“ Measuring Antigen Specific Tcells using Surface and
Intracellular Staining
Polychromatic Flow Cytometry ”
4 th Annual CFAR Flow Cytometry Wet-Workshop
13-17 October, 2013
Janet Staats
Flow Cytometry Core Facility
Center for AIDS Research
Duke University Medical Center
E-mail: jotti@duke.edu

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Memory CD4 T Cell Response to Ag
IL-2 IL-4
Rantes
Apoptosis
IFN g
TNF a
APC-T cell interactions
From H. Maecker
Cytokine/Chemokine expression
Proliferation/
Death
Duke University Medical Center

Ag
Whole protein
APC MHC
II
MHC I
CD4
CD4 +
T cell cytokines
Optimal peptide
T, B, or APC
MHC I
From H. Maecker peptide
CD8
CD8 +
CTL cytokines
Duke University Medical Center

Response to CMVpp65 Peptide Mix
CD4 pp65 protein
0.27% peptide mix
0.27%
A2 peptide
0.04%
CMV lysate
7.41%
CD8
0.19% 2.03% 1.14% 0.87%
From H. Maecker

15 a.a.
CMV pp65: pool of 138 peptides
HIV p55: pool of 120 peptides
11 a.a.
Duke University Medical Center

Sampson Clinical Trial:
11-Color Maturation/Function Panel
Basic Subset Markers:
• CD3 (T-cells)
• CD4 (T-Helper Subset)
• CD8 (T-Suppressor Subset)
Exclusion Markers:
• CD14 (Monocytes)
• CD19 (B-cells)
• vAmine (Dead cell marker)
Maturational Markers:
• CD45RO
–
CD45RA
•
CD27
–
CD197 (CCR7)
• CD57
Functional Markers:
• CD107
• IFNg
• TNF a
• IL-2
Duke University Medical Center

Overview of 11-Color Assay
Monday
1. Thaw
Tuesday Wednesday
Thursday
- Friday
2. Stimulate
Brefeldin
Monensin
3. Surface Stain 4. Lyse/Fix
5. Permeabilize 6. IC Stain
7. Acquisition
8. Analysis
Wash Wash Wash
7+g+M+
M+ g+M+
Rest 6 hrs
6 h
CD107 cytokine lymphocyte erythrocyte
Costim
SEB
CMVpp65
Amine
CD45RA
CD197
Wash
CD107
CD3
CD4
CD8
IFN g
IL2
TNF
CD8 + CM Response
Duke University Medical Center

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Instrument Performance:
Gel smeared on flow cell
Green E
Green C
Green A
Green D
Green B
10000
1000
100
10
1
0 200 400 voltage
600 800 1000
12

Instrument Performance:
Low Staining Index

Instrument Performance:
Variance in noise

Instrument Calibration:
Use of target channels

Instrument calibration vs biological variation
Note: instrument fluorescence detectors are set using established target channel values, allowable range <5% variance daily

Instrument calibration vs biological variation
1Mar13: DP06_008 9Nov12: 009 & 010 14Nov12: DP06_013 20Nov12: DP06_014 & 017

Instrument calibration vs
6Nov12: DP06_012 16Feb13: DP06_020 23Jan13: DP06_019
1Mar13: DP06_008 9Nov12: DP06_009 14Nov12: DP06_013 20Nov12: DP06_014
9Nov12: DP06_010 20Nov12: DP06_017
Note: instrument fluorescence detectors are set using established target channel values, allowable range <5% variance daily

Batch Processing Error
CD38 vs HLA-DR Staining on Ctrl 5L
28Feb08
5L CD8+
Lot 05262
10
5
1.01
5.8
10
4
10
5
1.18
04Marb08
5L CD8+
Lot 05262
5.8
10
4
06Mar08
5L CD8+
Lot 05262
10
5
1.11
7.2
10
4
10
5
1.11
11Mar08
5L CD8+
Lot 05262
6.94
10
4
10
3
0
10
5
21.2
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.52
72
10
5
0.058
10
4
10
3
0
98.2
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
1.17
10
5
10
3
10
3
0
10
5
22
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.63
71
10
5
0.032
10
4
0
10
5
21.9
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
69.8
10
5
0.8
0.014
10
4
10
3
0
10
5
20.1
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
71.8
10
5
0.61
0.016
10
4
10
3
0
98.7
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.63
10
5
10
3
0
98.9
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.27
10
5
10
3
0
99
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.39
10
5
Duke University Medical Center

Batch Processing Error
CD38 vs HLA-DR Staining on Ctrl 5L
10
5
1.05
26Feb08
5L CD8+
Lot 05262
5.38
10
4
28Feb08
5L CD8+
Lot 05262
10
5
1.01
5.8
10
4
10
3
0
10
5
29.4
64.1
0
0.16
10
3
10
4
<Green E-A>: CD38 PE
10
5
1.27e-3
10
4
10
3
0
10
5
21.2
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.52
72
10
5
0.058
10
4
10
3
0
99.8
0
0.058
10
3
10
4
<Green E-A>: CD38 PE
10
5
10
3
0
98.2
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
1.17
10
5
10
5
1.18
04Marb08
5L CD8+
Lot 05262
5.8
10
4
06Mar08
5L CD8+
Lot 05262
10
5
1.11
7.2
10
4
10
5
1.11
11Mar08
5L CD8+
Lot 05262
6.94
10
4
10
3
10
3
0
10
5
22
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.63
71
10
5
0.032
10
4
0
10
5
21.9
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
69.8
10
5
0.8
0.014
10
4
10
3
0
10
5
20.1
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
71.8
10
5
0.61
0.016
10
4
10
3
0
98.7
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.63
10
5
10
3
0
98.9
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.27
10
5
10
3
0
99
0 10
2
10
3
10
4
<Green E-A>: CD38 PE
0.39
10
5
Duke University Medical Center

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Titration: objective results

Titration: subjective results

Optimization using Spillover Assessments: Using
Titration Files to Assess Spreading Error
Blue
Laser
Violet
Laser
Red
Laser
Green
Laser
CD3AC (5ug/ml) Spillover assessment:
• After compensation CD3AC showed spilllover into Blue-B detector (FITC channel)
Violet G- CD3 AmCyan
•
•
•
Ottinger, et. al., Poster #28, 23rd Annual Clinical Cytometry Meeting (2008)
Mahnke, et. al. Clin Lab Med. 2007 September; 27(3): 469-v.
Lamoreaux, et. al., Nature Protocols 1, 1507-1516 (2006) on line 9 November 2006
Duke University Medical Center

Spillover Assessments:
CD3 AmCyan (5µg/mL) Spillover into CD27 (0.32µg/mL)
& CD57 FITC (1.8µg/mL)
Unstained
CD27
FITC
CD3
AmCyan
0.13
66.3
4.58
Blue B
Unstained
CD57
FITC
CD3
AmCyan
9.8e-4
20.5
0.047
• Spillover from CD3AC interferes with detection of dim CD27 pos cells
• Spillover from CD3AC does not interfere with detection of CD57
• Spillover is acceptable if it does not interfere with proper classification of events
• mAb concentration may be varied to reduce spillover as long as frequency is unaffected
Blue B
Duke University Medical Center

Tandems Degrade!
Maecker, et. al.
• Ice
• Dark
• Fix
• Controls
• 6 hours
Duke University Medical Center

Ship tandems at 4ºC

Panel Development: FS Test

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Why is Reproducibility Important?
CFSE Standardization Results (13 EXPERT IM Labs):
Very high inter-laboratory variability.
High background in some laboratories.
Responses to Gag and Nef peptide pools were detected in HIV negative (control) donors!
Example Gag stimulation
HIV negative donor
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 1
Laboratory
Example CMVpp65 stimulation
CMV positive donor
40
30
20
10
0
70
60
50
1 2 3 4 5 6 7 8 9 1
Duke University Medical Center

Pe pt ide
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
JH E
0.08
0.04
0.02
1 .3 2
0 .1 9
0.01
0.02
0.03
0.02
0.05
JO
0.07
0.03
0.02
1.03
0.02
0.01
0.03
0 .0 6
0.02
0.07
N M
0.08
0.05
0.01
LAD
0.08
0 .0 8
0.03
0.96
0 .8 5
0.07
0.01
0.01
0.01
0.04
0.02
0.02
0.03
0.02
0 .0 9
0 .0 4
0.06
2 8 / 4 9 d
JY
0 .0 5
0.03
0.00
1.13
0.01
0.00
0.01
0.01
0.01
0.04
Spr e a d
0.03
0.05
0.02
0.47
0.18
0.01
0.02
0.05
0.08
0.03
An a ly z e r
CD 1 0 7 a +
I FN g+
JH E
0.76
1.57
0.12
I L2 +
M I P1 b+ 3 .4 3
TN Fa + 0 .5 9
CD 1 0 7 a + 0.02
I FN g+
0.20
I L2 +
0.23
M I P1 b+
0.03
TN Fa + 0.48
JO
0.74
1.62
0.10
2.99
0.38
0.02
0.22
N M
0.80
1.51
LAD
0 .8 8
0 .0 4
2 .2 1
0.38
2 .3 3
0.10
2.93
0.55
0.02
0 .0 4
0 .1 8
0.20
0.22
0.22
0.03
0.55
0 .1 9
0.48
JY
GAGB
0 .6 3
1.70
0.07
3.33
0.41
Spr e a d
0.25
0.82
0.08
1.22
0.21
0 .0 0
0 .1 6
0.19
0.02
0.55
0.04
0.06
0.06
0.17
0.12
0 .2 5
0.04
0 .6 0
Pe pt ide
An a ly z e r JH E
CD 1 0 7 a + 0.29
I FN g+
0.54
I L2 +
0.05
M I P1 b+ 2 .1 2
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
0 .3 1
0.01
0.06
0.10
0.01
0.17
An a ly z e r JH E
CD 1 0 7 a + 0.21
I FN g+
0.40
I L2 +
0.04
M I P1 b+
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
1 .6 0
0 .3 4
0.02
0.07
0.12
0.02
0.18
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
JH E
0.12
0.15
0.01
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
1 .5 8
0 .1 1
0.02
0.04
0.02
0.02
0.06
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
An a ly z e r
CD 1 0 7 a +
I FN g+
JH E
0.10
0.16
0.02
2 .8 1
0 .1 5
0.00
0.02
0.03
0.13
0.07
JH E
0.15
0.19
I L2 +
M I P1 b+
0 .0 2
2 .3 4
TN Fa + 0 .1 2
CD 1 0 7 a + 0.01
I FN g+
0.02
I L2 +
0.02
M I P1 b+
0.09
TN Fa + 0.05
JO
0.27
0.55
0.03
1.78
0.11
0.01
0.07
0 .1 2
0.01
0 .2 1
JO
0.21
0.37
0.03
1.29
0.14
0.01
0.09
0 .1 4
0.01
0 .2 4
JO
0.12
0.11
0.00
1.05
0.02
0.01
0 .0 7
0 .0 4
0.01
0.07
JO
0.10
0.15
0.01
1.20
0.03
0.00
0.03
0 .0 5
0.05
0 .0 9
JO
0.14
0.17
0.01
1.38
0.02
0.01
0.03
0.03
0.03
0.06
N M
0.30
0.50
0.01
1 .3 6
0.14
0.01
0.04
0.08
0.01
0.19
N M LAD
0.21
0 .2 5
0.38
0.01
0 .6 6
0.04
1 .0 4
0.17
0.01
0.05
0.10
0.01
0.20
N M
0.12
0.00
1.02
0.03
0.02
0.02
N M
0.11
0.17
0.01
1.37
0.06
0.01
N M
0.15
0.22
0.00
1.32
0.04
0.01
0.01
0.01
0.04
0.04
1.31
0.21
0.02
0.07
0.11
0 .0 5
0.17
LAD
0 .1 4
0.14
0 .2 2
0.01
1.12
0.04
0.03
0.04
0.02
0.02
0.01
0 .1 4
0.05
0.05
LAD
0.11
0 .2 2
0.01
1.36
0.06
0.01
0.02
0.02
0.02
0.04
0.05
0 .7 0
0.07
0.08
LAD
0.16
0 .2 8
0.01
1.42
0.05
0.02
0.02
0.02
0 .4 2
0.05
POLB1
LAD JY
0.29
0 .2 3
0 .7 6
0.05
0.53
0.01
1.68
0.16
1.91
0.09
0 .0 2
0.00
0.06
0 .0 3
0.09
0.09
0 .0 7
0.18
0.01
0.19
Spr e a d
0.07
0.26
0.04
0.76
0.22
0.01
0.04
0.04
0.06
0.04
POLB2
JY
0 .1 7
0.37
0.01
1.42
0.13
0 .0 0
0.04
0.10
0.01
0.20
JY
JY
JY
Spr e a d
0.08
0.29
0.03
0.56
0.21
0.01
0.06
0.04
0.05
0.07
EN V A
0 .1 0
0.11
0.00
1.14
0.01
0 .0 0
0.01
0.02
0.00
0.05
Spr e a d
0.04
0.11
0.01
0.56
0.10
0.03
0.05
0.02
0.14
0.02
EN V B
0 .0 8
0.14
0.00
1.40
0.03
0.00
0.02
0.02
0.02
0.06
Spr e a d
0.03
0.08
0.02
1.61
0.12
0.01
0.01
0.03
0.68
0.03
EN V C
0 .1 1
0.15
0.00
1.57
0.01
0.00
0.01
0.01
0.01
0.04
Spr e a d
0.05
0.13
0.01
1.02
0.11
0.02
0.02
0.02
0.41
0.02
Pe pt ide SEB
An a ly z e r JH E JO N M LAD JY
CD 1 0 7 a + 5.43
I FN g+
I L2 +
M I P1 b+
TN Fa +
5.25
5.32
5 .7 6
10.80 10.80
9.89
3 .0 5
2.92
16.20 14.00
6.27
6.06
1 .8 6
1 0 .8 0
5 .2 9
1 4 .3 0
2.20
14.70 15.40
6.52
4 .6 5
11.30
2.66
6.38
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
0.33
6.59
0.26
6.65
0.34
6.33
0.40
6.56
0 .0 0
5.93
13.50 13.40 13.40
1 4 .5 0 1 4 .6 0
0.57
1 6 .4 0
0.57
0.47
24.20 23.00
1 .1 4
1 5 .9 0
0.41
22.60
Spr e a d
1.11
4.41
1.19
5.40
1.23
0.40
0.72
1.20
0.73
8.30
M SD * * - 1 * * + 1
0.07
0.01
0.06
0.05
0.02
0.02
0.01
0.01
0.00
1.06
0.18
0.88
0.10
0.01
0.09
0.17
0.03
0.14
0.01
0.01
0.00
1.63
0.67
0.96
0.06
0.05
0.01
0.00
0.00
0.00
0.02
0.01
0.02
0.03
0.01
0.02
0.09
0.07
0.03
1.24
0.07
0.07 -0.01
0.14
0.01
0.01
0.00
0.02
0.02
0.01
0.01
0.03
0.02
0.01
0.03
0.05
0.03
0.03 -0.01
0.06
0.05
0.01
0.04
0.06
M
0.76
0.09
0.67
1.75
0.33
1.41
0.09
0.03
0.05
2.98
0.48
2.50
0.46
0.10
0.36
0.02
0.01
0.01
0.20
0.03
0.17
0.22
0.02
0.19
0.24
0.06
0.07 -0.01
0.13
0.53
0.05
0.48
M SD * * - 1 * * + 1
0.28
0.03
0.25
0.58
0.10
0.47
0.03
0.02
0.01
1.77
0.28
1.49
0.16
0.09
0.08
0.01
0.01
0.00
0.05
0.01
0.04
0.10
0.01
0.08
0.30
0.68
0.05
2.05
0.25
0.01
0.07
0.11
0.02
0.03 -0.01
0.05
0.19
0.01
0.17
0.20
M
0.21
0.03
0.18
0.44
0.13
0.31
0.03
0.01
0.01
1.33
0.20
1.13
0.20
0.09
0.11
0.01
0.01
0.01
0.06
0.02
0.04
0.11
0.02
0.09
0.02
0.02
0.00
0.20
0.03
0.17
0.24
0.56
0.04
1.54
0.28
0.02
0.09
0.13
0.04
0.22
M SD * * - 1 * * + 1
0.12
0.01
0.11
0.13
0.15
0.05
0.10
0.01
0.00
0.00
1.18
0.23
0.95
0.04
0.04
0.00
0.02
0.01
0.01
0.04
0.02
0.01
0.19
0.01
1.41
0.08
0.03
0.06
0.02
0.01
0.02
0.03
0.04
0.06 -0.02
0.09
0.06
0.01
0.05
0.07
M
0.19
0.29 -0.10
0.48
0.07
0.01
0.06
0.08
M
SD * * - 1 * * + 1
0.85
2.08
0.12
3.46
0.56
0.03
0.22
0.58
SD * * - 1 * * + 1
SD * * - 1 * * + 1
0.11
0.20
0.02
2.29
0.11
0.01
0.03
0.04
SD * * - 1 * * + 1
0.14
0.02
0.12
0.20
0.05
0.15
0.01
0.01
0.00
1.61
0.42
1.19
0.05
0.04
0.01
0.01
0.01
0.00
0.02
0.01
0.01
0.16
0.25
0.01
2.03
0.09
0.02
0.03
0.02
0.01
0.01
0.03
0.12
0.17 -0.05
0.29
0.05
0.01
0.04
0.06
M SD * * - 1 * * + 1
5.28
0.40
4.88
5.69
11.42 1.69
9.73
13.11
2.54
0.50
2.04
3.04
14.22 2.08 12.14 16.30
6.10
0.49
5.62
0.27
0.16
0.11
6.59
0.42
6.41
0.30
6.12
6.71
13.88 0.61 13.27 14.49
0.63
0.29
0.34
0.92
20.42 3.95 16.47 24.37
Pe pt ide
An a ly z e r JH E
CD 1 0 7 a + 0.12
I FN g+
0.10
I L2 +
0.03
M I P1 b+
TN Fa +
1.60
0.14
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
0.08
0.05
0.04
0.06
0.06
Pe pt ide
An a ly z e r JH E
CD 1 0 7 a + 0.93
I FN g+
2.08
I L2 +
0.14
M I P1 b+
4.16
TN Fa + 0.68
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
0.09
0.25
0.23
0.11
0.48
Pe pt ide
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
JH E
0.35
0.74
0.05
2.59
0.28
0.04
0.08
0.10
0.03
0.17
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
Pe pt ide
JH E
0.30
0.61
0.05
2.00
0.34
0.06
0.10
0.12
0.03
0.18
JO
0.12
0.07
0.03
1.10
0.02
0.07
0.10
0.07
0.07
0.07
JO
0.84
1.77
0.11
3.16
0.41
0.11
0.37
0.27
0.21
0.63
JO
0.32
0.60
0.04
1.86
0.11
0.05
0.13
0.13
0.05
0.22
JO
0.27
0.45
0.03
1.37
0.15
0.05
0.17
0.15
0.05
0.24
Sp r e a d
0.00
0.03
0.01
0.50
0.12
0.01
0.05
0.03
0.02
0.02
0.73
0.17
0.01
0.05
0.03
0.02
0.05
0.16
0.02
0.63
0.19
0.01
0.07
0.03
0.01
0.06
2 8 / 4 9 d
Sp r e a d
0.09
0.31
0.03
1.00
0.27
0.02
0.12
0.04
0.10
0.15
Sp r e a d
0.03
0.14
0.01
Sp r e a d
0.03
M
0.03
1.35
0.08
GAGB
SD * * - 1 * * + 1
0.12
0.00
0.12
0.08
0.02
0.06
0.00
0.35
0.08
0.03
1.00
0.00
0.07
0.00
0.07
0.07
0.03
0.04
0.06
0.02
0.03
0.06
0.01
0.05
0.06
0.01
0.05
0.12
0.10
0.03
1.70
0.16
0.08
0.10
0.08
0.08
0.08
M SD * * - 1 * * + 1
0.89
0.06
0.82
1.93
0.22
1.71
0.95
2.14
0.13
0.02
0.10
3.66
0.71
2.95
0.55
0.19
0.35
0.10
0.01
0.09
0.31
0.08
0.23
0.25
0.03
0.22
0.16
0.07
0.09
0.56
0.11
0.45
0.15
4.37
0.74
0.11
0.39
0.28
0.23
0.66
POLB1
M SD * * - 1 * * + 1
0.34
0.02
0.31
0.36
0.67
0.10
0.57
0.04
0.01
0.03
2.23
0.52
1.71
0.20
0.12
0.07
0.04
0.00
0.04
0.11
0.03
0.07
0.11
0.02
0.09
0.04
0.01
0.02
0.20
0.04
0.16
POLB2
0.77
0.05
2.74
0.32
0.05
0.14
0.14
0.05
0.23
M SD * * - 1 * * + 1
0.29
0.02
0.26
0.31
0.53
0.11
0.42
0.04
0.01
0.03
1.69
0.45
1.24
0.64
0.06
2.13
0.25
0.13
0.11
0.06
0.01
0.05
0.14
0.05
0.09
0.14
0.02
0.11
0.04
0.01
0.03
0.21
0.04
0.17
0.38
0.06
0.18
0.16
0.05
0.25
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
JH E
0.16
0.20
0.02
M I P1 b+
TN Fa +
1.88
0.10
CD 1 0 7 a + 0.07
I FN g+
0.07
I L2 +
0.03
M I P1 b+
0.06
TN Fa + 0.06
Pe pt ide
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
JH E
0.19
0.21
0.02
3.21
0.12
CD 1 0 7 a + 0.07
I FN g+
I L2 +
M I P1 b+
TN Fa +
0.04
0.04
0.23
0.07
JO
0.17
0.15
0.01
1.14
0.02
0.06
0.11
0.04
0.05
0.07
JO
0.16
0.17
0.02
1.36
0.03
0.06
0.06
0.06
0.12
0.09
Sp r e a d
0.01
0.05
0.01
0.74
0.08
0.02
0.04
0.01
0.01
0.01
Sp r e a d
0.03
0.04
0.01
1.85
0.09
0.00
0.02
0.02
0.11
0.02
EN V A
M SD * * - 1 * * + 1
0.17
0.01
0.16
0.17
0.18
0.04
0.14
0.01
0.00
0.01
1.51
0.52
0.99
0.06
0.06
0.00
0.06
0.09
0.03
0.06
0.07
EN V B
0.01
0.03
0.01
0.01
0.01
0.05
0.06
0.02
0.05
0.06
0.21
0.02
2.03
0.12
0.08
0.12
0.04
0.06
0.08
M SD * * - 1 * * + 1
0.18
0.02
0.15
0.20
0.19
0.03
0.16
0.02
0.00
0.02
2.29
1.31
0.98
0.08
0.06
0.01
0.22
0.03
3.59
0.14
0.06
0.00
0.06
0.05
0.01
0.04
0.05
0.01
0.03
0.18
0.08
0.10
0.07
0.01
0.07
0.07
0.07
0.06
0.25
0.08
Pe pt ide
An a ly z e r JH E
CD 1 0 7 a + 0.19
I FN g+
0.23
I L2 +
0.02
M I P1 b+
2.68
TN Fa + 0.10
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
0.06
0.03
0.03
0.14
0.05
Pe pt ide
An a ly z e r
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
CD 1 0 7 a +
I FN g+
I L2 +
M I P1 b+
TN Fa +
JO
0.19
0.18
0.01
1.49
0.02
0.06
0.06
0.04
0.08
0.06
JH E
5.66
JO
5.40
12.20 11.60
2.86
2.93
17.40 14.90
6.88
6.37
0.39
6.64
0.32
6.85
Sp r e a d
0.26
0.60
0.07
2.50
0.51
0.07
0.21
14.40 14.70
14.70
0.81
0.86
0.05
16.20 24.20
8.00
Sp r e a d
0.00
0.05
0.01
1.19
0.08
0.00
0.03
0.01
0.06
0.01
EN V C
M SD * * - 1 * * + 1
0.19
0.00
0.19
0.21
0.04
0.17
0.19
0.24
0.01
0.00
0.01
2.09
0.84
1.24
0.06
0.06
0.00
0.06
0.00
0.05
0.05
0.02
0.03
0.03
0.01
0.02
0.11
0.04
0.07
0.06
0.01
0.05
0.02
2.93
0.12
0.06
0.07
0.04
0.15
0.06
SEB
M SD * * - 1 * * + 1
5.53
0.18
5.35
5.71
11.90 0.42 11.48 12.32
2.90
0.05
2.85
2.94
16.15 1.77 14.38 17.92
6.63
0.36
6.26
6.99
0.36
0.05
0.31
6.75
0.15
6.60
0.40
6.89
14.55 0.21 14.34 14.76
0.84
0.04
0.80
0.87
20.20 5.66 14.54 25.86
Intra-Operator
Comparison

ICS Proficiency Testing
Results: March 2007
Duke University Medical Center

History of Flow-based
Proficiency/Standardization Efforts
Duke University Medical Center

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Improperly annotated data

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics

Markers:
CD3
CD4
CD8
IL-2+IFNg
(FSC)
(SSC)
Duke University Medical Center

• ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision (<20% C.V.), that improves as the frequency of responding cells increases.
• Gating is a significant source of variability, and can be reduced by centralized analysis and/or use of standardized gating.
• Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood.
• Use of pre-aliquoted lyophilized reagents for stimulation and staining can reduce variability.
BMC Immunology 2005, 6:13 http://www.biomedcentral.com/1471-2172/6/13
Duke University Medical Center

Gating bias in proficiency panel results
Unstim
0.02
%
CEF
0.01
%
CMV pp65
0.16
%
0.03
%
0.02
%
0.17%
0.02
%
0.03
%
0.21
%
CD4 FITC
Duke University Medical Center

Compensation:
False Positive CD4 Response to CEF Pepmix mSA
EOLm

Compensation:
False Positive CD4 Response to CEF Pepmix

Backgating:
Include CD3dim+ cells in gate

Backgating:
Include CD8dim+ in gate

C (3.1%) B (3.4%) J (4.8%) A (6.8%)
K (9.4%) D (10.2%) H (10.2%) F (10.5%)
I (12.7%) E (13.4%) G (16.9%) • Here labs are listed in order of their total TNF a response. It is visually apparent that, while all labs had overcompensation, it is worst in labs with the lowest cytokine responses.

C (3.1%)
K (9.4%)
B (3.4%) J (4.8%)
10
5
10
4
10
3
0
D (10.2%)
0 10
3
10
<Alexa 700-A>: CD3
4
H (10.2%)
10
5
A (6.8%)
F (10.5%)
I (12.7%) E (13.4%) G (16.9%) • Here labs are listed in order of their total TNF a response. It is visually apparent that, while all labs had overcompensation, it is worst in labs with the lowest cytokine responses.

110 labs participate d and there were
110 different approache s to gating

McNeil et. a. Cytometry A.
2013 Aug;83(8):728-38

Reproducible analysis allows us to measure an expansion of CD4+ CM cells post vaccination with some degree of confidence
Pre-Vaccination Post-Vaccination
17% 8%
17%
33%
2%
2%
27%
CM
21%
EM
25%
TE N E
48%
Duke University Medical Center

2xSD?
>0.05%?
Outside
Normal Range
RCV?
“Another aspect of analysis of ICS experiments that affects reproducibility across laboratories is the question of how to define a positive response. Criteria for determining a positive response can range from simply subtracting background to twofold, threefold, or even fourfold above background to more complex statistical analyses
”
Roederer. Cytometry Part A, 73A:384-385 (2008)
Horton et. al. J Immuno Methods, 323:39-54 (2007)
Maecker et. al. Cytometry Part A, 69A:1037-1042 (2006)
Comin-Anduix et. al. Clin Cancer Res, 12(1):107-116 (2006)
Duke University Medical Center

Workshop Overview
• ICS Assay
• Instrument Qualification
• Reagent Qualification
– Titration
– Spillover
– Panel Development / Assay Qualification
• Operator Qualification
– Training
– Proficiency Testing
• Data annotation
• Data Analysis
– Compensation
– Gating
– Back-gating
– Uniform/Standardized Gating
• Bioinformatics and Statistics – Death-by-excel!

Panel
Description
#Colors
# Gating Generations
# Endpoints/Sample
Endpoint Increase Over 4-Color (Complexity of Analysis)
Avg File Size/Tube (mb)
Avg Size/Sample (mb)
Annual Storage in TB (based on 6003 tubes/yr)
Acute
Maturation &
Activation
4
3
18
0.00
0.20
1.20
0.01
Roche
Maturation &
Quantitative
Activation
9
6
149
727.78
20.37
40.74
0.24
Sampson
Maturation &
ICS
12
8
1440
7900.00
91.86
367.44
2.21
Duke University Medical Center

Endpoints for 11-Color Maturation/Function Panel
DEATH BY EXCEL ……..
Basic (3)
CD4 + CD8 -
CD4 + CD8 +
CD4 CD8 +
Maturation (5) Function
Naïve
Central Memory
Effector Memory
Effector
Terminal Effector
CD107
IFNg
IL-2
TNFa
Boolean (16)
7+g+2+T+
7+g+2+T-
7+g+2-T+
7+g-2+T+
7-g+2+T+
7+g+2-T-
7+g-2+T-
7+g-2-T+
7-g+2+T-
7-g+2-T+
7-g-2+T+
7+g-2-T-
7-g+2-T-
7-g-2+T-
7-g-2-T+
7-g-2-T-
Basic (3) X Maturation (5) X Boolean (16) = 240/stim
X 3 Stimulations/Sample (CoStim, SEB, CMVpp65) = 720 Endpoints/Sample
720 Endpoints/Sample x 200 Samples (192 Participants + 8 Controls) = 144,000 Endpoints/Trial
Note 1: Frequency of parent only, reporting units of #cells/µL doubles the total EP/trial
Duke University Medical Center

for all 143,280 data points!
Study ID
Method
Assay Name
Batch #
Operator
Sample ID
Visit ID
Accession #
% Viable (Flow)
% Viable (Guava)
Recovery
Checking:
X1 - for electronic data
X3 - for manual entry
Requires STRONG statistical support:
• Quickly exceeds limits of excel
•
Format data for statistical analysis
• FJ: column (gates) vs row (file)
• CSV: column (identifiers) vs row (single value)
• Check data
• Manual check: 8sec/value x 143280 = 49 days!!!
CD4 count
CD8 count
Gate Name (Parameter Names)
Tube Name
File Name
Error Code (1-11)
Duke University Medical Center

Specimen processing matters
N=60 samples, stained across 5 batches
Duke University Medical Center

VRC
Steve Perfetto
Laurie Lamoureaux
Mario Roederer
EQAPOL
Duke CFAR
Kent Weinhold
Jennifer Enzor
Twan Weaver
Cliburn Chan
Scott White Duke Tisch Brain Tumor Center
Gary Archer
Duane Mitchell
John Sampson
CVC
Sylvia Janetski
Lisa McNeil
Duke University Medical Center