SCYM Data Acquisition & Analysis 4 — Questions and Answers
Question 1: What is the effect of overcompensation in a flow cytometry experiment?
- The negative population appears above the positive population
- The negative population is pulled below the baseline (false negative shift) (Correct answer)
- Scatter parameters are distorted
- The CV of all peaks increases
Correct answer: The negative population is pulled below the baseline (false negative shift)
Overcompensation subtracts too much signal from the channel, causing the unstained population to appear with lower fluorescence than the instrument baseline.
Question 2: A log-scale biaxial plot in flow cytometry is preferred over a linear scale when displaying fluorescence data because:
- It compresses high-signal populations for clarity
- It allows simultaneous visualization of dim and bright populations spanning several decades (Correct answer)
- It automatically applies compensation
- It eliminates autofluorescence
Correct answer: It allows simultaneous visualization of dim and bright populations spanning several decades
Logarithmic display accommodates the wide dynamic range of fluorescence intensity, enabling dim and bright populations to be resolved on the same plot.
Question 3: Which analysis approach is BEST suited for identifying unexpected cell populations without prior hypothesis?
- Manual sequential gating
- Unsupervised high-dimensional clustering algorithms (e.g., FlowSOM, t-SNE, UMAP) (Correct answer)
- Fixed quadrant gates from a reference sample
- Compensation-only correction
Correct answer: Unsupervised high-dimensional clustering algorithms (e.g., FlowSOM, t-SNE, UMAP)
Unsupervised algorithms explore all parameters simultaneously and can reveal novel or unexpected populations that manual gating strategies might miss.
Question 4: During acquisition, a sudden increase in event rate without a corresponding increase in sample volume most likely indicates:
- Laser misalignment
- A clog partially releasing and causing a bolus of cells (Correct answer)
- Compensation error
- PMT voltage too high
Correct answer: A clog partially releasing and causing a bolus of cells
Partial clogs can trap cells and then release them suddenly, causing a transient spike in event rate that skews data.
Question 5: When designing a multicolor flow cytometry panel, bright fluorochromes should be paired with:
- Highly expressed antigens
- Lowly expressed or dim antigens to maximize sensitivity for those targets (Correct answer)
- Antigens on dead cells
- Antigens requiring highest compensation
Correct answer: Lowly expressed or dim antigens to maximize sensitivity for those targets
Bright fluorochromes provide enough signal to detect dimly expressed antigens above background, while dimmer fluorochromes are sufficient for highly expressed antigens.
Question 6: The 'stain index' (SI) is a metric used to evaluate fluorochrome performance and is calculated as:
- Mean of positive / mean of negative
- (Mean positive − Mean negative) / (2 × SD of negative) (Correct answer)
- Standard deviation of positive / mean of positive
- Peak height positive / peak height negative
Correct answer: (Mean positive − Mean negative) / (2 × SD of negative)
Stain index = (MFI positive − MFI negative) / (2 × SD negative), providing a robust measure of the separation between positive and negative populations.
Question 7: What is the primary reason for using viability dyes (e.g., DAPI, 7-AAD, Live/Dead fixable dyes) in flow cytometry panels?
- To increase fluorescence intensity of surface markers
- To exclude dead cells, which non-specifically bind antibodies and generate false-positive signals (Correct answer)
- To improve light scatter resolution
- To reduce autofluorescence
Correct answer: To exclude dead cells, which non-specifically bind antibodies and generate false-positive signals
Dead cells have compromised membranes and non-specifically bind antibodies, so excluding them with a viability dye prevents false-positive staining in the live population.
SCYM Data Acquisition & Analysis 4
What is the effect of overcompensation in a flow cytometry experiment?