SCYM Instrument Operation 2 — Questions and Answers
Question 1: What is the primary purpose of the sheath fluid in a flow cytometer?
- To lyse red blood cells before analysis
- To hydrodynamically focus cells into a single-file stream (Correct answer)
- To fluorescently label surface antigens
- To cool the laser during operation
Correct answer: To hydrodynamically focus cells into a single-file stream
Sheath fluid creates laminar flow that hydrodynamically focuses the sample stream so cells pass through the laser interrogation point one at a time.
Question 2: Which parameter is used to distinguish doublets from single cells during flow cytometric analysis?
- FL1 area vs FL2 area
- FSC height vs FSC area (or width) (Correct answer)
- SSC height vs FSC height
- FL3 area vs SSC area
Correct answer: FSC height vs FSC area (or width)
Doublet discrimination is performed by comparing FSC-H (height) vs FSC-A (area); doublets produce disproportionately higher area relative to height.
Question 3: When performing daily QC on a flow cytometer using fluorescent beads, a sudden rightward shift in bead peak CV is most likely caused by:
- Laser power fluctuation (Correct answer)
- Incorrect scatter voltage settings
- Clogged sample line reducing flow rate
- Expired sheath fluid
Correct answer: Laser power fluctuation
Laser power instability causes variable fluorescence excitation, broadening the bead peak and increasing CV.
Question 4: In a spectral flow cytometer, unmixing algorithms require which reference material?
- Unstained cells only
- Single-color controls (single-stained references) for each fluorochrome (Correct answer)
- A compensation matrix from a previous run
- FMO controls for every fluorochrome
Correct answer: Single-color controls (single-stained references) for each fluorochrome
Spectral unmixing requires single-color reference spectra for each fluorochrome to mathematically separate overlapping emission signals across detectors.
Question 5: The time delay setting in a cell sorter is used to:
- Set the interval between successive droplet formations
- Synchronize the deflection charge with the droplet containing the target cell (Correct answer)
- Control the sample injection pressure
- Adjust the laser pulse frequency
Correct answer: Synchronize the deflection charge with the droplet containing the target cell
The time delay (sort delay) accounts for the transit time from the laser interrogation point to the droplet break-off point so the correct droplet is charged.
Question 6: Which action should be taken first if the sample flow rate on a benchtop cytometer drops unexpectedly mid-run?
- Replace the sample tube immediately
- Run the instrument cleaning protocol
- Check for a clog in the sample line and perform a backflush if needed (Correct answer)
- Increase sheath pressure by 10 PSI
Correct answer: Check for a clog in the sample line and perform a backflush if needed
A sudden flow rate drop typically indicates a partial clog; checking and performing a backflush resolves most sample-line obstructions before escalating.
Question 7: What does a low coefficient of variation (CV) for a fluorescent bead peak indicate during instrument QC?
- The detector PMT voltage is set too high
- The instrument is performing consistently with tight measurement precision (Correct answer)
- Excessive background fluorescence is present
- The laser is operating below target power
Correct answer: The instrument is performing consistently with tight measurement precision
A low CV on bead peaks indicates that the instrument is producing highly reproducible fluorescence measurements, reflecting good optical and electronic performance.
SCYM Instrument Operation 2
What is the primary purpose of the sheath fluid in a flow cytometer?