3+IFN-γ+Area under % ROC curve（AUC）For 90.6%, cutoff value 0.15%, sensitivity 0.828 is special
The opposite sex is 0.864, positive predictive value 0.938, negative predictive value 0.667.
CD3+TNF-α+% AUC is 77.3%, cutoff value 0.20%, sensitivity 0.813, specificity 0.667, positive pre-
Measured value 0.858, negative predictive value 0.597.
CD4+TNF-α+% AUC is 86.8%, cutoff value 0.35%, sensitivity 0.793, specificity 0.852, positive pre-
Measured value 0.930, negative predictive value 0.622.
CD4+ IFN-γ+% AUC is 89.8%, cutoff value 0.15%, sensitivity 0.754, and specificity 0.914 is positive
Predicted value 0.956, negative predictive value 0.601.
CD4+TNF-α+% AUC is 86.8%, cutoff value 0.35%, sensitivity 0.793, specificity 0.852, positive pre-
Measured value 0.930, negative predictive value 0.622.
CD3+(IFN-γ/TNF-α)+% AUC is 87.6%, cutoff value 0.35%, sensitivity 0.900, specificity
0.741, positive predictive value 0.896, negative predictive value 0.750.
CD4+ (IFN-γ/TNF-α)+% AUC is 89.3%, cutoff value 0.35%, sensitivity 0.847, specificity
0.852, positive predictive value 0.934, negative predictive value 0.690.
Built-up pattern diagnosis efficiency：
With IFN-γ+CD4+% >=0.15% or/and TNF-α+CD4+% >=0.35% is cutoff values, and sensitivity 0.867 is specific
For 0.800, positive predictive value 0.703, negative predictive value 0.917.
With (IFN-γ/TNF-α)+CD4+% >=0.35% or/and IFN-γ+CD3+% >=0.15% is cutoff values, sensitivity
For 0.867, specificity is 0.800, positive predictive value 0.703, negative predictive value 0.917.
Such as with IFN-γ+CD4+%≥0.15%；TNF-α+CD4+%≥0.35%；(IFN-γ/TNF-α)+CD4+%≥
0.35%；IFN-γ+CD3+% >=0.15% is cutoff values, has one or more to be more than cutoff values in four indexs, then
Diagnostic sensitivity is 0.867, and specificity is 0.764, positive predictive value 0.667, negative predictive value 0.713.
With CD3+(IFN-γ/TNF-α)+% >=0.35% is cutoff values, and sensitivity 0.833, specificity is 0.764,
Positive predictive value 0.658, negative predictive value 0.894.
28 tuberculosis patients carry out TB.FACS simultaneously and detect and be analyzed with TB.IGRA, TB.FACS with
IFN-γ+CD4+ % >=0.15% or/and TNF-α+CD4+% >=0.35% are that positive criterion is that then TB.FACS methods is quick
Perceptual rate 89.3%；And the sensitiveness 78.6% of IGRA methods.
Row TB.FACS and TSPOT.TB are detected and are analyzed 36 tuberculosis patients simultaneously,
TB.FACSTB.FACS using IFN-γ+CD4+ % >=0.15% or/and TNF-α+CD4+% >=0.35% be positive criterion as,
The then sensitiveness 91.7% of TB.FACS methods；And TSPOT.TB methods sensitiveness is 80.6%.
To sum up, TB.FACS has the following advantages that：1. directly using CD4+ T lymphocytes as detection object, while analyze knot
The IFN-γ and TNF-α of core T lymphocyte specific release, significantly improve sensitivity.2. detection technique is simpler, influence
Factor is reduced, and giving full play to flow cytometry can quantify, be specificity analysis cell population of interest, quick and can single Samples detection
Advantage.
Raw materials used in the present invention, equipment, it is the conventional raw material, equipment of this area unless otherwise noted；In the present invention
Method therefor, it is the conventional method of this area unless otherwise noted.
It is described above, only it is presently preferred embodiments of the present invention, not the present invention is imposed any restrictions, it is every according to the present invention
Any simple modification, change and the equivalent transformation that technical spirit is made to above example, still fall within the technology of the present invention side
The protection domain of case.
Claims (4)
- A kind of 1. method based on the double cell factors of Flow cytometry tuberculosis specificity, it is characterised in that including following step Suddenly：1）It is prepared by PMNC：Selected object peripheral blood 4-6mL is gathered with vacuum anticoagulant heparin pipe, with lymphocyte Separating liquid presses 1:1.8-2.2 ratio is added in centrifuge tube, and wherein lymphocyte separation medium first adds centrifugation bottom of the tube, and blood adds On lymphocyte separation medium upper strata, centrifugal treating；Peripheral blood mononuclear cells layer is drawn to new centrifuge tube, adds RPMI1640 to cultivate Base 9-11mL wash centrifugal treating, abandoning supernatant, again plus RPMI1640 culture mediums 9-11mL piping and druming mix, wash again from Heart processing, abandoning supernatant, adds the RPMI1640 culture mediums containing hyclone, and mononuclearcell number is adjusted to 1.8-2.2 ×106/mL；2）Tuberculosis specific lymphocyte is induced to produce the foundation of the cell culture system of IFN-γ and TNF-α：It will be resuspended in and contain The PMNC of hyclone culture medium is divided into 3 pipes, often pipe 0.4-0.6mL；Wherein, the first pipe negative control pipe：Add CD28 and CD49d each 0.8-1.2ug/mL, coban 9-11ug/mL；The Two pipe positive control pipes：PMA9-11ug/mL, each 0.8-1.2ug/mL of ION 0.8-1.2ug/mL, CD28 and CD49d are added, not Can rhzomorph 9-11ug/mL；3rd pipe measure pipe：It is each to add ESAT-6 and CFP-10 each 4.5-5.5ug/mL, CD28 and CD49d 0.8-1.2ug/mL, coban 9-11ug/mL；36-38 DEG C is put into, 4-6%CO216~18h is cultivated in incubator；3）Flow cytometry CD4+T lymphocyte intracellular IFN-γs and TNF-α cell factor：Cell after culture is centrifuged Processing, abandoning supernatant, adds CD3-PerCP 4.5-5.5uL, CD8-APC 2-3uL, lucifuge 12-18min；Add the liquid of rupture of membranes agent I 18-22uL, lucifuge 12-18min；Centrifugal treating, abandoning supernatant, add the liquid 18-22uL of rupture of membranes agent II, jog 4-6min, add IFN-γ-FITC and TNF-α-PE each 4.5-5.5uL, lucifuge 25-35min；Adjust forward direction and lateral scattering in flow cytometer The voltage of light, irises out lymphocyte populations, and SS and CD3 scatter diagram is built as door, irises out CD3+Cell mass, using this group as Door, SS and CD8 scatter diagram is built, irises out CD8- cell masses, the scatter diagram of IFN-γ and TNF-α is built as door, is drawn The percentage of two cytokine-positive groups, determining the value of pipe, to subtract the value of negative control pipe be testing result.
- 2. a kind of method based on the double cell factors of Flow cytometry tuberculosis specificity as claimed in claim 1, it is special Sign is, step 1）In, the parameter of centrifugal treating is 2200rpm first, centrifuges 17min；The parameter of second of centrifugal treating is 1600 rpm, 7min；The parameter of third time centrifugal treating is 1300 rpm, 7min.
- 3. a kind of method based on the double cell factors of Flow cytometry tuberculosis specificity as claimed in claim 1, it is special Sign is, step 1）In, hyclone content is 9-11wt% in the RPMI1640 culture mediums containing hyclone.
- 4. a kind of method based on the double cell factors of Flow cytometry tuberculosis specificity as claimed in claim 1, it is special Sign is, step 3）In, the parameter of first time centrifugal treating is 2500rpm, 5min；The parameter of second of centrifugal treating is 2000rpm, 5min.
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