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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 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor®, Texas Red®, and Qdot® are registered trademarks, and Pacific Blue™ is a trademark of Molecular Probes, Inc. Cy™ is a trademark of Amersham Biosciences Corp. Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2011 BD For More Information…. If you have further questions: Contact Technical Service (US) at: 877-232-8995, Prompt 3, 2 or email: [email protected] Please visit our multicolor resource site at: bdbiosciences.com/colors