ISAC Equipment Operation and Maintenance 2 — Questions and Answers
Question 1: What is the purpose of the sheath fluid in a flow cytometer?
- To stain cells for fluorescence detection
- To hydrodynamically focus cells into a single-file stream (Correct answer)
- To lyse red blood cells before analysis
- To calibrate the laser power output
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 measure cell granularity in flow cytometry?
- Forward scatter (FSC)
- Side scatter (SSC) (Correct answer)
- Fluorescence channel 1 (FL1)
- Time-of-flight (TOF)
Correct answer: Side scatter (SSC)
Side scatter (SSC) measures light scattered at roughly 90 degrees and correlates with internal complexity and granularity of cells.
Question 3: When performing daily QC on a flow cytometer, which action is most important to perform FIRST?
- Run a sample of cells to check gating
- Warm up the laser for the manufacturer-specified time (Correct answer)
- Replace all filters in the optical bench
- Adjust the PMT voltage to maximum
Correct answer: Warm up the laser for the manufacturer-specified time
Lasers require a warm-up period to reach stable power output before any QC beads or samples are run.
Question 4: A flow cytometer's CV (coefficient of variation) for a fluorescent bead population is suddenly much higher than usual. What is the MOST likely cause?
- The sheath fluid tank is nearly empty
- Partial clogging of the flow cell or sample injection tube (Correct answer)
- The computer software needs updating
- Antibody staining was performed incorrectly
Correct answer: Partial clogging of the flow cell or sample injection tube
Partial clogging causes unstable sample flow rate, leading to increased variation in signal intensity and a higher CV.
Question 5: What does 'backflushing' the flow cytometer accomplish during maintenance?
- It increases laser power to clean optical surfaces
- It reverses the flow direction to clear clogs from the sample line (Correct answer)
- It purges residual fluorescent dye from PMT detectors
- It resets the instrument's electronic baseline
Correct answer: It reverses the flow direction to clear clogs from the sample line
Backflushing reverses sheath or DI water flow through the sample injection tube to dislodge and clear partial blockages.
Question 6: In a spectral flow cytometer, what replaces the traditional bandpass filter-based detector array?
- A single avalanche photodiode
- A prism or diffraction grating coupled to a multi-detector array (Correct answer)
- A CCD camera mounted at 90 degrees
- A time-correlated single photon counting module
Correct answer: A prism or diffraction grating coupled to a multi-detector array
Spectral instruments use a prism or grating to disperse emitted light across an array of detectors, capturing the full emission spectrum.
Question 7: Which of the following BEST describes the role of a neutral density (ND) filter in flow cytometry optical design?
- It selects a specific wavelength band for detection
- It attenuates laser light intensity uniformly across wavelengths (Correct answer)
- It converts UV excitation to visible fluorescence
- It blocks backscattered laser light from the forward scatter detector
Correct answer: It attenuates laser light intensity uniformly across wavelengths
Neutral density filters reduce overall light intensity by a fixed factor without altering the spectral distribution, allowing adjustment of signal levels.
ISAC Equipment Operation and Maintenance 2
What is the purpose of the sheath fluid in a flow cytometer?