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Panel Design: Multi-Sizing Your Multi-Color William Godfrey, Ph.D. Manager, Reagent & Application Development Beckman Coulter, Miami CYTO 2010 For more information: www.CoulterFlow.com Slide 2 Multi-Sizing into Your Multi-Color Fluor selection Tandem dyes Considerations for cocktail design Krome Orange - A new violet fluor Slide 3 How Many Colors Needed? CD3-PC5 CD4-PE CD45-FITC Side Scatter TetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 with normal blood Slide 4 How Many Colors Needed? T-reg cells:CD4 + CD25 + FoxP3 + CD127 lo/- Up to 6-8 colors with addition of CD45 and markers for other leukocyte subpopulations CD4 Side Scatter Forward Scatter Side Scatter CD127 CD25 CD4 FoxP3 Slide 5 Labor Efficiency: Fewer Steps Correlated Data: Defined Populations High Complexity Multi-Color Flow Benefits 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 Slide 6 Prerequisites and Pitfalls for 8+ Colors Specific / avid monoclonal antibodies (clone selection) Bright fluorochromes (high extinction coefficients; high quantum yields) with range of Stokes 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 Monocyte binding of Cyanine dye CD3 ECD CD3 PC5 CD3 PC7 CD56-PE CD56-APC-AF750 Slide 7 Fluorochrome Landscape Intrinsic Characteristics Extinction Coefficient Quantum Yield Emission Spectral Overlap Instrument Optics Filter Selection PMT Sensitivity Laser Power Comparative Intensities of CD8 Conjugates FITC PE ECD PC5 PECy5.5 PC7 APC Alexa 700 APCAlexa 700 APC-H7 APCAlexa 750 Pacific Blue Pacific Orange Excitation Blue Red Violet +Qdot Nanocrystals ( ) ( ) Brighter Fluors Slide 8 Dye Options: 10-Colors 405nm Pacific Blue Pacific Orange 488nm 635nm FITC PE ECD PECy7 PECy5.5 APC or AF647 APC-AF700 APCCy7 or APC-AF750 400500600700 800 Slide 9 Dye Options: Conventional Fluors 405nm Pacific Blue Pacific Orange 488nm 635nm FITC PE APC or AF647 400500600700 800 Slide 10 R-PE(565/576), = 2,000 K APC: (650/662), = 700 K Stokes Shift FITC (495/518) = 78 K Spectra of Common Fluorochromes Critical Fluorescence Properties Extinction Coefficient Quantum Efficiency Stokes Shift Consider Available excitations Emission filters Stokes Shift 488 635 Slide 11 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 Slide 12 F/P 1.8 F/P 6.8 F/P 5.2 F/P 3.5 F/P 8.4 CD3-Alexa Fluor 488 F/P 3.4 F/P 13.1 F/P 9.1 F/P 6.4 F/P 16.3 CD4-Alexa Fluor 488 Performance Impact: Organic Dye Ratio Slide 13 Dye Options: Tandem Dyes 405nm 488nm 635nm ECD PECy7 PECy5.5 APC-AF700 APCCy7 or APC-AF750 400500600700 800 Slide 14 Definition Excitation is transferred from donor to acceptor without emission of a photon. Donor / acceptor molecules must be in close proximity (10100 ). 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 Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002 Fluorescence Resonance Energy Transfer Wavelength (nm) Fluorescence Intensity 488 nm PE Texas Red Slide 15 Definition Excitation is transferred from donor to acceptor without emission of a photon. Donor / acceptor molecules must be in close proximity (10100 ). 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 Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002 Fluorescence Resonance Energy Transfer Limitations Lot-to-lot variation -Fluorescence sensitivity -Energy transfer efficiency -Non-specific binding to myeloid populations Stability: Photostability & Chemical interactions PE-Cy7 Conjugates Multiple Vendors, Multiple Conjugates Slide 16 Patented Tandem Dye Process Native State Phycobiliprotein Unfold Protein Couple Acceptor Dye Refold to Native State Conjugation process delivers optimum fluorescence intensity Slide 17 HIC Purification Step PC7 Pre-HIC purification PC7 Post-HIC purification HIC discarded fraction 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 Patented Tandem Dye Process Slide 18 MUST treat different vendor tandems as different fluorochromes for compensation set-up! BCI Vendor 2 BCI Vendor 2 Under Over Impact on Compensation Slide 19 1.5% Comp 47.5% Comp Tandem Dye Selection: Dual Laser Minimized Spectral Overlap = Better Resolution Slide 20 0 Hours 2 Hours6 Hours FL4-FL5 Compensation = 10.5% CD3-APC-AF 750 APC FL4-FL5 Compensation = 10.5% CD3-APC-Cy7 APC-Alexa Fluor 750 Photostability Enhanced on photo-stability of APC-Alexa Fluor 750 conjugate regardless of paraforaldehyde Slide 21 Performance Impact: Antibody & Dye Non-Specific Binding Conjugate/Dye Aggregation CD14-PECy5 CD3-PECy5 Binding due to Fc receptor Binding due to Cy dye binding CD15-FITC (IgM) 0.25 g0.125 g0.063 g0.031 g Slide 22 Monocyte binding of Cyanine dye Pre-FormulationPost-Formulation Company B Company C Low background fluorescence on negative populations Optimal signal to noise Low affinity binding of cyanine dyes to monocyte populations eliminated Proprietary Chemistry / Enhanced Specificity CD3 PE-TxRed CD3 PE-Cy5 CD3 PE-Cy7 CD3 PE-Cy5 CD3 PC7 CD3 PC5 CD3 ECD Beckman Coulter Slide 23 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 Slide 24 Instrument Contributions Channels available Sensitivity in channels GALLIOSFC500 GALLIOS cytometer 3 lasers 10 colors Slide 25 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 High density Ag/ Dim dye CDx Low density Ag/ Bright dye CDz Slide 26 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 Slide 27 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 2X1x x CDyy 2X2,22,12, 1x1,21,11, X, 2 , 1 , Slide 28 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 PEECDPC7 CD45-ECD vs CD45-PC7 Overlap into PE Impact on co-expressed antigens Spectral Overlap Slide 29 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 Sakeenah Hicks, Chris Ibegbu, John Altman, February 19, 2002 A2/CMV - PE + CD4 PerCP No CD4 PerCP (PE-APC FRET) CD3 APC GatedCD8 APC GatedCD45 APC Gated Effect of Antigen Proximity Slide 30 Interferants: Washing Kappa/Lambda Resolution Requires high sensitivity Dependant on sample preparation methodology Pre-wash required to remove plasma immunoglobulins 1x 2x 3x Slide 31 Beckman Coulter Solastra Panels* Aligned with Bethesda Recommendations * Not available for sale in US Aligned with Bethesda Recommendations * Not available for sale in US FITCPEECDPC5.5PC7 KappaLambda CD19CD5CD45 CD20CD10CD19CD38CD45 CD2CD56CD7CD5CD45 CD8CD4 CD3CD45 CD15CD11bCD16CD14CD45 HLADRCD56CD34CD117CD45 CD7CD13CD34CD33CD45 B-cell Kit T-cell Kit Myeloid Kit Slide 32 Peripheral Blood: Solastra B-cell Kits B-CLL #1: CD45 ++ /CD19 + /CD5 +/- / CD20 ++ /Kappa bright+ Tube 2 Tube 1 Slide 33 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 Slide 34 Krome Orange Spectrum Slide 35 Krome Orange Conjugation CD14 (RMO52)-Krome Orange SI = 101.3 Lymphocytes Monocytes CD16 (3G8)-Krome Orange SI = 18.6 Lymphocytes CD20 (B9E9)-Krome Orange SI = 51.2 Lymphocytes SI = 9.2 Lymphocytes CD19 (J4.119)-Krome Orange Slide 36 Krome Orange vs Other Violet Fluors UCHT1 SK71 SK3 S3.5 13B8.2 B9.11 3B5 SK1 RPA-T8 2D1 HI30 J.331 Slide 37 Krome Orange vs Other Violet Fluors Relative compensation values Slide 38 Krome Orange: 4-Color Stain 0.0% 1.5% 0.3% 1.0% 19.4% 1.0% 0.0% 1.5% 0.0% 1.0% 19.4% 1.0% FL9-%FL10 = 0.5% FL10-%FL9 = 5.9% FL9-%FL10 = 0.0% FL10-%FL9 = 5.8% Slide 39 CD8-Pacific Blue CD4-Krome Orange Gated on CD3+ CD3+ Side Scatter CD3-APC Side Scatter CD8-Pacific Blue CD4-Pacific Orange Gated on CD3+ CD3+ CD3-APC CD45-Krome Orange Lymphs Monos Grans CD14-PC5 CD45-Pacific Orange CD14-PC5 CD45-Krome Orange Monos CD45-Pacific Orange Lymphs Monos Grans Monos FL9 - %FL10FL10 - %FL9 Krome Orange1.59.1 Pacific Orange0.09.3 Side Scatter Krome Orange: 10-Color Stains Slide 40 Krome Orange: CD45/Side Scatter Data courtesy of F. Preijers, Radboud University Nijmegen Medical Center, The Netherlands CD45-V500 CD45-ECD CD45-Krome Orange CD45-Pacific Orange Slide 41 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 Summary Slide 42 Acknowledgementswww.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 Practical Flow Cytometry Howard Shapiro History of Flow Cytometry Spectrios Experiment Designer On-Line Knowledge Base Slide 43 Bone Yard Slide 44 Lymphocytic differentiation Slide 45 MFI: 14.7MFI: 44.7 CD8-APC-AF700CD8-AF700 Optimal excitation of Alexa Fluor 700 by APC energy transfer = to log brighter fluorescence Tandem Dye Selection: Alexa Fluor 700 Wavelength (nm) Fluorescence Intensity 635 nm APC Alexa Fluor 700 Slide 46 CD14 (RMO52)-Krome Orange SI = 101.3 Lymphocytes Monocytes CD20 (B9E9)-Krome Orange SI = 51.2 Lymphocytes CD16 (3G8)-Krome Orange SI = 18.6 Lymphocytes SI = 9.2 Lymphocytes CD19 (J4.119)-Krome Orange