Transcript Critical points in ICS Assays

Key Variables in ICS Assays
Holden T. Maecker
The original BD ICS assay protocol
1 ml whole blood +
Ag + CD28 + CD49d
+ Brefeldin
A
2h
+ FACSLyse,
10 min
+ EDTA/PBS
4h
15 min
Wash
Wash, fix,
analyze
CD69 PE
Centrifuge, aliquot into
staining tubes or freeze
anti-TNF FITC
FACS
Perm, 10 min
mAb staining,
30-60 min
3- or 4-color
Flow cytometry
96-well plate ICS protocol with
lyophilized reagents
PBMC or whole blood
Plate with lyophilized Ag+BFA
6h @ 37oC, EDTA, FACS Lyse, FACS Perm 2
Plate with lyophilized Ab
to flow cytometer
What are the key variables?
•
•
•
Stimulation
• PBMC vs. whole blood
• Resting PBMC before activation
• Activation/staining in plates vs. tubes
• Costimulatory Abs (CD28+CD49d)
• Stimulation time
• Brefeldin A and/or monensin
Processing and staining
• Use of EDTA or not
• Method of fixation/permeabilization
• Freezing of cells post-fixation
• Surface vs. intracellular staining for CD3, CD4, CD8, etc.
• Fluorochromes and fixation (APC-Cy7, AmCyan)
• Staining time, temperature, and agitation
• Number of washes (post-fix and post-stain)
Acquisition and analysis
• Number of events to collect
• Gating
• Interpretation of results, precision, comparisons with other assays
Effect of overnight rest on cryo PBMC
% IFNg+
MFI of IFNg+
3
750
2
500
1
250
0
100
10000
1000000
Reciprocal Dilution
0
100
10000
Reciprocal Dilution
No rest, 6 h peptide stimulation
Overnight rest, 6 h peptide stimulation
1000000
Correlation of tube vs. plate ICS
A. 96-well whole blood
Peptide
mix
% IFNg+ cells
CD4
% IFNg+ cells
SEB
CD4
CD8
CD8
1.25
2.5
4
4
1.00
2.0
3
3
0.75
1.5
2
0.50
1.0
r2= 0.95
p<0.0001
slope 0.94 0.5
2
r2 = 0.96
p < 0.0001 1
slope = 1.2
0.25
plate
B. 96-well PBMC
r2=0.87
p<0.0001
slope 0.88
0.00
0.0
0.000.250.500.751.001.25
0.0 0.5 1.0 1.5 2.0 2.5
10.0
7.5
5.0
2.5
0.0
0.0
14
12
10
8
6
r2=0.95
r2= 0.94
4
p<0.0001
p<0.0001
slope 0.93
slope 0.91 2
0
0 2 4 6 8 10 12 14
2.5 5.0 7.5 10.0
% IFNg+ cells
% IFNg+ cells
1
0
0
20
1
2
3
4
0
0
30
r2 = 0.73
p = 0.0002
slope = 1.1
1
2
3
4
20
10
tube
r2 = 0.74 10
p = 0.0002
slope = 0.78
0
0
10
% IFNg+ cells
20
0
0
r2 = 0.79
p < 0.0001
slope = 0.84
10
20
30
% IFNg+ cells
Similar IFNg MFI in tubes vs. plates
IFNg mean fluorescence
A. 96-well whole blood
300
B. 96-well PBMC
C. 24-well whole blood (HIV+)
300
300
tube
plate
200
200
200
100
100
100
0
0
CD4
CD8
0
CD4
CD8
CD4
CD8
Cell recovery in tubes vs. plates
75
50
25
Whole Blood
96-well
Tube
24-well
96-well
0
Tube
% CD3+ cell recovery
100
PBMC
Effect of costimulatory Abs (CD28+49d)
SEB
% of costimulated
response
pp65 peptide mix
125
125
100
100
75
75
50
50
25
25
0
0
froze n
fre sh
frozen
IFNg
IL-2
fresh
Monensin vs. Brefeldin Summary
• Brefeldin A preferred (10 mg/mL):
• TNF, IFNg, IL-2, IL-4, IL-12
• When combining any of the above with
CD107, TGFb, or IL-10:
• 5 mg/mL monensin + 5 mg/mL brefeldin A
What are the key variables?
•
•
•
Stimulation
• PBMC vs. whole blood
• Resting PBMC before activation
• Activation/staining in plates vs. tubes
• Costimulatory Abs (CD28+CD49d)
• Stimulation time
• Brefeldin A and/or monensin
Processing and staining
• Use of EDTA or not
• Method of fixation/permeabilization
• Freezing of cells post-fixation
• Surface vs. intracellular staining for CD3, CD4, CD8, etc.
• Fluorochromes and fixation (APC-Cy7, AmCyan)
• Staining time, temperature, and agitation
• Number of washes (post-fix and post-stain)
Acquisition and analysis
• Number of events to collect
• Gating
• Interpretation of results, precision, comparisons with other assays
Plates EDTA
Plates no EDTA
Tubes EDTA
Tubes no EDTA
% CD3+ CD8+ IFNg+
mean of triplicate samples
EDTA addition in plates and tubes
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
Fix/perm comparison: monocyte IL-12
Fix/perm comparison: T cell TGFb
TGFb PE
TGFb PE
TGFb PE
TGFb PE
BD FACS Lysing Solution
BD FACS Permeabilizing Sol’n 2
BD Cytofix/Cytoperm
BD Perm Wash
TGFb PE
TGFb PE
TGFb PE
TGFb staining and activation media
Freezing activated cells in FACS Lyse
Surface vs. IC staining for CD3
SEB stimulated PBMCs
surface stain
intracellular stain
CD3 Alexa 700
Clone SP34-2 @0.8mg/test
Surface vs. IC staining for CD4 & CD8
surface stain
intracellular stain
SEB stimulated PBMCs
CD8 APC-hilyte 750 @125ng
CD4 AmCyan @ 60ng
CD8 Qdot 605 @ 0.25ul
CD4 AmCyan @ 60ng
All events
CD3+ gate
CD8 APC-Cy7
Side Light Scatter
B
C
A
CD3 Pacific Blue
CD4 AmCyan
CD4+ CD8- gate
IFNg FITC
IFNg FITC
CD4- CD8+ gate
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD4+ IFNg+ IL-2+
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD45RA PE-Cy7
CD45RA PE-Cy7
CD27 APC
CD45RA PE-Cy7
CD27 APC
CD45RA PE-Cy7
CD45RA PE-Cy7
CD27 APC
Side Light Scatter
CD4+ IFNg+
CD8+ IFNg+ IL-2+
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD27 APC
IL-2 PE
Side Light Scatter
CD8+ IFNg+
Side Light Scatter
Side Light Scatter
IL-2 PE
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
Degradation of APC-Cy7 on stained cells
% spillover
APC-Cy7 -> APC
100
75
PBS/BSA
PBS/formaldehyde
50
25
In BDSF
0
no
storage
overnight overnight
room temp
4o C
BDSF and AmCyan don’t mix
AmCyan Signal:Noise
FITC Spillover
27.5
Spillover AmCyan ->
FITC
100
Signal:Noise
25.0
22.5
20.0
17.5
15.0
12.5
10.0
-0.5 0.0 0.5 1.0 1.5 2.0 2.5
75
50
25
0
-0.5 0.0 0.5 1.0 1.5 2.0 2.5
hours
hours
PBS/BSA
1% PFA
BDSF
IC staining time, volume, and mixing
160
30 min
140
60 min
30 min w /mix
IFNg FITC MFI
120
60 min w /mix
100
80
60
40
20
0
50
100
150
200
volume, ul
250
300
350
What are the key variables?
•
•
•
Stimulation
• PBMC vs. whole blood
• Resting PBMC before activation
• Activation/staining in plates vs. tubes
• Costimulatory Abs (CD28+CD49d)
• Stimulation time
• Brefeldin A and/or monensin
Processing and staining
• Use of EDTA or not
• Method of fixation/permeabilization
• Freezing of cells post-fixation
• Surface vs. intracellular staining for CD3, CD4, CD8, etc.
• Fluorochromes and fixation (APC-Cy7, AmCyan)
• Staining time, temperature, and agitation
• Number of washes (post-fix and post-stain)
Acquisition and analysis
• Number of events to collect
• Gating
• Interpretation of results, precision, comparisons with other assays
Number of events to acquire
CALCULATING SAMPLE SIZE FOR RARE EVENT ANALYSIS
Enter the estimated values in the grey fields:
% bkgd=
%pos=
0.1
0.2
For power of 90%, p<0.05,
For power of 99%, p<0.005,
For power of 80%, p<0.05,
p(av)=
delta=
0.0015
0.001
N=
N=
N=
25761
71592
18572
_______________________________________________________________
________# events to collect__________
% bkgd
lowest % positive
90% power, p<0.05
99% power, p<0.005
_______________________________________________________________
0.01
0.02
260,000
720,000
0.01
0.05
32,000
90,000
0.01
0.1
12,000
32,000
0.02
0.05
67,000
190,000
0.02
0.1
16,000
45,000
0.03
0.05
170,000
480,000
0.03
0.1
23,000
63,000
0.04
0.1
33,000
93,000
0.05
0.1
52,000
140,000
0.06
0.1
86,000
240,000
0.07
0.1
160,000
450,000
0.08
0.2
17,000
46,000
0.1
0.2
26,000
72,000
_______________________________________________________________
Importance of gating dim cells
Multi-site lyophilized control cell data
Individual (Manual) Analysis
32%
29%
7%
Central (Automated) Analysis
14%
7%
20
10
0
30
% cytokine+ cells
% cytokine+ cells
30
20
10
0
3%
3%
3%
Gating
Manual analysis
ungated
Side Scatter
Side Scatter
R2
10 0
1000
CD8 PerCP-Cy5.5
gated on R1
10 1
gated on
R1&R2
&R3
R4
10 3
10 4
10 1
10 2
CD3 APC
10 3
10 4
10 0
10 1
0
200
400
600 800
Forward Scatter
10 2
IFNg FITC
10 3
1000
ungated
0.55%
CD69 PE
R3
10 2
CD3 APC
10 4
100
R4
101
102
CD3 APC
gated on
R1&R3
&R5
R5
100
101
102
CD3 APC
103
R6
103
104
0.56%
CD69 PE
400
600 800
Forward Scatter
CD8 PerCP-Cy5.5
200
10 0
R3
R2
R1
R1
0
gated on R1
Side Scatter
ungated
Side Scatter
ungated
Batch analysis with dynamic gates
104
R7
10 0
10 1
10 2
IFNg FITC
10 3
10 4
What are the key variables?
•
•
•
Stimulation
• PBMC vs. whole blood
• Resting PBMC before activation
• Activation/staining in plates vs. tubes
• Costimulatory Abs (CD28+CD49d)
• Stimulation time
• Brefeldin A and/or monensin
Processing and staining
• Use of EDTA or not
• Method of fixation/permeabilization
• Freezing of cells post-fixation
• Surface vs. intracellular staining for CD3, CD4, CD8, etc.
• Fluorochromes and fixation (APC-Cy7, AmCyan)
• Staining time, temperature, and agitation
• Number of washes (post-fix and post-stain)
Acquisition and analysis
• Number of events to collect
• Gating
• Interpretation of results, precision, comparisons with other assays
Holden’s rules for ICS (and life)
• Validate your assay, but don’t validate that the
earth is round
• take advantage of others’ data if it’s sound
• Know the inherent variability in your assay
• don’t talk about significant differences unless you
run at least triplicate samples
• Avoid comparing apples and oranges
• any changes to the assay can produce variability,
so keep protocols constant and run samples in
parallel whenever possible
Acknowledgements
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Laurel Nomura
Smita Ghanekar
Maria Jaimes
Margaret Inokuma
Sonny Bhatia
Joyce Ruitenberg
Maria Suni
Jack Dunne
Skip Maino
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Brinda Emu
Rebecca Hoh
Steve Deeks
Jeff Martin
Mike McCune
Doug Nixon