“Measuring Antigen Specific T-cells using Surface and Intracellular Staining Polychromatic Flow Cytometry” 3 rd Annual CFAR Flow Cytometry Workshop 6-10 May, 2013 Janet Staats Flow Cytometry Core Facility Center for AIDS Research Duke University Medical Center E-mail: [email protected]
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“Measuring Antigen Specific T- cells using Surface and Intracellular Staining Polychromatic Flow Cytometry” 3 rd Annual CFAR Flow Cytometry Workshop 6-10.
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“Measuring Antigen Specific T-cells using Surface and
Simplified Presentation of Incredibly Complex Evaluations
Dr. Mario RoedererImmunotechnology SectionVRC / NIAID / NIH
Duke University Medical Center
Part 2 of 3
PFC Challenges
Duke University Medical Center
Challenges…
• Instrument - optical configuration, optimization, standardization, and calibration
• Reagent - optimization and standardization
• Sample processing• Staining protocols• Data Analysis - compensation &
gating• Operators• Volume of data (death-by-excel!)
Duke University Medical Center
Consistency across batchesCD38 vs HLA-DR Staining on Ctrl 5L
28Feb085L CD8+
04Marb085L CD8+
11Mar085L CD8+
06Mar085L CD8+
Duke University Medical Center
uncompensated
compensationFSC/SSC settings
PMT settings
highlow
Difficulties in doing Automated Analysis related to Instrument Settings
CD4
CD
3
IFNg
CD
69
CD4
SS
C
FSC
SS
C
optimal optimal
Duke University Medical Center
Challenges…
• Instrument - optical configuration, optimization, standardization, and calibration
• Reagent - optimization and standardization
• Sample processing• Staining protocols• Data Analysis - compensation &
gating• Operators• Volume of data (death-by-excel!)
Duke University Medical Center
Optimization using Spillover Assessments: Using Titration Files to Assess Spreading Error
Violet G- CD3 AmCyan
<B
lue
B-A
><
Vio
let
H-A
><
Red
C-A
>
<R
ed B
-A>
Red
A-A
<G
reen
E-A
>
<G
reen
D-A
>
<G
reen
C-A
>
<G
reen
B-A
>
<G
reen
A-A
>
CD3AC (5ug/ml) Spillover assessment:
• After compensation CD3AC showed spilllover into Blue-B detector (FITC channel)
Blue Laser
Violet Laser
Red Laser
Green Laser
<B
lue
A-A
>
• 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 2006Duke University Medical Center
Spillover Assessments:CD3 AmCyan (5µg/mL) Spillover into CD27 (0.32µg/mL)
& CD57 FITC (1.8µg/mL)
• 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
CD27 FITC
Blue B
SS
C
CD3AmCyan
9.8e-4Unstained
SS
C
0.047
Blue B
Unstained
66.3
4.58
0.13
CD57 FITC
CD3AmCyan
20.5
Duke University Medical Center
Is this positive???
CMV pp65 stimulated sample
Maecker, et. al.Duke University Medical Center
Tandems Degrade!
• Ice• Dark• Fix• Controls• 6 hours
Maecker, et. al.Duke University Medical Center
Challenges…
• Instrument - optical configuration, optimization, standardization, and calibration
• Reagent - optimization and standardization
• Sample processing• Staining protocols• Data Analysis - compensation &
gating• Volume of data (death-by-excel!) Duke University Medical Center
9-Color Activation/Maturation Using Cryo-preserved PBMC
Duke University Medical Center
Batch Processing ErrorCD38 vs HLA-DR Staining on Ctrl 5L
28Feb085L CD8+Lot 05262
04Marb085L CD8+Lot 05262
11Mar085L CD8+Lot 05262
06Mar085L CD8+Lot 05262
26Feb085L CD8+Lot 05262
Duke University Medical Center
Challenges…
• Instrument - optical configuration, optimization, standardization, and calibration
• Reagent - optimization and standardization
• Sample processing• Staining protocols• Data Analysis - compensation &
gating• Operator• Volume of data (death-by-excel!)
Duke University Medical Center
How would you gate?
Markers:CD3CD4CD8IL-2+IFNg(FSC)(SSC)
Duke University Medical Center
N CM EM TE E
Pre-Vaccination
33%
21%
27%
2%
17%
Post-Vaccination
8%
48%25%
2%
17%
Duke University Medical Center
Reproducible analysis allows us to measure an expansion of CD4+ CM cells post vaccination with
some degree of confidence
ICS Standardization Conclusions
• 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
CIC ICS Gating Panel
110 labs participated and there were 110 different approaches to gating
BeforeBackgate
AfterBackgate
IFNgBackgate
CD3 AmCyan
Exc
lusi
on
0.38 5.74
CD4 Gated CD8 Gated
5.230.27
IFNg PE-Cy7
CD
4 P
erC
P-C
y5.5
CD
8 A
PC
-Cy7
BeforeBackgate
AfterBackgate
A
B
BACKGATING: purity & recovery
Duke University Medical Center
Gating bias in proficiency panel results
CD4 FITC
IL2+
IFN P
E
Unstim CEF CMV pp65
0.02%
0.01%
0.16%
0.03%
0.02%
0.17%
0.02%
0.03%
0.21%
Duke University Medical Center
We NEED better analysis tools!!!Manual (Expert) vs. Automated Analysis of
4-Color ICS Data File (CMVpp65)
0.21%0.18%
CD4 FITC
1.9%1.65%
CD8 PerCP-Cy5.5
IFN
- +
IL-2
PE
Expert GatingManual
Cluster GatingAutomated
Duke University Medical Center
Would you know a positive if you saw one?
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)
2xSD?>0.05%?
OutsideNormal Range
RCV?
Duke University Medical Center
Challenges…
• Instrument - optical configuration, optimization, standardization, and calibration
• Reagent - optimization and standardization
• Sample processing• Staining protocols• Data Analysis - compensation &
gating• Operator• Volume of data (death-by-excel!)
Duke University Medical Center
Assay Complexity
Duke University Medical Center
Endpoints for 11-Color Maturation/Function Panel DEATH BY EXCEL ……..
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 stimulationHIV negative donor
Example CMVpp65 stimulationCMV positive donor
% C
D8
+ C
FS
E lo
w
LaboratoryDuke University Medical Center
History of Flow-based Proficiency/Standardization Efforts
Duke University Medical Center
The number of measurements outside the optimal range established by the GS was determined for each laboratory. Each laboratory performed a total of 54 measurements (27 for CD4+ cells and 27 for CD8+ cells). The red line represents 50% (=27) of the total measurements. Laboratories above this line had over 50% of their measurements outside the optimal range. The green line represents 20% of total measurements. The laboratories below this line had over 80% of their measurements within the optimal range.
ICS Proficiency Testing Results: March 2007
Duke University Medical Center
DAIDS ICS Proficiency:Round 6, 26Jun09 (CMVpp65)
CD4-CD8+ CD4+CD8-
IFN
g +
IL-2
PE
CD3 APC-Cy7
Rep #1
Rep #2
Rep #3
Duke University Medical Center
Acknowledgements
Duke University Medical Center
Duke CFARKent WeinholdJennifer EnzorTwan WeaverJianling ShiCliburn Chan
Patricia D’Souza (DAIDS) CFSE Standardization:
Claire Laundry (NIML)
EQAPOL
Duke Tisch Brain Tumor CenterGary ArcherDuane MitchellJohn Sampson