Transcript No Slide Title

Optimizing Multiparameter Flow
Cytometry in 6-12 Colors
Holden T. Maecker
Outline
• Choosing fluorochrome combinations
and filter sets
• Matching antibody specificities with
fluorochromes
• Instrument setup and quality control
• Experiment setup and controls
• Analysis of multiparameter data sets
Outline
• Choosing fluorochrome combinations
and filter sets
• Choose bright fluorochromes
• Minimize spillover between channels
“Bright” = good resolution sensitivity
D
W1
W2
Stain Index (SI)  D
W
Various fluorochromes-stain index
Reagent
Clone
Filter
Stain Index
PE
RPA-T4
585/40
356.3
Alexa 647
RPA-T4
660/20
313.1
APC
RPA-T4
660/20
279.2
PE-Cy7
RPA-T4
780/60
278.5
PE-Cy5
RPA-T4
695/40
222.1
PerCP-Cy5.5
Leu-3a
695/40
92.7
PE-Alexa 610
RPA-T4
610/20
80.4
Alexa 488
RPA-T4
530/30
75.4
FITC
RPA-T4
530/30
68.9
PerCP
Leu-3a
695/40
64.4
APC-Cy7
RPA-T4
7801/60
42.2
Alexa 700
RPA-T4
720/45
39.9
Pacific Blue
RPA-T4
440/40
22.5
AmCyan
RPA-T4
525/50
20.2
Spillover affects resolution sensitivity
FITC background
contributes noise to
PE measurement
www.bdbiosciences.com/spectra
Choices for 6-, 8-, 10-, and more colors
6-color
8-color
10-color
Additional
FITC or Alexa 488
FITC or Alexa 488
FITC or Alexa 488 FITC or Alexa 488
PE
PE
PE
PE
PE-Texas Red or
PE-Alexa 610
PE-Texas Red or
PE-Alexa 610
PerCP-Cy5.5
PerCP-Cy5.5
PerCP-Cy5.5
PerCP-Cy5.5
PE-Cy7
PE-Cy7
PE-Cy7
PE-Cy7
APC or Alexa 647
APC or Alexa 647
APC or Alexa 647
APC or Alexa 647
Alexa 680 or 700
Alexa 680 or 700
APC-Cy7
APC-Cy7
APC-Cy7
AmCyan
AmCyan
AmCyan
Pacific Blue
Pacific Blue
Pacific Blue
APC-Cy7
Q-dot 655, 705…
Outline
• Choosing fluorochrome combinations
and filter sets
• Matching antibody specificities with
fluorochromes
• Instrument setup and quality control
• Experiment setup and controls
• Analysis of multiparameter data sets
Fluorochrome selection considerations
• “Bright” antibodies go on “dim”
fluorochromes
• Avoid spillover from bright cell populations
into channels requiring high sensitivity
• Beware of tandem dye degradation
Data spread of fluorescent signals
uncompensated
compensated
data
spread
uncompensated
compensated
FITC mean fluorescence
---------------------------negative
positive
-------------------123
3541
123
3564
PE mean fluorescence
---------------------------negative
positive
-------------------184
1648
134
136
HT Maecker, T Frey, LE Nomura, J Trotter: Selecting fluorochrome
conjugates for maximum sensitivity. Cytometry A 2004, 62:169-73.
Data spread of fluorescent signals
A. With CD8 APC-Cy7 and CD4 PE-Cy7:
Gating scheme
B. Without CD8 APC-Cy7:
CD8 APC-Cy7+ cells
CD4 PE-Cy7+ cells
Data spread in APC
channel reduced in
absence of APC-Cy7
Data spread in
PE channel
remains
Effect of Spillover on Double Stained Cells
CD45-FITC
Dim CD4-PE
CD45- PerCP
Dim CD4-PE
Compensated data:
CD45 FITC makes
dim CD4+CD45+ difficult to
Distinguish due to FITC spillover
Into PE and resultant “spread”
CD45 PerCP allows
same dim CD4+CD45+ cells to be
distinguished from background
(PerCP contributes little spillover
into PE)
Spillover affects resolution senstivity
8-color Antigen-Specific
Immunophenotyping
Ab Conjugate
Laser
CD28 PerCP-Cy5.5
488
CD45RA PE-Cy7
488
CD27 APC
633
Surface
CD8 APC-Cy7
633
staining
CD3 Pacific Blue
405
CD4 AmCyan
405
Anti-IFNg FITC
488
Intracellular
Anti-IL-2 PE
488
staining
Outline
• Choosing fluorochrome combinations
and filter sets
• Matching antibody specificities with
fluorochromes
• Instrument setup and quality control
• Experiment setup and controls
• Analysis of multiparameter data sets
Instrument setup
• Finding PMT voltages that maximize
resolution sensitivity
• Compensating for fluorescence spillover
How to set PMT voltages?
• Put unstained cells in first log decade
• Autofluorescence varies by channel
• Leads to highly variable setup
• No guarantee that performance on
stained cells will be optimal
• Use dim particles to find voltages that
minimize their CV, thus maximizing
resolution sensitivity
A suitably dim particle
• Spherotech Rainbow Calibration Particles
(3.0-3.4 mM)
• Peak 2 of 8-peak bead set
• Catalog # RCP-30-5A-2
PMT characterization to minimize CV
When to change baseline voltages?
• Stained cells go off-scale
• Should lower voltages so there are no
events in the highest channel
• Background is very high
• Can lower voltages, but risk losing
resolution sensitivity
Target channels using mid-range beads
Compensation controls with capture beads
Outline
• Choosing fluorochrome combinations
and filter sets
• Matching antibody specificities with
fluorochromes
• Instrument setup and quality control
• Experiment setup and controls
• Analysis of multiparameter data sets
Types of controls
• Instrument setup controls
• PMT voltage settings
• Compensation
• Gating controls
• Isotype controls
• FMO controls
• Biological controls
• Unstimulated samples
• Healthy donors
Comparison of controls
Potential problems of tandem dyes (I)
• Multiple specificities of a given tandem may
require individual compensation
• Due to variations in tandem conjugation
• The same tandem reagent may require
different compensation lot-to-lot and
experiment-to-experiment
• Due to partial degradation of tandem
• Solution: compensate on the same reagents
used for each experiment (for tandems)
Potential problems of tandem dyes (II)
• Degradation of conjugate in bottle
• Degradation of conjugate on stained cells
• Aggravated by exposure to:
• Light
• Elevated temperature
• Formaldehyde-based fixation
• Worst for APC-Cy7, then PE-Cy7
False positives due to tandem degradation
A. With CD8 APC-Cy7 and CD4 PE-Cy7:
Gating scheme
B. Without CD8 APC-Cy7:
CD8 APC-Cy7+ cells
CD4 PE-Cy7+ cells
False positives in
APC channel reduced
in absence of APC-Cy7
False positives
in PE channel
remain
Degradation of APC-Cy7 on stained cells
% spillover
APC-Cy7 -> APC
100
75
PBS/BSA
PBS/formaldehyde
50
25
In Fixation Stabilizing Buffer
0
no
storage
overnight overnight
room temp
4o C
Standardization using lyophilized reagents
• Lyophilization provides increased stability,
even at room temperature or 37oC
• One batch of reagents can be used for an
entire longitudinal study
• Pre-configured plates can avoid errors of
reagent addition
• Complex experiments (multiple stimuli,
multiple polychromatic staining cocktails)
become easier
• Lyophilized cell controls can provide run-torun standardization
Lyophilized Reagent ICS Protocol
PBMC or whole blood
Plate with lyophilized Ag+BFA
6h @ 37oC, EDTA, FACS Lyse, FACS Perm 2
Plate with lyophilized Ab
to flow cytometer
Liquid vs. Lyophilized Reagents
CD3+CD8+
CD3+CD8-
0.97%
MFI 489
0.59%
MFI 608
CD69 PE
Liquid Ag
Liquid Ab
0.95%
MFI 420
0.58%
MFI 608
Lyophilized Ag
Lyophilized Ab
IFNg FITC
Multi-Site Data with Lyoplates
Fixed
Activated
Blood
Shipped
Whole
Blood
Cryopreserved
PBMC
Cryopreserved
PBMC with
Lyoplates
Number of samples
12
12
24
9
Mean % CV
24%
31%
23%
18%
Assay Type
Outline
• Choosing fluorochrome combinations
and filter sets
• Matching antibody specificities with
fluorochromes
• Instrument setup and quality control
• Experiment setup and controls
• Analysis of multiparameter data sets
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
Facilitating Data Organization
Batch
analysis
with
dynamic
gating
FCS
files
Statistics files
Custom
Excel
spreadsheet
Donor
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168 - May 6,
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168 - May 6,
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168 - May 6,
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
CMV sero Well Gate
Label
% Gated
+
A01 CD4 IFNg+CD69+
0.01
+
A02 CD4 IFNg+CD69+
0.36
+
A03 CD4 IFNg+CD69+
0.15
+
A04 CD4 IFNg+CD69+
0.07
+
A05 CD4 IFNg+CD69+
0.07
+
A06 CD4 IFNg+CD69+
0.06
+
A07 CD4 IFNg+CD69+
0.08
+
A08 CD4 IFNg+CD69+
0.02
+
A09 CD4 IFNg+CD69+
0.01
+
A10 CD4 IFNg+CD69+
0.03
+
A11 CD4 IFNg+CD69+
0.05
+
A12 CD4 IFNg+CD69+
0.09
+
B01 CD4 IFNg+CD69+
0.05
+
B02 CD4 IFNg+CD69+
2.69
+
B03 CD4 IFNg+CD69+
0.15
+
B04 CD4 IFNg+CD69+
0.05
+
B05 CD4 IFNg+CD69+
0.03
+
B06 CD4 IFNg+CD69+
0.03
+
B07 CD4 IFNg+CD69+
0.1
+
B08 CD4 IFNg+CD69+
0.03
+
B09 CD4 IFNg+CD69+
0.04
+
B10 CD4 IFNg+CD69+
0.03
+
B11 CD4 IFNg+CD69+
0.03
+
B12 CD4 IFNg+CD69+
0.11
Web
database
4000
CD4+ IFNg+
CD4+ IFNg+ IL-2+
CD8+ IFNg+
CD8+ IFNg+ IL-2+
HIV- subjects
pp65+IE-1
Cytokine+ cells/ml blood
2000
0
9000
HIV+ subjects
pp65+IE-1
2000
2000
0
4000
HIV+ subjects
gag+env
2000
0
CD27:
+ + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - -
CD28:
+ + + + - - - - + + + + - - - - + + + + - - - - + + + + - - - -
CD45RA: + - - + - + - + + - - + - + - + + - - + - + - + + - - + - + - +
Conclusions
• Polychromatic flow cytometry is not impossible
• Select fluorochromes for brightness and least spillover
• Optimize antibody panels by taking into account reagent
brightness and data spread
• Stabilize longitudinal experiments with proper QC
• Put systems into place to allow streamlined and
reproducible analysis
• Some solutions that can help
• Lyophilized reagent plates
• Fixation stabilizing buffer
• Beads for calibration and compensation
• Maecker lab weblog for references, ICS protocols, tools:
• http://maeckerlab.typepad.com
Acknowledgements
•
•
•
•
•
•
•
Laurel Nomura
Margaret Inokuma
Maria Suni
Smita Ghanekar
Daiva Gladding
Jack Dunne
Skip Maino
• Joe Trotter
• Dennis Sasaki
• Perry Haaland
• Eugene Veteska