Download Multicolor Flow Cytometry

Multicolor Flow Cytometry
Setup and Design
Presented by
Maria C. Jaimes, MD, Scientist, BD Biosciences
23-13501-00
Overview
•
Multicolor flow – successful application
prerequisites for immunophenotyping:
A. Careful reagent selection and sample
preparation.
B. Proper cytometer performance, setup, and data
collection.
C. Proper classification (analysis) of multiple
combinations of positive and negative CD
markers.
Immunophenotyping by Flow Cytometry (1)
• Ultimate Goal
Resolve distinct subsets of leucocytes expressing one or
more unique key markers.
• Method
The proper classification of many of the large number of
distinct leucocyte subsets (phenotypic and functional)
often requires simultaneous labeling with several
markers: a multicolor panel.
Immunophenotyping by Flow Cytometry (2)
• Typical Problems/Challenges
– Some markers are highly expressed, others are expressed at low levels.
– Some dyes are much brighter than others.
– Significant emission spillover from non-primary fluorescent reagents
contributes to optical background, which can often diminish the resolution of
dim markers (due to spread after compensation).
– Some markers may be available only in certain colors.
– New fluorochromes are not as bright or stable as the original ones.
– Instrument setup considerations.
– Additional complexity increases error possibilities.
Immunophenotyping by Flow Cytometry (3)
• As a result, multicolor flow assays and reagent panel
combinations need to be very carefully assembled to
obtain reliable and interpretable data.
• What factors affect reagent performance on a cytometer
and what is the best approach for assembling a
multicolor panel?
TNK as an Example of
Multiparametric Flow Cytometry Assays
Several markers used to define T- and NK-cell
subsets:
- T-cell markers: CD3, CD8, and CD7
- NK-cell markers: CD16, CD56, and CD57
- CD8 and CD7 are also expressed by some
subsets of NK cells
Principles of Panel Design – Reagent Selection
1
Check for reagent availability – clone selection.
2
Match fluorochromes by brightness (values from
stain index) according to antigen density and
distribution (published values or TDS).
3
Minimize spectral overlap.
4
Use tandem dyes with consideration of their
technical limitations.
5
Run appropriate controls.
Choices for 6, 8, 10, and More Colors Depending
1
on Instrument Configuration
6-color
8-color
10-color
Additional
FITC or
Alexa Fluor® 488
FITC or
Alexa Fluor® 488
FITC or
Alexa Fluor® 488
FITC or
Alexa Fluor® 488
PE
PE
PE
PE
PE-Texas Red®
PE-Texas Red®
PE-Cy™5/PerCP/
PerCP-Cy™5.5
PE-Cy5/PerCP/
PerCP-Cy5.5
PE-Cy5/PerCP/
PerCP-Cy5.5
PE-Cy5/PerCP/
PerCP-Cy5.5
PE-Cy™ 7
PE-Cy7
PE-Cy7
PE-Cy7
APC or
Alexa Fluor® 647
APC or
Alexa Fluor® 647
APC or
Alexa Fluor® 647
APC or
Alexa Fluor® 647
Alexa Fluor® 700
Alexa Fluor® 700
APC-H7/APC-Cy7
APC-H7/APC-Cy7
APC-H7/APC-Cy7
BD Horizon™ V500 or
AmCyan
BD Horizon V500 or
AmCyan
BD Horizon V500 or
AmCyan
BD Horizon™ V450
BD Horizon V450
BD Horizon V450
APC-H7/APC-Cy™7
Qdots®
1 Checking Reagent Availability
• New online tool available at
bdbiosciences.com/paneldesigner
• Clone selection. Consider:
– Cell type (example: CD16 staining for NK vs
granulocytes)
– Sample preparation (LW vs LNW)
– Check TDS and literature
Principles of Panel Design – Reagent Selection
1
2
Check for reagent availability – clone selection.
Match fluorochromes by brightness (values from
stain index) according to antigen density and
distribution (published values or TDS).
3
Minimize spectral overlap.
4
Use tandem dyes with consideration of their
technical limitations.
5
Run appropriate controls.
2 Determine Antigen/Fluorochrome Combos (1)
• Use the brighter fluorochromes for dimly
expressed markers.
• Use the dimmer fluorochromes for more
highly expressed markers.
2 Determine Antigen/Fluorochrome Combos (2)
•
Classify the antigens you would like to measure.1
1. Primary: Well characterized, easily classified as positive or
negative (CD3, CD4, CD8, etc.). Get these in as many colors
as possible if you need to do testing.
2. Secondary: Well characterized, also expressed at a higher
density, often over a continuum (CD27, CD28, CD45RA/RO,
IFN-γ). Get some of these.
3. Tertiary: Expressed at low levels only (CD25), also
uncharacterized antigens. Often available in only one or two
colors.
1
Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay.
Clin Lab Med. 2007 September,27:469–485,v.
2 Glossary:
Antigen Density
• Level of antigen expression
on a cell:
– Antigen expression can vary
due to cell activation level and
functional differences
– Antigen density can be a
range (ie, smeared
population)
2
Glossary: “Bright” = Good Resolution Sensitivity
Stain Index
D
W1
W2
Stain Index (SI) = D
W
D = difference between positive and negative peak medians
W = the spread of the background peak (= 2X rSDnegative)
Resolution sensitivity = the ability to resolve a dim positive signal from
background
2 Various Fluorochrome Stain Indexes
Measured on the BD™ LSR II
Estimated Stain Index Ranking Available from
2
TDS
• Review antibody/fluorochrome combinations in TDS
– Visually compare all antigens conjugated to the same
fluorochromes.
CD5
CD8
2 Example
“Bright” antibodies go on “dim” fluorochromes
Example:
CD8 “bright”
V450 (SI = 80)
CD7 “less bright” PE (SI = 302)
CD8 = 90K molecules/cell
CD7 = 20K molecules/cell
Principles of Panel Design – Reagent Selection
1
2
Check for reagent availability – clone selection.
Match fluorochromes by brightness (values from
stain index) according to antigen density and
distribution (published values or TDS).
3
Minimize spectral overlap.
4
Use tandem dyes with consideration of their
technical limitations.
5
Run appropriate controls.
3
Fluorescence Spillover
• The single most important factor affecting resolution
sensitivity (SI) in multicolor flow cytometry experiments.
• Fluorescence spillover from other channels:
– Directly and irreversibly reduces the resolution sensitivity
of that channel
– Contributes to background
• This “background” is subtracted in the process called
compensation.
3 Spillover Decreases Resolution Sensitivity
Population resolution for a
given fluorescence
parameter is decreased by
increased spread due to
spillover from other
fluorochromes.
This spread is NOT eliminated by compensation
More colors = more spillover = higher background
•
The dim CD4+ cells when negative for CD8 (orange ball) are easily
resolved from the double negative.
• The same dim CD4+ cells when positive for CD8 cannot be resolved
from CD4–, CD8+ cells.
• To improve resolution (sensitivity) of subpopulations, including dim
subpopulations you want to minimize the amount of spillover from
other fluorochromes.
3 % Spillover of Fluorochromes – BD FACSCanto™
Spillover Column into Row
(calculated using BD FACS™ 7-color setup beads and BD
FACSCanto™ software)
Detector
FITC
PE
FITC
PE
PerCP
PE-Cy7
APC
—
1.57%
0%
0.22%
0.01%
18%
—
0.32%
2.06%
0.01%
—
4.00%
1.05%
PerCP
2.67% 16.16%
PE-Cy7
0.32%
1.44%
10.40%
—
0.19%
0%
0.12%
5.04%
0.08%
—
APC
Before designing a multicolor experiment, KNOW ALL the %
spillovers for the fluorescence parameters to be used in your
experiment.
3
Dual Excitation Reduces Resolution
Fluorochromes that are excited by more than 1 laser
cause high spillover.
– AmCyan excited by violet and blue (FITC detector).
– PE-Cy5 spills into APC detector.
Without CD45 AmCyan:
With CD45 AmCyan:
CD19 FITC
Only an issue when the two markers (CD45
and CD19) are co-expressed
on the same cell population.
3
Strategies to Minimize Spillover Issues (1)
• Minimize the potential for spectral overlap
• Spillover estimates available in the spectrum viewer
3 Strategies to Minimize Spillover Issues (2)
If multiple antigens are present on a cell, spread them
across as many lasers as possible to minimize spillover.
Example:
CD3 “bright”
APC-Cy7 (SI = 42.2)
CD7 “less bright” PE (SI = 356.3)
Both antigens expressed on the same cell,
low spillover of CD3 into CD7 and vice
versa.
CD3 = 124K molecules/cell
CD7 = 20K molecules/cell
Principles of Panel Design – Reagent Selection
1
2
Check for reagent availability – clone selection.
Match fluorochromes by brightness (values from
stain index) according to antigen density and
distribution (published values or TDS).
3
Minimize spectral overlap.
4
Use tandem dyes with consideration of their
technical limitations.
5
Run appropriate controls.
4 Use Tandem Dyes with Consideration of
Their Technical Limitations
• Compensation requirements for tandem dye
conjugates can vary, even between two experiments
with the same antibody.
– Require compensation that is: lot-specific, experimentspecific, and label-specific.
– Treat compensation controls the same as sample cells.
• Certain tandem dye conjugates (APC-Cy7, PE-Cy7)
can degrade with exposure to light, elevated
temperature, and fixation.
– Minimize exposure to these conditions.
– Use BD™ Stabilizing Fixative for final fixation.
4 False Positives Due to Tandem Degradation
A.With CD8 APC-Cy7 and CD4 PE-Cy7
CD8 APC-Cy7+ cells
B.Without CD8 APC-Cy7
CD4 PE-Cy7+ cells
False positives in
APC channel reduced
in absence of APC-Cy7
False positives
in PE channel
remain
4 Effect of Light on Tandem Degradation
CD8
PE-Cy7
CD3
PE-Cy5
Time Sample
Left in Light
0 hours
PE
2 hours
22.5 hours
3 New Tandems Are More Stable
APC-H7 to replace APC-Cy7:
Comparison of Sample Stability
(in BD Stabilizing Fixative at RT)
250
% Spillover
200
CD4 APC-Cy7
150
CD8 APC-Cy7
CD4 APC-H7
100
CD8 APC-H7
50
0
0
1
2
4
6
8
Hours of light exposure
24
48
Principles of Panel Design – Reagent Selection
1
2
Check for reagent availability – clone selection.
Match fluorochromes by brightness (values from
stain index) according to antigen density and
distribution (published values or TDS).
3
Minimize spectral overlap.
4
Use tandem dyes with consideration of their
technical limitations.
5
Run appropriate controls.
5
What Controls Do You Need and Why?
– Instrument setup controls (eg, BD™ CompBead
particles)
– Gating controls (eg, FMO)
– Biological controls (eg, unstimulated samples)
•
This will allow you to:
–
–
–
Obtain consistent setup and compensation
Gate problem markers reproducibly
Make appropriate biological comparisons and
conclusions
5 Use FMO Controls for Accurate Data Analysis
• Fluorescence minus one (FMO) controls
contain all the lineage markers except the
one of interest.
• For low density or smeared populations (eg,
activation markers) FMOs allow accurate
delineation of positively vs negatively stained
cells.
5
FMO Example
Gated on lymphs, CD3+CD4–
Gated on lymphs, CD3+CD4+
Full 9-color
cocktail
FMO
AmCyan
5
Comparison of Gating Controls
Coming Back to T NK Panel
Fluorochrome
Fluorochrome Brightness
Brightness
Low
→ High
High
Low →
Antigen
Antigen Density:
Density:
High
→ Low
Low
High →
V500
CD45 V500
CD45
V450
CD8 V450
CD3
APC-Cy7
APC-H7
CD8
PerCP-Cy5.5
CD16
FITC
CD56
PE-Cy7
CD57
APC
CD7
PE
CD3 APC H7
CD16 PerCP-Cy5.5
CD57 FITC
CD56 APC
CD7 PE
T NK Panel
Overview
•
Multicolor flow – successful application
prerequisites for immunophenotyping:
A. Careful reagent selection and sample preparation.
B. Proper cytometer performance, setup, and data
collection.
C. Proper classification (analysis) of multiple
combinations of positive and negative CD
markers.
Cytometer Performance
and Setup
CS&T
BD™ Cytometer Setup &Tracking (CS&T) is a fully
automated software and reagent research system,
unique to BD digital cytometers, providing:
• Characterization
• Setup
• Tracking
CS&T Beads
• Two sizes and three intensities of uniform beads:
–
–
–
2-μm dim fluorescence intensity
3-μm mid fluorescence intensity
3-μm bright fluorescence intensity
• Excited and detected in all BD preconfigured cytometers.
Bright
Bright
& Mid
2 µm
3 µm
Dim
Dim Mid
FSC-A
(x 1,000)
FITC-A
Instrument Performance and Sensitivity
CS&T Baseline Report
• Instrument performance can have a significant impact on the
performance of an assay, especially for the far red channels.
• Instrument sensitivity is a function of Qr, Br, and SDEN.
– Increases in Br or decreases in Qr can reduce sensitivity and the
ability to resolve dim populations.
– On digital instruments, BD FACSDiva™ software v6 and CS&T
provides the ability to track performance data for all of these
metrics, allowing users to compare performance between
instruments.
Impact of Qr and Br on Assay Sensitivity
• Plots gated on CD3+ lymphocytes.
High Qr, Low Br
Low Qr, Low Br
Low Qr, High Br
.
Qr
Qr and
and Br
Br characterizations
characterizations are
are for
for both
both FITC
FITC and
and PerCP-Cy5.5
PerCP-Cy5.5 parameters
parameters
Setting Up Your Instrument
• Only two things need to be done to set up a single
instrument for a given type of assay.
1.Set the gain [PMT voltage], to achieve reproducible
fluorescence intensity (MFI)
2.Correct for background from fluorescence spillover
(compensation), which is instrument-dependent.
• For comparing results of assays to be run on multiple
instruments, it is important that the MFIs are consistent and
reproducible between the instruments.
Using CS&T Application Settings to
Standardize Instrument Setup
• The CS&T system is designed to set fluorescence gain to
optimize low-end sensitivity for each instrument.
• One of the most under-utilized features of BD FACSDiva 6/
CS&T software is the ability for users to create their own
application settings for each assay type.
– MFIs can be set by the user, saved, and reproducibly
reused.
• Using BD FACSDiva 6/CS&T Application Settings
functionality, it is possible to standardize multiple
instruments (different platforms and different sites) to give
equivalent fluorescence.
CS&T Saves Your Assay-Specific MFI Targets
• Run a CS&T Performance Check to standardize the instrument.
• Adjust the PMT voltages so that you have the fluorescence
intensities (MFIs) that are appropriate for your assay.
– Select Application Settings > Save (Right-click on Cytometer Settings)
• BD FACSDiva 6 / CS&T software
remembers the target MFI values.
– These settings can then be
applied to future experiments.
– Gives reproducible data
• Experiment to experiment
• Instrument to instrument
Class 1 Laser Product.
Factors to Consider for an Optimal Gain Setup
1. Electronic Noise can affect resolution sensitivity
9 A good minimal application PMT voltage would place the dimmest cells
(unstained) where electronic noise is no more than 10% to 20% of the total
variance.
2. Dynamic range assessment for each fluorescence parameter
a) Are the brightest populations within the linear range of the detector?
b) Are the compensation controls within the linear range of the detector?
c) Are the negatives (in a stained sample) too high?
3. An optimal cytometer gain setting is one for which both conditions are
met.
Electronic Noise (SDEN)
– Background signal due to electronics
• Contributed by
– PMT connections/PMT noise
– Cables too near power sources
– Digital error
– Broadens the distribution of unstained or dim particles
• Most important for channels with low cellular
autofluorescence
– APC-Cy7, PE-Cy7, PerCP-Cy5.5
– BD FACSDiva/CS&T software uses the SDEN to set
PMT voltages to minimize CV (spread) of negative/dim
populations
Correctly Setting PMT Voltage Gain
Improves Resolution
550 volts
CD4 dim monocytes
CD4 negative
CD4+ lymphocytes
Log: negative -100 V
650 volts
CD4 dim monocytes
CD4 negative
CD4+ lymphocytes
Log: negative opt V
750 volts
CD4 dim monocytes
CD4 negative
CD4+ lymphocytes
Log: negative +100 V
Effect of Non-Linearity on Compensation
A
B
C
D
FITC MFI
68
1,796
5,921
73,000
PE MFI
80
75
80
365
• Samples are BD CompBead particles stained with varying levels
of FITC-Ab.
• Compensation is calculated using samples A and C.
• This cytometer has a poor linear range, with >2% deviation from
linearity above the Max Linearity Channel at 50,000.
Conclusions
• Multiple tools available from BD for reagent selection
(BD FACSelect™ Multicolor Panel Designer,
Fluorescence Spectrum Viewer, TDS).
• CS&T is a unique system for instrument
characterization, setup, and monitoring.
• Application settings are a powerful tool that enhances
data quality (sensitivity) and reproducibility.
• Reproducible setup across instruments is a reality.
• Data sets are more predictable and therefore more
amenable for automated data analysis.
Tools section of
bdbiosciences.com/colors
Step 1: Select Target Species
Step 2: Select Markers
List markers by
specificity or clone
Add additional markers
by clicking on the
Acknowledgements
•
•
•
•
•
•
•
Mark Edinger
Ming Yan
Alan Stall
Joe Trotter
Skip Maino
Margaret Inokuma
Bob Hoffman
•
•
•
•
•
•
•
Pat Collins
Joerg Hildmann
Holden Maecker
Mirion Schultz
Barny Abrams
Laurel Nomura
Dennis Sasaki
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For More Information….
If you have further questions:
Contact Technical Service (US) at:
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or email: [email protected]
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