SCYM Instrument Operation 3 — Questions and Answers
Question 1: A technologist observes that FSC and SSC populations appear dimmer than usual and events are fewer than expected. The most likely instrument issue is:
- Incorrect PMT voltage for FL channels
- Laser misalignment or reduced laser power (Correct answer)
- Compensation matrix applied incorrectly
- Sample tube loaded upside down
Correct answer: Laser misalignment or reduced laser power
Reduced FSC/SSC signal intensity combined with lower event counts strongly suggests laser misalignment or power loss at the interrogation point.
Question 2: In electrostatic cell sorting, cells are deflected into collection tubes by:
- Mechanical valves that open when a target cell is detected
- Charged droplets passing through an electrostatic deflection field (Correct answer)
- Acoustic waves that push cells off-axis
- Pressure differentials applied to the sample stream
Correct answer: Charged droplets passing through an electrostatic deflection field
In electrostatic sorting, droplets containing target cells are given an electrical charge and deflected by high-voltage plates into collection vessels.
Question 3: Which type of flow cytometer detector is most commonly used to measure fluorescence signals?
- Charge-coupled device (CCD)
- Photomultiplier tube (PMT) (Correct answer)
- Photodiode array
- CMOS sensor
Correct answer: Photomultiplier tube (PMT)
PMTs are the standard fluorescence detectors in flow cytometry because of their high sensitivity and ability to amplify low-level photon signals.
Question 4: What is the recommended maximum number of cells per milliliter in the sample tube to avoid coincident events in most standard flow cytometers?
- 500,000–1,000,000 cells/mL (Correct answer)
- 5,000,000–10,000,000 cells/mL
- 50,000–100,000 cells/mL
- 10,000,000–20,000,000 cells/mL
Correct answer: 500,000–1,000,000 cells/mL
Most protocols recommend 0.5–1 × 10⁶ cells/mL to minimize coincidence (two cells entering the laser beam simultaneously) and preserve data accuracy.
Question 5: When comparing the MFI of a test sample to a control, the operator must ensure that:
- Both samples were acquired on the same PMT voltage settings (Correct answer)
- Both samples used identical staining volumes regardless of cell count
- The control sample was always acquired first
- Compensation was applied after all samples were acquired
Correct answer: Both samples were acquired on the same PMT voltage settings
MFI comparisons are only valid across samples acquired with identical voltage settings, as changing voltages shifts the fluorescence scale.
Question 6: During a sort, the operator notices that the yield is very low but purity is acceptable. This is most likely due to:
- Sort gate set too broadly
- Abort rate is high due to coincident events (Correct answer)
- Deflection plates voltage set too high
- Sample flow rate set too low
Correct answer: Abort rate is high due to coincident events
A high abort rate, caused by coincident events where two cells arrive simultaneously, causes the instrument to reject events, reducing sort yield while maintaining purity.
Question 7: To verify laser alignment on a flow cytometer, a technologist should use:
- Unstained peripheral blood cells
- Alignment beads or standard fluorescent microspheres (Correct answer)
- A known positive control antibody panel
- Freshly prepared red blood cell suspension
Correct answer: Alignment beads or standard fluorescent microspheres
Alignment (or calibration) beads provide bright, consistent fluorescent signals used to optimize and verify laser alignment and optical configuration.
SCYM Instrument Operation 3
A technologist observes that FSC and SSC populations appear dimmer than usual and events are fewer than expected.
The most likely instrument issue is: