Instrument Characterization and PerformanceTracking for Digital Flow CytometersBD Biosciences Cytometer Set-up & Tracking (CS&T) SystemFor Research Use Only. Not for use in diagnostic or therapeutic procedures.Class I (1) Laser Products23-9335-00

Key Performance Factors in High Quality FlowCytometry Data Relative measured values of fluorescence Linearity and accuracy Resolution of subpopulations, including dim subpopulations Sensitivity Reproducibility of results and cytometer performance Tracking Comparison of results across time and among laboratories Standardization2

A Breakthrough for Flow Cytometry The Cytometer Setup & Tracking (CS&T) system is afully automated software and reagent system for BDdigital flow cytometers Functions of the CS&T system Defines and characterizes baseline performance Optimizes and standardizes cytometer setup Tracks cytometer performance Benefits of the CS&T system Provides consistent, reproducible data every day Simplifies design of multi-color experiments Yields higher quality data from multi-color experiments Identifies any degradation of cytometer performance early3

Cytometer Setup & Tracking (CS&T) System:The Software BD FACSDiva 6.0 software Has Unique Cytometer Setup & Tracking module Compatible with BD FACSCanto , BD FACSCanto II,BD FACSAria , and BD LSR II digital cytometers One tube–one click simplicity Newly developed BD CS&T beads Very flexible system using user-defined: Cytometer configurations to support any number of lasersand parameters Labeling - Fluorochrome parameters, filters, and mirrorsincluding Q-dots and new fluorochromes Alarm boundaries for tracking performance Application settings – linked to CS&T setup4

Cytometer Setup & Tracking (CS&T) System:The Beads The CS&T Bead set consists ofuniform beads of 3 differentintensities designed to fullycharacterize the flow cytometerBright Bright Beads - negligiblephotoelectron contribution toCV Mid Beads - largephotoelectron contribution toCV Dim Beads - significantbackground contribution to CV5DimMid

Determining the Intrinsic CV of BeadsIn order to estimate bead photoelectron statistics we firstneed to know the intrinsic (non-photoelectron) CVs102448Rainbow Bead Peak 2CV 6.28 Mean 96.88Count3624SSC-H768Rainbow Bead Peak 4CV 2.15 Mean -H7681024Measurethe difference 2in CV2Particle CVIntrinsic CVParticle CVLEDOf the beads against a “perfect particle”,LED pulsesPeak 2 CVIntrinsic 6.282 4.582 4.29610110210310RH000620.001Rainbow Lin2.01PMT640RGB FACScan 120 mW laserM11500FSC-HLED pulsesCV 4.51 Mean 99.58300LED pulses010FL2-H4500512512Peak4 CV 2.15%LED CV at channel 800 predicted 1.59%Peak4 non-photoelectonCV 1.48%Peak3 CV 3.24%,LED CV at mean 255 predicted 2.82%Peak 3 non photoelectron CV 1.59Peak2 CV 6.28%LED CV at mean 97 4.58%Peak2 non-photoelectron CV 4.294

Performance: Using the 3 bead setBy correcting for the bead intrinsic CVs, and for alignment and illuminationeffects at each step, we can calculate the photoelectron statistics.If we know the variance we can determine performance.100.00CV (%)CV without Bkg or SampleCV with BkgCV with Bkg and SampleDim BeadMid BeadBright n CVIs proportional to1Signal Background .

The Cytometer Setup & Tracking (CS&T) System:Beads FlexibilityThe beads are designed to be excitedby all currently supplied lasers andemit in the respective detectors forvirtually any fluorescent dye.FITCBrightMid2 µm83 µmPEPE-TRAmCyanQdot 655Violet (407 nm)ExcitationPerCPCy5.5PE-Cy7Blue (488 nm)ExcitationDimFSCPacificBlueAPCAlexa 700APC-Cy7Red (633 nm)ExcitationIndo-1VioletIndo-1BlueUV (362 nm)Excitation

The Cytometer Setup & Tracking (CS&T)System: Multicolor work flowDefine a Cytometer Configuration (Lasers, Detectors, Parameter names and Filters)Run CS&T Baseline [ 16 minutes]For(Defines Cytometer Baseline; Baseline Performance with Beads)Eachconfiguration Define Application Settings [Optional](User-defined Application settings bound to CS&T performance)Daily orbefore eachexperimentEachexperiment9Run CS&T Daily Performance Tracking [ 3 minutes](Standardizes Cytometer Setup; Records Performance)Create withinFACSDiVa a. Specify Configurationb. Specify ApplicationRun appropriatecompensationcontrolsAcquireData

Defining Cytometer ConfigurationsTotal User Flexibility Any number of lasers Octagon and trigon opticalbenches Any number of fluorochromeparameters Any number of bandpassfilters Any number of dichroicmirrorsSimply drag parameters,filters, and mirrors ontooctagon and trigon opticalbenches10Alexa Flour 700

Characterizing the Cytometer Baseline Perform ONCE for every new cytometer configuration or if you change or add aconfiguration (lasers, PMTs or filters) For each fluorescence detector, BD FACSDiva 6.0 software’s CS&T systemautomatically determines the following performance parameters Laser Delays (and adjust) – uses bright beadLinear Range ( 2%) – uses mid and bright beadsFluorescence Detector Efficiency (Qr) - Mid Bead (with adjustments Optical Background (Br) - dim bead (with adjustments from mid bead Electronic Noise (SDEN) – uses dim bead performanceBaseline PMT Voltage Settings: Minimal Effects from ElectronicNoise – based on dim bead performance (using SDEN)Creates Target Values for Ongoing Standardization - bright bead 11from bright and dim bead performance)performance)

Performance Parameter: Linearity Define Linearity as proportionality of output toinput (signal : number of photons) Important for accurate fluorescencecompensation Important for quantitative measurements DNA measurements Antigen - Antibody binding The BD FACSDiva 6.0 CS&T module usesa robust and reliable method (dual signal ratio)12

Linearity: Using RatiosLinearity means proportionality5000OutputR11 R22 R33 R44Y2 Y1 X 2 X1Proportionality60004000Or equivalently30002000100000100020003000Y2 X 2 Y1 X1InputThe detector is linear from 2500 to 5000 units13

Linearity: Effect on Compensation Compensation of data in the last decade involves subtraction of largenumbers Errors (non-linearity) in one or both large numbers can cause a largeabsolute error in the resultADetectorFITCPEBCDMedian Fluorescence Intensity (MFI)681796592173,000807579365BD CompBeads stained with varying levels of FITC-Ab.Compensation was set using samples A and C.This cytometer had a 2% deviation from linearity above 50,000 units.14

Linearity: CS&T Baseline Report BD FACSDiva 6.0CS&T moduleautomatically generates aCytometer BaselineReport Linearity range chartedfor every fluorescencedetector Default trackingboundary of 2.0%deviation Entire reports or chartscan be printed15

Performance Parameter: Sensitivity Defining sensitivity Threshold - Degree to which a flow cytometer can distinguishdimly stained particles from particle free background. Usually usedto distinguish populations on the basis of Molecules EquivalentSoluble Fluorochrome (MESF). Resolution - Degree to which a flow cytometer can distinguishunstained and dimly stained particles in a mixture. Can be verycomplicated in a polychromatic scenario. What is good sensitivity? Generally cytometer threshold sensitivity is high – thresholddefinition (50–200 MESF FITC, for example) Good threshold sensitivity does not necessarily guarantee goodresolution of dim cells from unstained cells Detector efficiency (Q) Contributes to sensitivity (both kinds) and is predetermined byfactors such as laser power and optical design Goal: Optimize cytometer setup for the best resolution sensitivity byusing enough detector gain to place measured populations above noise16

Sensitivity: Resolution vs. BackgroundNegativePopulationNegative population has low signal and low CVPopulations well resolvedNegative population has high signalPopulations not resolvedNegative population has low signal and high CVPopulations not resolvedThe ability to resolve populations is a function of both therelative signal levels and spread of the populations17PositivePopulation

Sensitivity: Factors Impacting ResolutionIncreased )SampleNon(Fluorescence(Fluorescence OptimizedDetectionDetectionPMT GainsEfficiency)Efficiency)Low QCytometer

Performance Parameter: Q Measures the relative efficiency of a given detector to measurefluorescence Photoelectrons detected per equivalent fluorochrome molecule Fewer photons detected per molecule lower Q higher CV increased spread lower resolution sensitivityAccepted standard in the flow cytometry communityBD FACSDiva 6.0 CS&T module calculates a relative Q (Qr) All fluorescence detectorsBased upon the median and robust CV (rCV) values for each of theCS&T fluorescent bead typesThe following affect Q: 19Laser power and laser alignmentCleanliness of optics (cuvette, lenses, mirrors, etc )PMT spectral sensitivity: PMTs become less sensitive towards the red;therefore, Q is lower for these detectors (e.g. PE-Cy7 lower than FITC)Optical Design: number of dichroic mirrors, N.A. of collection lens, etc

Q: Relationship to Resolution SensitivitySample of multi-intensity beadsanalyzed on the BD FACSCalibur cytometerLaserPowerQ30 mW.030Vary laser power which affects QPMT voltages adjusted to keep brightbead median constant15 mW.018Resolution of dim populationsdepends more on the width thanthe mean of the distributions7.5 mW.0083.75 mW.004Lower laser power Fewer photons per fluorochrome Lower Q Decreased Resolution Sensitivity20FL1-H Green FL

Performance Parameter: B Optical background B is a measure of optical background signal in the detector Baseline restoration electronics subtract constantbackgrounds during current to pulse conversion Baseline restoration electronics subtract background signallevels, but cannot remove measurement error when theirlevels are high (works like compensation)Increased optical background broadens the distribution of allpopulations and results in decreased resolution sensitivity,most evident when measuring dim populations. Background Contributing Factors Free fluorescent dye in sample Raman scattering Ambient light Damaged optics21

Performance Parameter: Electronic Noise (SDEN) Effects due to 9 electronics signal connections, PMTnoise, ADC noise, digital error, etc Broadens the distribution of all populations, mostnoticeable at the low end of the scale: dim populations Increases in electronic noise results in decreasedresolution sensitivity (most noticeable at the low end) Used by the BD FACSDiva 6.0 CS&T module toestablish baseline targets that minimize the spread ofnegative and dim populations Baseline Target value of dim bead 10 x SDEN Set application voltage in DiVa so that MFI of negative cellsremains at CS&T baseline target or above22

Electronic Noise (SDEN): DeterminingBaseline PMT VoltagesThe BD FACSDiva 6.0 CS&T module analyzes dim particles, which aresimilar to dim cells’ brightness, allowing relevant detector baselines to bevisualized by plotting fluorescence intensity vs (PMT gain, CV, and SD)PE: Detailed Performance PlotDim Bead For this detector, the SDEN 18 Fluorescence intensity of dim bead 10 x SDEN 1801810110100100010000100100000Median Fluorescence Intensity180PMT VoltageCV or SD500 V1000100 As PMT voltage is increasedCV unchanged Æ resolution unchanged231000PMT Voltage Determine PMT voltage required toset the dim beads at 180 500 volts baseline voltage As PMT voltage is lowered, CVincreases Æ resolution decreasesCVStandard Deviation10000

PMT Voltages: Optimal Gains Can ReduceClassification Errors12.0%CD4 dim monocytes650 VCD4 negativeCD4 lymphocytes% Negative in CD4 Gate550 V% Negative in CD4 Monocyte Gate10.0%8.0%6.0%4.0%2.0%750 V0.0%-1000100PMT Voltage Offset24

Performance Parameters: Summary Linearity Lets you know where potential errors in compensation may occurVisually inspect linear range ( 2%, 1% or 0.5% error)Avoid reagents with medians outside the linearity range Detector Efficiency (Q) Optical Background (B) Electronic Noise (SDEN) All performance parameters affect resolution sensitivity Important dim fluorescent markers should be measured in detectorswith high Q values, low B, and low SDEN Changes in these parameters can indicate cytometer problems25 Increases in SDEN Æ bad PMT connections or other electronic problem Decreases in Q Æ low laser power, dirty flowcell, alignment or filter issue Increases in B Æ fluorescent contaminant, failing laser or filter problems

Cytometer Baseline: ReportCytometer InformationBead InformationDetectorsBead Medians and rCVsLinearityQr and BrSD Electronic NoiseBaseline PMT VoltageBright Bead Target ValuesMedian Fluorescence26

Cytometer Baseline Report: Qr, Br, and SDENData from a BD FACSCanto cytometerBackground from Raman Scattering; PE and FITCLower detector efficiency in far red; PE-Cy727

Cytometer Baseline Report:Performance Plots Report providesperformance plots forevery detector Shows data for dim beads Shows baseline targetand PMT voltage Recommended as astarting PMT voltagebased on beadperformance, can beoverridden by theoperator28

Daily Performance ChecksPerform at least once a day or before each experimentFor each fluorescence detector, BD FACSDiva 6.0CS&T module will automatically: Check and adjust laser delays Check and adjust area scaling factors Adjust detector PMT voltages to place bright beads attheir target values in each detector (linked toapplication settings) Measure performance parameters Detector efficiency (Qr), background (Br), rCVs, andPMT voltages 29Record and track performance parameters with LeveyJennings Graphs

Daily Performance Check: ReportDetectorsBead Medians andrCVsPMT VoltageΔ from BaselineQr and BrDetector Pass/Fail30

Performance Tracking: Levey-Jennings Graphs All measured performance parameters aretracked Laser Alignment (CVs), detection efficiency (Qr),optical background (Br) PMT voltages Fluorescence levels (target values) are held constant,changes in PMT voltages can reflect changes in laseroutput, cleanliness, laser alignment and opticalalignment Data can be analyzed in Levey-Jennings graphs All data points are directly linked to their respective dailyperformance reports31

Performance Data: ReportsAll data is directly traceable back tothe original Baseline portsReports

Applications: Cytometer Settings Automated flow cytometer setup software hasoffered users two basic application settings HuPBL – Lyse/Wash HuPBL – Lyse/No Wash However, some applications may havereproducible high-fluorescence backgrounds Cell lines, activated cells, etc Intra-cellular staining (e.g. cytokine or BD Phosflow) In these cases, PMT voltages can be decreasedwithout adversely affecting resolution sensitivity33

Application Settings: User Defined The BD FACSDiva 6.0 software provides customers a means to defineproper application settings and link to CS&T performance dataA pre-defined application settings Worksheet provides visual guides to settingPMT voltages for the low end of the scale for stained samples Crosshair indicates theminimum recommended targetvalue for the negativepopulation 10 x SDEN The gray box indicates the 97thpercentile of the dim beads For maximum low endresolution sensitivity, PMTvoltages should be adjusted toplace the negative populationwithin or above the gray box Actual target center is at thecrosshair – note asymmetrydue to log scale34

Summary BD Cytometer Setup & Tracking (CS&T) beads work withBD FACSDiva 6.0 software on the BD FACSCanto ,BD FACSCanto II, BD FACSAria , and BD LSR II platforms Any number of lasers or fluorescence parametersCompatible with novel fluorochromesThe BD FACSDiva CS&T module will: Fully characterize the cytometer’s performance Linearity, detector efficiency (Qr); background fluorescence (Br); electronic noise(SDEN); and laser alignment (rCV) Optimize cytometer settings Laser delays; Area scaling factors; PMT voltages Set up your cytometer with reproducible performance Obtain consistent, reproducible experimental data from day to day Define application settings in FACSDiVa and link to automated setup Track cytometer performance Detect component failures and alterations Provide graphical representations of performance trends over time35

Conclusion This Cytometer Setup & Tracking System Provides you with the flexibility to optimally configure and useyour cytometer to meet your specific research requirementscytometerperformance high-leveland reproducible Insures consistentDesigningandperformingsetup for multi-colorexperimentsis no longer Better resolutionof dim populations Fewer compensation artifactssolimited by the complexity of Provides you with the information needed to simplify the designcytometersetupof complex multicolorexperiments Assist with choosing parameters providing the best low levelresolution Assist with avoiding potential problems with compensation36

Acknowledgments 37Alan StallBob HoffmanMing YanDennis SasakiMajid MehrpouyanAndrea NguyenDavin Jutila Lalet SharmaZen GongChak DunnaMaria SemyonovaNaichuan NadkarniDon Fourby

A Breakthrough for Flow Cytometry The Cytometer Setup & Tracking (CS&T) system is a fully automated software and reagent system for BD digital flow cytometers Functions of the CS&T system Defines and characterizes baseline performance Optimizes and standardizes cytometer setup Tracks