CYTO 2010
Panel Design:
“Multi-Sizing” Your Multi-Color
William Godfrey, Ph.D.
Manager, Reagent & Application Development
Beckman Coulter, Miami
For more information:
www.CoulterFlow.com
“Multi-Sizing” into Your Multi-Color
•
•
•
•
Fluor selection
Tandem dyes
Considerations for cocktail design
Krome Orange™ - A new violet fluor
How Many Colors Needed?
CD4-PE
Side Scatter
TetraCHROME™ CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 with normal blood
CD45-FITC
CD3-PC5
How Many Colors Needed?
CD25
Forward Scatter
Side Scatter
Side Scatter
T-reg cells: CD4+
CD25+
FoxP3+
CD127lo/-
FoxP3
CD4
CD127
Up to 6-8 colors with addition of CD45 and markers for
other leukocyte subpopulations
CD4
High Complexity Multi-Color Flow
Benefits
Correlated Data:
Labor Efficiency:
Defined Populations
Fewer Steps

Single cell interrogation / multiple markers

Higher throughput / fewer tubes

Expanded utility (Research / Clinical)

Minimize sample volume
Challenges

More Colors = Greater Complexity

Requires optimization

Complex antibody combinations

Requires greater expertise

Selecting fluorochromes that work together
Requires
validation
Prerequisites and Pitfalls for 8+ Colors
• Specific / avid monoclonal antibodies
(clone selection)
CD56-PE
MY31 Clone
CD56-PE
NKH-1 Clone
• Bright fluorochromes (high extinction
coefficients; high quantum yields) with
range of Stoke’s shifts
• “Well-behaved” conjugates (stable
binding; low spectral overlap, low
background)
• Higher-plex flow cytometers with
efficient light paths
• BD Canto™ cytometer (8-colors)
CD56-APC-AF750
CD56-PE
CD3
ECD
CD3
PC5
CD3
PC7
• BC Cyan™ cytometer (9-colors)
• BC GALLIOS™ cytometer (10-colors)
Monocyte binding
of Cyanine dye
Fluorochrome Landscape
Instrument Optics
• Filter Selection
• PMT Sensitivity
• Laser Power
Intrinsic Characteristics
• Extinction Coefficient
• Quantum Yield
• Emission Spectral Overlap
Excitation
Comparative Intensities of CD8 Conjugates
Blue
FITC

Red
APC
ECD

Alexa 700
PC5
PECy5.5

APCAlexa 700
APC-H7

APCAlexa 750

Pacific Blue
Violet
PE
()
Pacific Orange
+Qdot Nanocrystals
Brighter Fluors
PC7
()
Dye Options: 10-Colors
APC or AF647
APC-AF700
APCCy7 or APC-AF750
635nm
FITC
PE
ECD
PECy5.5
PECy7
488nm
Pacific Blue
Pacific Orange
405nm
400
500
600
700
800
Dye Options: Conventional Fluors
APC or AF647
635nm
FITC
PE
488nm
Pacific Blue
Pacific Orange
405nm
400
500
600
700
800
Spectra of Common Fluorochromes
R-PE(565/576), Є = 2,000 K
• Critical Fluorescence Properties
Stokes
Shift
• Extinction Coefficient
• Quantum Efficiency
• Stoke’s Shift
488
• Consider
• Available excitations
• Emission filters
FITC (495/518) Є = 78 K
APC: (650/662), Є = 700 K
Stokes
Shift
488
Stokes
Shift
635
Conjugation Chemistry
Brightness: Optimization of F/P molar ratio
• Minimize impact on antibody binding affinity
• Maximize fluorescence at saturation dosing
Performance: Influenced by multiple factors
• Site of covalent linkage to the antibody
- Fc – minimal impact on binding affinity
- F(ab) Region – competition with antigen binding
• Molecular weight (size) of dye molecule
• Hyperconjugation
- Fluorescence quenching due to close coupling proximity
- Non-specific binding
- Dye/Cell aggregation
Performance Impact: Organic Dye Ratio
CD4-Alexa Fluor 488
CD3-Alexa Fluor 488
F/P 8.4
F/P 6.8
F/P 5.2
F/P 3.5
F/P 1.8
F/P 16.3
F/P 13.1
F/P 9.1
F/P 6.4
F/P 3.4
500
25
250
20
200
25
Flow Cytometry
15
200
10
100
5
0
0.00
5.00
10.00
15.00
CD4 F/P Molar Ratio
0
20.00
Flow Cytometry
20
150
15
100
10
50
5
0
0
2
4
6
CD3 F/P Molar Ratio
8
0
10
Flow MFI
300
Emission Intensity
Fluorimeter
Flow MFI
Emission Intensity
Fluorimeter
400
Dye Options: Tandem Dyes
APC-AF700
APCCy7 or APC-AF750
635nm
ECD
PECy5.5
PECy7
488nm
405nm
400
500
600
700
800
Fluorescence Resonance Energy Transfer
• Definition
•
•
•
•
Excitation is transferred from donor to acceptor without emission of
a photon.
Donor / acceptor molecules must be in close proximity (10–100 Å).
Absorption spectrum of the acceptor must overlap emission
spectrum of the donor
Donor and acceptor transition dipole orientations must be
approximately parallel.
• Advantages
•
•
Fluorescence Intensity
•
Expands fluorochrome choices from single laser source
Minimize cost and complexity of instrumentation – up to 8 colors
using 2 lasers
Enhanced fluorescence intensity compared to organic dyes with
equivalent emission
Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002
488 nm
PE
Texas Red
Wavelength (nm)
Fluorescence Resonance Energy Transfer
• Definition
•
•
•
•
Excitation is transferred from donor to acceptor without emission of
a photon.
Donor / acceptor molecules must be in close proximity (10–100 Å).
Absorption spectrum of the acceptor must overlap emission
spectrum of the donor
Donor and acceptor transition dipole orientations must be
approximately parallel.
• Advantages
•
•
•
Expands fluorochrome choices from single laser source
Minimize cost and complexity of instrumentation – up to 8 colors
using 2 lasers
Enhanced fluorescence intensity compared to organic dyes with
equivalent emission
• Limitations
•
•
Lot-to-lot variation
400
350
300
- Fluorescence sensitivity
250
- Energy transfer efficiency
200
- Non-specific binding to myeloid
populations
150
Stability: Photostability & Chemical interactions
Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002
PE-Cy7 Conjugates
Multiple Vendors,
Multiple Conjugates
100
50
0
550
650
750
850
Patented Tandem Dye Process
Native State
Phycobiliprotein
Unfold
Protein
Couple
Acceptor Dye
Refold to Native
State
Conjugation process delivers optimum fluorescence intensity
600
1.2
500
1.0
400
0.8
300
0.6
200
0.4
Optimal
Ratio
100
Cross-Over Ratio
Dye Coupling
Step
Cy7 Fluorescence Intensity
Patented Tandem Dye Process
Finished Tandem Dye
Three Lot Comparison of PC5
Process controls variability
0.2
0.0
0
2
3
4
5
6
7
250
Fluorescence Intensity
Cy7/PE (A/D) Ratio
HIC
Purification
Step
200
Lot # 3021-101
Lot # 1828-47
Lot # 1828-95
150
100
50
0
550
PC7 Pre-HIC
purification
HIC discarded
fraction
PC7 Post-HIC
purification
600
650
700
Emission Wavelength (nm)
750
800
Impact on Compensation
BCI
BCI
Vendor 2
Under
Vendor 2
Over
MUST treat different vendor tandems as different fluorochromes
for compensation set-up!
Tandem Dye Selection: Dual Laser
Emission Intensity
700
600
47.5%
Comp
500
400
300
200
100
0
500
700
Emission Intensity
PECy5
488nm excitation
633nm excitation
APC
600
600
700
Wavelength (nm )
800
PECy5.5
488nm excitation
633nm excitation
APC
1.5%
Comp
500
400
300
200
100
0
500
600
700
Wavelength (nm )
800
Minimized Spectral Overlap = Better Resolution
APC-Alexa Fluor® 750 Photostability
2 Hours
6 Hours
280
240
200
160
120
CD3-APC-Cy7
80
FL4-FL5 Compensation = 10.5%
40
0
0
6
12
18
24
Time (hours) Exposed to Light (80 Lumens)
APC
% Change FL4/FL5 MFI Ratio
0 Hours
CD3-APC-AF 750
FL4-FL5 Compensation = 10.5%
Enhanced on photo-stability of APC-Alexa Fluor 750
conjugate regardless of paraforaldehyde
Performance Impact: Antibody & Dye
Non-Specific Binding
Binding due to
Fc receptor
Binding due to
Cy dye binding
CD14-PECy5
CD3-PECy5
Conjugate/Dye Aggregation
0.25 µg
CD15-FITC (IgM)
0.125 µg
0.063 µg
0.031 µg
Proprietary Chemistry / Enhanced Specificity
Pre-Formulation
Post-Formulation
Monocyte
binding of
Cyanine dye
Beckman Coulter
CD3
ECD
CD3
PC5
CD3
PC7
CD3
PE-Cy5
CD3
PE-Cy7
CD3
PE-Cy5
CD3
PE-Cy7
Company “B”
• Low background fluorescence on
negative populations
• Optimal signal to noise
• Low affinity binding of cyanine dyes to
monocyte populations eliminated
Company “C”
CD3
PE-TxRed
Fluor Choice?
• Detection of Surface Antigens
• Large selection of conjugates
• Limited by detection sensitivity and proximity of coexpressed antigens
• Tandem dyes provide  sensitivity over organic dyes
• FITC, Alexa Fluor dyes, violet-excited dyes
- Constitutively expressed antigens
- Subset gating
• Detection of Intracellular Antigens
• Cytoplasmic antigens
- Phycobiliproteins and organic dyes may be better
- Alexa Fluor 488 better than FITC – lower background
• Nuclear antigens
- Phycobiliproteins or tandem dyes hindered due to
conjugate size?
- Close proximity can lead to FRET between dyes
Instrument Contributions
• Channels available
• Sensitivity in channels
Comparative Flurochrome Sensitivity between Platforms
800
700
FITC
PE
ECD
PECY5
PECY5.5
PECY7
APC
Alexa Fluor 700
APCA700
APCA750
Pacific Blue
Pacific Orange
GALLIOS™ cytometer
• 3 lasers
• 10 colors
Signal to Noise
600
500
400
300
200
100
0
GALLIOS
CRS
FC500
FC500
Optimizing the Combination
• Determine fluorochrome/conjugate strategy
• Organic dyes to maximize spectral separation for gating reagents
• PE & APC used for antigens with continuum of expression
• Tandems dyes for mid-density → bright antigens
• Perform titration curves for each conjugate
• Determine Signal/Noise Ratio
• Choose optimal dose: Saturation, Highest S/N
Low density Ag/
Bright dye
• Prepare combination, verify performance
CDz
• Always use controls – approach can vary
- Negative Control
-
Internal negative population
FMO
Isotype controls
- Positive controls
-
Each antibody as single color
Known positive control material
• Evaluate performance for major interactions
High density Ag/
Dim dye
CDx
What Can Go Wrong?
• Potential conjugate interactions
• Non-specific binding
- Aggregate formation between conjugates
- Cyanine & Alexa Fluor dye binding to myeloid populations
• Steric Hindrance
Charge
- Ligand – receptor binding blocked
due to physical interference
• Fluorescence Quenching
- Over conjugation of antibody
- Concentration and proximity on the cell
• FRET Potential
Hyperconjugation
Size
Concentration &
Proximity
Dose Optimization: Multi-Step Process
Single color titrations:
• Optimal S/N
• Saturation binding when possible
CD127-APC
20.0
Fluorescencence
Fluorescencence
20.0
15.0
S/N
Positive MFI
Negative MFI
10.0
5.0
15.0
S/N
Positive MFI
Negative MFI
10.0
5.0
0.0
0.0
0
0.2
0.4
0.6
0.8
1
0
0.2
0.6
0.8
1
Dose (ug/test)
Dose (ug/test)
Combination Matrix to finalize dosing
• Target optimal S/N dose for each component
• Evaluate for potential interactions
• Evaluate multiple doses:
Simple matrix or DOE
0.4
CDxx
CDyy
2X
1x
½x
2X
2,2
2,1
2, ½
1x
1,2
1,1
1,½
½X
½, 2
½, 1
½, ½
Spectral Overlap
Impact on co-expressed antigens
PE ECD
PC7
CD45-ECD vs CD45-PC7 Overlap into PE
S/N=22
S/N=19
• Spectral Overlap/Compensation
• Loss of low end resolution
• Display artifacts
CD45-ECD
• If bright signal overlaps into
PMT containing dimmer signal
• Increased “noise”
• Spread of the negative population
• Difficulty in accurate determination of
low level positivity
CD7-PE
S/N=38
CD45-PC7
CD7-PE
CD19-PE
S/N=27
CD19-PE
Effect of Antigen Proximity
• APC conjugates of CD3, CD8, and CD45 versus PE-labeled tetramer
• FRET from PE to APC results in FL3 signal (PerCP channel)
• CD3 & CD8 close to TCR; CD45 antigen spatially separated from TCR
CD3 APC Gated
CD8 APC Gated
A2/CMV - PE
+ CD4 PerCP
No CD4 PerCP (PE-APC FRET)
Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002
CD45 APC Gated
Interferants: Washing
Kappa/Lambda Resolution
• Requires high sensitivity
• Dependant on sample preparation methodology
• Pre-wash required to remove plasma immunoglobulins
1x
2x
3x
Beckman Coulter Solastra™ Panels*
B-cell Kit
FITC
PE
ECD
PC5.5
PC7
Kappa Lambda CD19 CD5
CD45
CD20
CD10
CD19 CD38
CD45
CD2
CD56
CD7
CD5
CD45
CD8
CD4

CD3
CD45
CD15
CD11b
T-cell Kit
Myeloid Kit
CD16 CD14
CD45
HLADR CD56
CD34 CD117
CD45
CD7
CD34 CD33
CD45
CD13
Aligned with Bethesda Recommendations
* Not available for sale in US
Peripheral Blood: Solastra B-cell Kits
B-CLL #1:
CD45++/CD19+/CD5+/-/
CD20++/Kappabright+
Tube 1
Tube 2
New Violet-Excitable Dye
• GALLIOS™ Configuration:
3 laser 10 color instrument
• 405nm laser – 2 colors
• 488nm laser – 5 colors
• 635nm laser – 3 colors
• Krome Orange™ dye
Second violet-excitable
fluor to pair with Pacific
Blue™ dye
See Poster #P346
Krome Orange Spectrum
405 nm
550/40
100%
Relative Intensity
80%
KO
V500
Pac Or
AmCyan
60%
40%
20%
0%
350
400
450
500
550
600
Wavelength (nm)
650
700
750
CD45-Krome
Orange Titration
Krome Orange
Conjugation
CD45-Krome Orange Titration Curve
Median Fluorescence
30
F:P 17.3
F:P 15.9
F:P 14.0
F:P 10.8
25
F:P =
F:P =
F:P =
F:P =
F:P =
20
15
F:P 6.1
10
6.1
10.8
14.0
15.9
17.3
SI = 51.2
Lymphocytes
5
0
0.00
0.50
1.00
1.50
2.00
2.50
µg Conjugate per Test
SI = 101.3
CD20 (B9E9)-Krome Orange
SI = 18.6
SI = 9.2
Lymphocytes
Lymphocytes
Lymphocytes
Monocytes
CD14 (RMO52)-Krome Orange CD16 (3G8)-Krome Orange
CD19 (J4.119)-Krome Orange
Krome Orange vs Other Violet Fluors
CD3
Krome Orange Dye
(550/40)
(standard FL10
filter on Gallios
cytometer)
UCHT1
CD4
SI = 33.2
13B8.2
SI = 36.6
CD8
CD45
B9.11
SI = 81.7
J.331
23.20
29.65
6.75
7.16
Pacific Orange Dye
(575/26)
UCHT1
SI = 26.1
S3.5
SI = 13.5
3B5
SI = 34.7
HI30
16.03
9.92
5.19
AmCyan
(525/50)
SK71
SI = 43.5
2.92
SK3
SI = 20.6
4.19
SK1
SI = 48.5
13.32
8.67
V500 Dye
(525/50)
RPA-T8
3.54
SI = 12.0
2D1
21.20
Krome Orange vs Other Violet Fluors
Relative compensation values
FL10-%FL9 = 5.9%
0.0%
1.5%
CD4-Pacific Orange
FL9-%FL10 = 0.5%
CD19-PE
CD19-PE
19.4%
1.0%
CD8-Fluorescein
CD4-Pacific Orange
CD19-PE
0.0%
1.0%
CD8-Fluorescein
0.3%
1.0%
19.4%
1.0%
CD19-PE
FL10-%FL9 = 5.8%
0.0%
1.5%
CD4-Krome Orange
FL9-%FL10 = 0.0%
CD4-Krome Orange
Krome Orange: 4-Color Stain
CD8-Fluorescein
CD8-Fluorescein
Lymphs
Side Scatter
Monos
Monos
Side Scatter
Grans
CD45-Pacific Orange
Side Scatter
Krome Orange: 10-Color Stains
CD3+
CD3+
CD3-APC
CD3-APC
Lymphs
Monos
Gated on CD3+
CD8-Pacific Blue
CD45-Krome Orange
Gated on CD3+
CD4-Pacific Orange
Monos
CD45-Krome Orange
Side Scatter
Grans
CD4-Krome Orange
CD14-PC5
CD45-Pacific Orange
CD8-Pacific Blue
CD14-PC5
FL9 - %FL10
Krome Orange™
1.5
Pacific Orange™
0.0
FL10 - %FL9
9.1
9.3
CD45-ECD
CD45-Pacific Orange
CD45-V500
CD45-Krome Orange
Krome Orange: CD45/Side Scatter
Data courtesy of F. Preijers, Radboud University Nijmegen Medical Center, The Netherlands
Summary
• Multi-parametric flow analysis provides a powerful tool
• Dissection of complex cell populations
• Identification of underlying mechanisms and alterations in
disease states
• Increased efficiency in laboratory testing
• Optimal design is critical for scientifically valid results
• Match fluorochrome choices to the platform capability
• Optimize sensitivity by pairing dye intensity with antigen
density
•  colors =  complexity
• Violet-excited fluors can easily add 2 parameters
• Validate, validate, and validate your application
specificity prior to initiating studies
Acknowledgements
Miami: Reagent Development
Ravinder Gupta
Sireesha Kaanumalle
David Bloodgood
Meryl Foreman
Jeffrey Cobb
Marseille: Reagent Development
Laura Nieto Gligorovsky
Franck Gaille
Emmanuel Gautherot
Felix Montero
Detroit: Organic Chemistry
Hashem Akhavan-Tafti
Robert Eickholt
Mark Sandison
Rhonda Federspiel
Collaboration
Frank Preijers
(Nijmegen Medical Center)
www.CoulterFlow.com
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