Establishing PMT Adjustment Tolerances Protocol

Posted: April 26, 2018

  1. Dilute blank and dyed beads at 1 drop of each in 1.6 mL of lab use diluent and mix via vortex or tapping.
  2. Set the PMT voltages for all fluorescence channels of interest to those used for daily QC to ensure that all peaks are able to be seen.
  3. Place the tube on the cytometer and run the beads, and record the lab standard number of events (typically 10,000).
  4. Record the median fluorescence intensity (MFI) for several of the peaks, with markers placed at full width, half maximum (FWHM), along with the PMT voltage for each fluorescence channel. Performing measurements at the low/mid-range/high intensities are recommended at a minimum, as this will provide understanding as to how the instrument responds at the edges and center of the PMT linear response. Recording the MFI of more peaks will give a better idea of the overall PMT response however.
  5. Increase the PMT voltage by 1 V for each of the fluorescence channels and record another run. Record the MFI again for each fluorescence channel for each peak being observed.
  6. Repeat Step 5 twice more, going 3 V above the daily QC PMT setpoints.
  7. Return the PMT voltages to their daily QC setpoint and record another run at this level. Rerunning will remove any hysteresis effects on PMT voltage change.
  8. Decrease the PMT voltage by 1 V for each of the fluorescence channels and record another run. Record the MFI again for each fluorescence channel for each peak being observed.
  9. Repeat Step 8 twice more, going 3 V below the daily QC PMT setpoints.
  10. Once the data has been tabulated, determine the change in MFI between each step for each channel for each peak being observed.
  11. Average these values to determine the MFI per PMT voltage change seen at each bead intensity level. These values will allow PMT setpoint control limits from being too restrictive.

 The tolerances determined for each channel should factor into the daily PMT setpoint testing to ensure that the target range specified is not less than tolerance. If the target range is indeed less than the rate of change found in that detector channel, then it can become difficult to meet the specification for that channel.