SCYM Instrument Operation 5 — Questions and Answers
Question 1: A technologist is troubleshooting a flow cytometer and finds that all fluorescence channels show elevated background, but scatter parameters are normal. The most likely cause is:
- Laser power is too low
- Ambient light entering the optical system or a contaminated flow cell (Correct answer)
- PMT voltages set too low across all channels
- Sample concentration is too low
Correct answer: Ambient light entering the optical system or a contaminated flow cell
Elevated background across all fluorescence channels with normal scatter typically points to stray light contamination, either from ambient light or a fluorescent residue in the flow cell.
Question 2: In a high-speed cell sorter operating with a vibrating nozzle, the droplet break-off frequency is determined by:
- The sheath fluid viscosity alone
- The piezoelectric crystal oscillation frequency applied to the nozzle (Correct answer)
- The sample pressure setting
- The deflection plate voltage
Correct answer: The piezoelectric crystal oscillation frequency applied to the nozzle
The piezoelectric crystal in the sort head vibrates at a defined frequency, creating regular droplet break-off from the stream.
Question 3: Which of the following is NOT a standard daily instrument performance check for a flow cytometer?
- Running calibration beads to verify laser alignment and detector performance
- Checking sheath fluid level and waste container
- Recalibrating the electrostatic deflection plates (Correct answer)
- Verifying that the instrument's CV for bead peaks meets laboratory specifications
Correct answer: Recalibrating the electrostatic deflection plates
Recalibrating deflection plates is not a routine daily check; it is an instrument-specific maintenance task performed less frequently or during troubleshooting.
Question 4: When using an acoustic focusing cytometer (e.g., Attune), how is cell focusing achieved differently from conventional hydrodynamic focusing?
- Cells are focused using electric fields instead of sheath fluid
- Acoustic waves push cells to the center of the channel, reducing or eliminating sheath fluid requirements (Correct answer)
- A laser beam physically guides cells into single file
- Gravity is used to settle cells into the interrogation zone
Correct answer: Acoustic waves push cells to the center of the channel, reducing or eliminating sheath fluid requirements
Acoustic focusing cytometers use ultrasonic standing waves to position cells in the center of the microchannel, enabling faster sample rates with less sheath fluid.
Question 5: Which instrument setting is most critical to adjust when switching from immunophenotyping peripheral blood to analyzing small particles such as platelets or extracellular vesicles?
- Compensation matrix values
- Trigger threshold lowered and scatter voltages optimized for small particles (Correct answer)
- Increasing sample acquisition speed to high
- Changing fluorochrome panel to include additional channels
Correct answer: Trigger threshold lowered and scatter voltages optimized for small particles
Small particles like platelets and extracellular vesicles require a lower trigger threshold and optimized scatter voltages to be detectable above instrument noise.
Question 6: What is the significance of the 'coefficient of variation' (CV) reported for QC bead peaks?
- It measures the percentage of beads that are fluorescent
- It quantifies the spread of fluorescence measurements relative to the mean, reflecting instrument precision (Correct answer)
- It defines the ratio of live to dead cells in the sample
- It calculates the number of cells passing through the laser per second
Correct answer: It quantifies the spread of fluorescence measurements relative to the mean, reflecting instrument precision
CV = (standard deviation / mean) × 100; a lower CV indicates tighter, more reproducible fluorescence measurements, signifying better instrument performance.
Question 7: If a laboratory switches to a new lot of sheath fluid, which step is essential before resuming patient sample acquisition?
- Reorder all antibody reagents to match the new sheath lot
- Re-run the full QC bead panel and verify that all parameters remain within established performance specifications (Correct answer)
- Perform a sort purity check with the new sheath fluid
- Replace all PMT filters to compensate for the new refractive index
Correct answer: Re-run the full QC bead panel and verify that all parameters remain within established performance specifications
A new sheath fluid lot may have subtle differences; re-running QC beads ensures all optical and scatter parameters still meet performance specifications before clinical use.
SCYM Instrument Operation 5
A technologist is troubleshooting a flow cytometer and finds that all fluorescence channels show elevated background, but scatter parameters are normal.
The most likely cause is: