amina kariminia ph.d. oncology lab, child & family research institute, department of pediatrics,...
TRANSCRIPT
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Amina Kariminia Ph.D.
Oncology Lab, Child & Family Research Institute,Department of Pediatrics, Faculty of Medicine
University of British Columbia
ICIA 2014 Milad Tower, Tehran
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Outlines• A glance to multi-color flow cytometry
• Tregs in whole blood sample
• Kinetic of NFkB phosphorylation in pre B-cell leukemia
• Monitoring immuno-suprressor drugs
• Multi-color flow cytometry for normal human B-cell sub-populations
• MIFlowCyt
• Introducing Cy-TOF: mass-cytometry
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Types of controlsTypes of controls
• Instrument set up control– PMT voltage settings– Compensation
• Gating controls– Isotype controls– FMO controls
• Biological controls– Unstimulated samples– Healthy donors
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Fluorescence Minus One is not always an ideal controlFluorescence Minus One is not always an ideal control
FMO FMO quadrant copy/pasted CD4- quadrant setting
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CD3
Gal-Ser tetramer
No FRET with proper panel of conjugated Abs
No FRET with proper panel of conjugated Abs
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Flow cytometry of rare populations Flow cytometry of rare populations
Minimal Residual Disease
GFP
PBL stem cells
BM stem cells
Tetramers: Ag specific T cells
Minimal Residual Disease
GFP
PBL stem cells
BM stem cells
Tetramers: Ag specific T cells
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Rare population & Flow cytometryPractical Considerations
Rare population & Flow cytometryPractical Considerations
Number of events should be increased for statistical accuracy 200,000-1,000,000
Proper controls
Proper instrument setting
Number of events should be increased for statistical accuracy 200,000-1,000,000
Proper controls
Proper instrument setting
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Intracellular stainingIntracellular staining
Cytokine
&
Phospho protein
Cytokine
&
Phospho protein
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Intracellular stainingIntracellular staining• Cytokines
Type of stimulator Time point Type of protein transport inhibitor Level of production Background level Type of Fix/perm Color selection
• Cytokines
Type of stimulator Time point Type of protein transport inhibitor Level of production Background level Type of Fix/perm Color selection
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Intracellular stainingIntracellular staining
• TH1/2/17
Frozen PBMCs from healthy donors PMA/I stimulation for 6 hr , CO2 incubator In presence of monensin 6 color staining BD fix/perm 500,000 cells acquired
• TH1/2/17
Frozen PBMCs from healthy donors PMA/I stimulation for 6 hr , CO2 incubator In presence of monensin 6 color staining BD fix/perm 500,000 cells acquired
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CD3
CD4
IFN-g
IL-17
IFN-g
IL-4
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Phospho-protein detectionPhospho-protein detection
• Phospho-protein
Type of stimulator Time point Level of production Background level Type of Fix/perm Color selection
• Phospho-protein
Type of stimulator Time point Level of production Background level Type of Fix/perm Color selection
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B-cell Acute lymphoblastic leukemia cell linesB-cell Acute lymphoblastic leukemia cell lines
• Phospho-protein
TLR2 ligand Pam2CSK
Pre B-cell leukemia cell lines
Phosphorylation of NFkB & degradation of IkB
• Phospho-protein
TLR2 ligand Pam2CSK
Pre B-cell leukemia cell lines
Phosphorylation of NFkB & degradation of IkB
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Time 0
Time 20Time 10
Time 5
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380
0
5
10
15
20
25
30
0 0.01 0.1 1 10
TLR-2 agonist ug/ml
% of pNFkB+IKBa-
Pam2
Pam3
OP-1
0
5
10
15
20
25
30
35
40
45
0 0.01 0.1 1 10
TLR-2 agonist
% of pNFkB+IKBa-
Pam2
Pam3
TLR1
TLR-2
TLR-6
TLR1
TLR-2
TLR-6
TLR1
TLR-2
TLR-6
Dose response curve and phospho-protein
kinetic analysis
Dose response curve and phospho-protein
kinetic analysis
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B-cell acute lymphoblastic leukemia: IL-7R expression
B-cell acute lymphoblastic leukemia: IL-7R expression
CD19+CD10+
0 102 103 104 105
<PE-A>
0
102
103
104
105
Sample %
Specimen_001_379-CD127.fcs 10.7
Specimen_001_1279-CD127.fcs 3.14
IL-7Ra
Ig Light chainHealthy control
B-ALL relapse
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YB-1YB-1
Interruption of host immune system recognitionBy HLA-DO up-regulation/decreasing
Ag-presentation Prevention of apoptosis by Survivin
Blocking the effect of cytotoxic drugsThrough IL-7 signaling and multi-drug resistance -1 (MDR-1) up-regulation
YB-1 in acute lymphoblastic leukemia YB-1 in acute lymphoblastic leukemia
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Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1
Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1
0 102 103 104 105
<PE-Cy7-A>
0
102
103
104
105 0.0141 0.243
94.65.09
0 102 103 104 105<PE-Cy7-A>
0
102
103
104
105 0 0.0237
94.85.17
CD19
pYB-1 No IL-7 IL-7 stimulated
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Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647
Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647
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Immunophenotyping on preserved whole blood sample usingCyto-Chex BCT tube
Immunophenotyping on preserved whole blood sample usingCyto-Chex BCT tube
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0 102 103 104 105
<Qdot 800-A>: CD27
0
102
103
104
105
0 102 103 104 105
<APC-A>: CD10
0
102
103
104
105
51.5
11.8
6.98
0 102 103 104 105
<V500-A>: IgD
0
102
103
104
105
0 102 103 104 105
<Qdot 800-A>: CD27
0
102
103
104
105
B-cell ImmunophenotypingB-cell Immunophenotyping
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0 102 103 104 105
PE-A
0
102
103
104
105
29.9
0 102 103 104 105
PE-A
0
102
103
104
105
0 102 103 104 105
APC-A
0
5
10
15
20
25
CD27-PE
IgD-FITC
CD19+ (Pacific Blue) gated cells
CD10-APC
IgD-FITC
CD27-PE
Bone marrow Peripheral Blood
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Whole blood Treg phenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450
Whole blood Treg phenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450
0 102 103 104 105
<V450-A>: FOXP3
0
102
103
104
105
0 102 103 104 105
<V450-A>: FOXP3
0
102
103
104
105
Cyto-Chex tube EDTA tubeCD3+CD4+ gated T cells
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0 102 103 104 105
<Alexa 647-A>
0
102
103
104
105
4.26
0 102 103 104 105
<Alexa 647-A>
0
102
103
104
105
1.82
FOXP3-AF647
CD25-PE
Treg phenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647
Treg phenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647
HD IPEX
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0 102 103 104 105
<Pacific Blue-A>
0
102
103
104
105
0 102 103 104 105
<Pacific Blue-A>
0
102
103
104
105
CD25-PE
CD127-BV421
HD IPEX
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Immuno-supressor Monitoringdetection of surface CD69 up-regulation
upon CD2 activation (IPEX patient)
Immuno-supressor Monitoringdetection of surface CD69 up-regulation
upon CD2 activation (IPEX patient)
CD3+CD4+ gated T cells
Un-stimulated
Corticosteroid Sirolimus
0 102 103 104 105
V450-A: FOXP3
0
102
103
104
105 7.04 1.07
17.374.5
0 102 103 104 105
V450-A: FOXP3
0
102
103
104
105 31.1 5.62
13.849.4
0 102 103 104 105
V450-A: FOXP3
0
102
103
104
105 11.1 2.31
10.576.1
Stimulated
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The Minimum Information about Flow cytometry Experiment
(MIFlowCyt)
The Minimum Information about Flow cytometry Experiment
(MIFlowCyt)
Ref: Cytometry part A
73A:926-030
2008
Ref: Cytometry part A
73A:926-030
2008
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MIFlowCytMIFlowCyt
Simplify cross-platform comparison and integration,
Simplify cross-platform comparison and integration,
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MIFlowCytMIFlowCyt• Experimental overview:
– Purpose/goal hypothesis
– Experiment variables
– Conclusions
– Quality control
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MIFlowCytMIFlowCyt
• Flow sample (specimen):
– Material
– Source/organism/location
– Treatment
– Reagent/analyte/detector/reporter
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MIFlowCytData presentation
MIFlowCytData presentation
• Experimental and Sample Information:
– Experimental design
– Sample preparation reagents
– Fluorescent reagents• Vendor• Catalog number• Clone
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MIFlowCytData presentation
MIFlowCytData presentation
• Data acquisition:
– Instrument configuration
– Instrument identification
– Fluidics configuration
– Optical configuration
– Electronic configuration
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MIFlowCytData presentation
MIFlowCytData presentation
• Data presentation:
– Flow plots• Label both axis• Fluorophore and antibody• Scale obvious
– Total events
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MIFlowCytData presentation
MIFlowCytData presentation
• Data presentation:
– Flow plots• Label both axis• Fluorophore and antibody• Scale obvious
– Total events
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CyTOF: mass -cytometry
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Bendall et al, Science, 2011
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Child & Family Research InstituteChild & Family Research Institute