SCYM Data Acquisition & Analysis 3 — Questions and Answers
Question 1: In a bivariate dot plot of FSC-A vs. FSC-H, a population appearing above the diagonal line (FSC-H lower than expected for its FSC-A) most likely represents:
- Single viable cells
- Doublets or aggregates (Correct answer)
- Dead cells
- Debris
Correct answer: Doublets or aggregates
Doublets have greater area (FSC-A) relative to height (FSC-H) because two cells passing together produce a widened pulse without proportionally increased peak height.
Question 2: Which gating strategy step should immediately follow excluding debris with an FSC/SSC gate in a standard immunophenotyping panel?
- Applying compensation
- Doublet exclusion using pulse geometry (Correct answer)
- Setting voltage
- Analyzing fluorescent channels
Correct answer: Doublet exclusion using pulse geometry
After the initial scatter gate removes debris, doublet exclusion ensures only single cells proceed to fluorescence analysis.
Question 3: The 'coefficient of variation' (CV) of a flow cytometry peak is calculated as:
- Mean divided by standard deviation × 100
- Standard deviation divided by mean × 100 (Correct answer)
- Median divided by mode × 100
- Peak height divided by peak width × 100
Correct answer: Standard deviation divided by mean × 100
CV = (SD / Mean) × 100; a lower CV indicates a tighter, better-resolved population.
Question 4: When analyzing rare events (e.g., circulating tumor cells at 1 per million), what is the most critical acquisition parameter to adjust?
- PMT voltage
- Total events collected—acquire far more events to detect rare populations (Correct answer)
- Threshold setting
- Compensation matrix
Correct answer: Total events collected—acquire far more events to detect rare populations
Statistical reliability for rare populations requires collecting a very large number of total events; at 1 per million, millions of cells must be acquired for meaningful counts.
Question 5: Which file format is the standard for storing flow cytometry list-mode data?
- CSV
- DICOM
- FCS (Flow Cytometry Standard) (Correct answer)
- XML
Correct answer: FCS (Flow Cytometry Standard)
FCS (e.g., FCS 3.1) is the ISAC-approved standard format encoding all event parameters and metadata for flow cytometry data.
Question 6: A researcher notices that the PE channel signal increases over a 30-minute acquisition run even though no new cells are added. The most likely cause is:
- Compensation drift
- Photobleaching of the fluorochrome
- Laser warm-up causing increasing power output (Correct answer)
- Clog in the sample line
Correct answer: Laser warm-up causing increasing power output
Many lasers, especially arc lamps and some solid-state lasers, increase in output power as they warm up, causing apparent signal drift during early acquisition.
Question 7: In spectral flow cytometry, unmixing replaces classical compensation because:
- It eliminates the need for single-stained controls
- It deconvolves the full emission spectrum of each fluorochrome using reference spectra rather than subtracting fixed spillover values (Correct answer)
- It uses scatter parameters instead of fluorescence
- It requires fewer PMTs
Correct answer: It deconvolves the full emission spectrum of each fluorochrome using reference spectra rather than subtracting fixed spillover values
Spectral unmixing uses least-squares fitting of complete reference spectra, which is more accurate than pairwise spillover subtraction, especially for highly overlapping fluorochromes.
SCYM Data Acquisition & Analysis 3
In a bivariate dot plot of FSC-A vs.
FSC-H, a population appearing above the diagonal line (FSC-H lower than expected for its FSC-A) most likely represents: