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Panel Design: “Multi-Sizing” Your Multi-Color
CYTO 2010 Panel Design: “Multi-Sizing” Your Multi-Color William Godfrey, Ph.D. Manager, Reagent & Application Development Beckman Coulter, Miami For more information:
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“Multi-Sizing” into Your Multi-Color
Fluor selection Tandem dyes Considerations for cocktail design Krome Orange™ - A new violet fluor
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How Many Colors Needed? TetraCHROME™ CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 with normal blood Side Scatter CD4-PE CD45-FITC CD3-PC5 3
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How Many Colors Needed? T-reg cells: CD4+ CD25+ FoxP3+ CD127lo/-
Side Scatter Side Scatter Forward Scatter CD25 CD4 FoxP3 CD127 Up to 6-8 colors with addition of CD45 and markers for other leukocyte subpopulations CD4 4
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High Complexity Multi-Color Flow
Benefits Correlated Data: Defined Populations Labor Efficiency: Fewer Steps Single cell interrogation / multiple markers Expanded utility (Research / Clinical) Higher throughput / fewer tubes Minimize sample volume Challenges More Colors = Greater Complexity Complex antibody combinations Selecting fluorochromes that work together Requires optimization Requires greater expertise Requires validation
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Prerequisites and Pitfalls for 8+ Colors
Specific / avid monoclonal antibodies (clone selection) 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) BC Cyan™ cytometer (9-colors) BC GALLIOS™ cytometer (10-colors) CD56-PE MY31 Clone CD56-PE NKH-1 Clone CD56-PE CD56-APC-AF750 CD3 ECD CD3 PC5 CD3 PC7 Monocyte binding of Cyanine dye 6
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Fluorochrome Landscape
Intrinsic Characteristics Extinction Coefficient Quantum Yield Emission Spectral Overlap Instrument Optics Filter Selection PMT Sensitivity Laser Power Excitation Comparative Intensities of CD8 Conjugates FITC PE ECD PC5 PECy5.5 PC7 Blue () Brighter Fluors APC Alexa 700 APCAlexa 700 APC-H7 APCAlexa 750 Red Pacific Blue Pacific Orange Violet +Qdot Nanocrystals
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Dye Options: 10-Colors 635nm 488nm 405nm 400 500 600 700 800
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
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Dye Options: Conventional Fluors
APC or AF647 635nm FITC PE 488nm Pacific Blue Pacific Orange 405nm 400 500 600 700 800
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Spectra of Common Fluorochromes
R-PE(565/576), Є = 2,000 K Critical Fluorescence Properties Extinction Coefficient Quantum Efficiency Stoke’s Shift Consider Available excitations Emission filters Stokes Shift 488 FITC (495/518) Є = 78 K APC: (650/662), Є = 700 K Stokes Shift Stokes Shift 488 635
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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
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Performance Impact: Organic Dye Ratio
F/P 3.4 F/P 13.1 F/P 9.1 F/P 6.4 F/P 16.3 CD4-Alexa Fluor 488 F/P 1.8 F/P 6.8 F/P 5.2 F/P 3.5 F/P 8.4 CD3-Alexa Fluor 488
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Dye Options: Tandem Dyes
APC-AF700 APCCy7 or APC-AF750 635nm ECD PECy5.5 PECy7 488nm 405nm 400 500 600 700 800
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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 Wavelength (nm) Fluorescence Intensity 488 nm PE Texas Red Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002
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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 PE-Cy7 Conjugates Multiple Vendors, Multiple Conjugates Limitations Lot-to-lot variation Fluorescence sensitivity Energy transfer efficiency Non-specific binding to myeloid populations Stability: Photostability & Chemical interactions Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002
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Patented Tandem Dye Process
Native State Phycobiliprotein Unfold Protein Couple Acceptor Dye Refold to Native State Conjugation process delivers optimum fluorescence intensity
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Patented Tandem Dye Process
0.0 0.2 0.4 0.6 0.8 1.0 1.2 Cross-Over Ratio Dye Coupling Step Optimal Ratio Fluorescence Intensity Emission Wavelength (nm) Finished Tandem Dye Three Lot Comparison of PC5 Process controls variability HIC Purification Step PC7 Pre-HIC purification PC7 Post-HIC purification HIC discarded fraction Two step process: Optimized coupling of the donor to acceptor dye Purification of heterogeneous dye mixture Graphs show the relationship of the A/D ratio on the dye fluorescence and energy transfer efficiency Optimization requires a balance between these two parameters Highest energy transfer = lowest Fluorescence Highest fluorescence = lowest energy transfer
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Impact on Compensation
BCI Vendor 2 BCI Vendor 2 Under Over MUST treat different vendor tandems as different fluorochromes for compensation set-up!
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Tandem Dye Selection: Dual Laser
47.5% Comp 1.5% Comp Minimized Spectral Overlap = Better Resolution
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APC-Alexa Fluor® 750 Photostability
0 Hours 2 Hours 6 Hours FL4-FL5 Compensation = 10.5% CD3-APC-AF 750 APC CD3-APC-Cy7 Enhanced on photo-stability of APC-Alexa Fluor 750 conjugate regardless of paraforaldehyde
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Performance Impact: Antibody & Dye
Non-Specific Binding CD14-PECy5 CD3-PECy5 Binding due to Fc receptor Binding due to Cy dye binding Conjugate/Dye Aggregation 0.25 µg 0.125 µg 0.063 µg 0.031 µg CD15-FITC (IgM)
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Proprietary Chemistry / Enhanced Specificity
Monocyte binding of Cyanine dye Pre-Formulation Post-Formulation Beckman Coulter CD3 ECD CD3 PC5 CD3 PC7 Company “B” CD3 PE-Cy5 CD3 PE-Cy7 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 CD3 PE-Cy5 CD3 PE-Cy7
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Fluor Choice? Detection of Surface Antigens
Large selection of conjugates Limited by detection sensitivity and proximity of co-expressed 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
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Instrument Contributions
Channels available Sensitivity in channels GALLIOS™ cytometer 3 lasers 10 colors GALLIOS FC500 24
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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 Prepare combination, verify performance 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 Low density Ag/ Bright dye CDz High density Ag/ Dim dye CDx
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Concentration & Proximity
What Can Go Wrong? Potential conjugate interactions Non-specific binding Aggregate formation between conjugates Cyanine & Alexa Fluor dye binding to myeloid populations Steric Hindrance Ligand – receptor binding blocked due to physical interference Fluorescence Quenching Over conjugation of antibody Concentration and proximity on the cell FRET Potential CDz CDx Charge CDz CDx Size Hyper-conjugation Concentration & Proximity
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Dose Optimization: Multi-Step Process
Single color titrations: Optimal S/N Saturation binding when possible Combination Matrix to finalize dosing Target optimal S/N dose for each component Evaluate for potential interactions Evaluate multiple doses: Simple matrix or DOE CDxx 2X 1x ½ x CDyy 2,2 2,1 2, ½ 1,2 1,1 1,½ ½ X ½, 2 ½, 1 ½, ½
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CD45-ECD vs CD45-PC7 Overlap into PE
Spectral Overlap Impact on co-expressed antigens PE ECD PC7 CD45-ECD vs CD45-PC7 Overlap into PE Spectral Overlap/Compensation Loss of low end resolution Display artifacts If bright signal overlaps into PMT containing dimmer signal Increased “noise” Spread of the negative population Difficulty in accurate determination of low level positivity
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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 CD45 APC Gated + CD4 PerCP A2/CMV - PE No CD4 PerCP (PE-APC FRET) Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002
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Interferants: Washing
Kappa/Lambda Resolution Requires high sensitivity Dependant on sample preparation methodology Pre-wash required to remove plasma immunoglobulins 1x 2x 3x
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Beckman Coulter Solastra™ Panels*
FITC PE ECD PC5.5 PC7 Kappa Lambda CD19 CD5 CD45 CD20 CD10 CD19 CD38 CD45 B-cell Kit T-cell Kit CD2 CD56 CD7 CD5 CD45 CD8 CD4 CD3 CD45 Myeloid Kit CD15 CD11b CD16 CD14 CD45 HLADR CD56 CD34 CD117 CD45 CD7 CD13 CD34 CD33 CD45 Aligned with Bethesda Recommendations * Not available for sale in US
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Peripheral Blood: Solastra B-cell Kits
B-CLL #1: CD45++/CD19+/CD5+/-/ CD20++/Kappabright+ Tube 1 Tube 2
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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
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Krome Orange Spectrum
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Krome Orange Conjugation
CD14 (RMO52)-Krome Orange SI = 101.3 Lymphocytes Monocytes CD16 (3G8)-Krome Orange SI = 18.6 CD20 (B9E9)-Krome Orange SI = 51.2 SI = 9.2 CD19 (J4.119)-Krome Orange
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Krome Orange vs Other Violet Fluors
UCHT1 13B8.2 B9.11 J.331 UCHT1 S3.5 3B5 HI30 SK71 SK3 SK1 2D1 RPA-T8
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Krome Orange vs Other Violet Fluors
Relative compensation values
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Krome Orange: 4-Color Stain
0.0% 0.0% 19.4% FL9-%FL10 = 0.0% FL10-%FL9 = 5.8% 1.5% 1.0% 1.0% 0.0% 0.3% 19.4% 1.5% 1.0% 1.0% FL9-%FL10 = 0.5% FL10-%FL9 = 5.9%
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Krome Orange: 10-Color Stains
CD3-APC Side Scatter Grans Monos Side Scatter Side Scatter CD45-Pacific Orange Monos CD3+ CD3+ Lymphs CD3-APC CD45-Pacific Orange CD14-PC5 Gated on CD3+ Gated on CD3+ Grans Monos CD4-Krome Orange CD4-Pacific Orange Side Scatter CD45-Krome Orange Monos Lymphs CD8-Pacific Blue CD8-Pacific Blue CD45-Krome Orange CD14-PC5 FL9 - %FL10 FL10 - %FL9 Krome Orange™ Pacific Orange™
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Krome Orange: CD45/Side Scatter
CD45-ECD CD45-Pacific Orange CD45-V500 CD45-Krome Orange Data courtesy of F. Preijers, Radboud University Nijmegen Medical Center, The Netherlands
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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
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Acknowledgements www.CoulterFlow.com
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) Research Tools On-Line Knowledge Base Spectrios Experiment Designer Practical Flow Cytometry Howard Shapiro History of Flow Cytometry
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Bone Yard
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Lymphocytic differentiation
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Tandem Dye Selection: Alexa Fluor® 700
635 nm MFI: 14.7 MFI: 44.7 CD8-APC-AF700 CD8-AF700 APC Alexa Fluor 700 Fluorescence Intensity Wavelength (nm) Optimal excitation of Alexa Fluor 700 by APC energy transfer = ½ to ¾ log brighter fluorescence
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SI = 101.3 SI = 9.2 CD14 (RMO52)-Krome Orange
Lymphocytes Monocytes SI = 9.2 Lymphocytes CD19 (J4.119)-Krome Orange CD20 (B9E9)-Krome Orange SI = 51.2 Lymphocytes CD16 (3G8)-Krome Orange SI = 18.6 Lymphocytes
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