Digital cytometer setup and QC Holden T Maecker

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 1 st to 2 nd laser Time from intersection of 1 st to 3 rd 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 10 X 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 Comp. Beads 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 Comp. Beads and calculate compensation • Record experiment-specific target values. • Run samples.

Comp. Beads as single-color controls Comp. Beads 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 Comp. Beads 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.