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Digital cytometer setup and QC Holden T. Maecker Outline • What do we care about in terms of cytometer performance? • What factors influence performance? • What can we measure and track over time? • What software adjustments are available? • What QC parameters to look at: • Initially on instrument setup • Periodically (daily) • With each experiment What’s important to cytometer performance? • Resolution Sensitivity: • Q = optical detection efficiency • B = background • Linearity: • Relative ability to detect fluorescence differences across the detection scale • Stability over time Factors influencing sensitivity, linearity, and stability • Fluidics: • System pressure and stability • Optics: • Laser type and performance • Optical path, filters • Electronics: • PMT type and performance • Signal processor performance What can we measure and track? • A set of beads of multiple intensities: • Resolution of dimmest bead populations and/or dim bead CV (sensitivity) • Brightness ratio of two bead populations across detection scale (linearity) • Voltage required to achieve a certain bead brightness (stability) • Old: 8-peak Rainbow beads (Spherotech) • Diva 6.0: CS&T beads (BD) Standard bead sets 8-peak Rainbow beads: CS&T beads: Bright 2 µm 3 µm Dim Mid FSC FITC What adjustments are available? • Laser delay • Window extension • Area scaling factor What is laser delay? Time from intersection of 1st to 2nd laser Time from intersection of 1st to 3rd laser • Dependent upon sheath velocity • Can be mitigated by larger window extensions What is a window extension? Time window over which a pulse signal is measured • Zero is the narrowest window, two is default • Larger window extensions can overcome minor imprecisions in laser delay setting What is “area scaling factor”? • A factor that is applied to the pulse area measurement to equalize the height and area scales • Should rarely need to be adjusted • Is important to keep bright events on-scale in both area and height What to measure and when? • Initial instrument characterization • Periodic (daily) performance check • Experiment-specific setup Initial instrument characterization • Objective 1: characterize instrument sensitivity and linearity • Old method: • • • • Run 8-peak rainbow beads Set voltages so brightest bead is ~105 Observe separation in each detector (no std) Manually adjust laser delay & area scaling factor • Diva 6.0: • Run baseline optimization routine 8-peak Rainbow beads CS&T Baseline Optimization Report Initial instrument characterization • Objective 2: find baseline PMT voltages that maximize resolution sensitivity • Old method: • • • • Run peak 2 beads at a series of voltages (350-800 volts) Export CV of tightly-gated singlet beads for each detector Plot CV versus voltage for all detectors Lowest plateau voltage is the minimum baseline voltage for that detector • Run mid-range (peak 3) beads at minimum baseline voltages, record mean in each detector (these are your initial baseline target values) • Diva 6.0: • Part of baseline optimization routine Manual determination of baseline PMT voltages Automated baseline PMT voltage determination in Diva 6.0 Determining Baseline PMT Voltages Baseline PMTV is set by placing the dim bead MFI to equal 10X SDEN 460 V SDEN Periodic (daily) performance check • Manual method: • Open Exp 1 (8-peak beads) • Run a new tube of 8-peak beads with same voltage settings • Compare histograms to initial run • Track at least one peak from each detector for MFI and CV over time • Diva 6.0: • Run system performance check Performance Tracking • All measured performance parameters are tracked: • Linearity, CVs, Q and B, laser alignment • PMT voltages required to hit target values • Data is analyzed in Levey-Jennings plots PMT Voltage 550 FITC Channel (Blue laser) 525 500 475 450 425 400 10/22/04 11/11/04 12/01/04 12/21/04 01/10/05 01/30/05 02/19/05 03/11/05 Time Experiment-specific setup for a new panel • Set voltages to achieve baseline target values • Run single-stained CompBeads to see if each bead is brightest in it’s primary detector • If not, increase voltage in the primary detector (!) • Run fully-stained cells and: • Decrease voltages for any detectors where events are off-scale • Increase voltages for any detectors where low-end resolution is poor (necessary?) • Re-run single-stained CompBeads and calculate compensation • Record experiment-specific target values. • Run samples. CompBeads as single-color controls CompBeads provide a convenient way to create single-color compensation controls: • Using the same Abs as in the experimental samples • Creating a (usually) bright and uniform positive fluorescent peak • Without using additional cells Experiment-specific setup for existing panel • Set voltages to achieve experiment-specific target channels. • Run single-stained CompBeads and calculate compensation. • Run samples. Why not put unstained cells in the first log decade? • Autofluorescence varies by detector (very low in far-red range of spectrum) • Leads to highly variable setup • No guarantee that performance on stained cells will be optimal Why use target channels rather than voltages? Once optimum voltages for a particular experiment are determined, the settings can be captured as target channels (median of mid-range beads in each detector). These target channels are more robust to instrument changes than are the voltages themselves. Summary • We want our instruments to have good sensitivity, linearity, and stability. • Many factors contribute to sensitivity, linearity, and stability, but we can track the net results using a set of standard beads. • Good setup and QC involves initial instrument characterization, daily performance checks, and experiment-specific setup. • The CS&T module in Diva 6.0 automates much of the QC workflow. Diva 6.0: Making instrument setup and QC so easy, a child can do it.* *Not an official BD claim. The child shown here has never run Diva 6.0, but she’s watched her daddy do it.