SCYM Data Analysis 4 — Questions and Answers
Question 1: In cytometry, what is the definition of 'median fluorescence intensity' (MFI), and why is it preferred over mean in skewed distributions?
- MFI is the most frequent value; it is preferred because it is computationally simple
- MFI is the middle value of ranked events; it is resistant to outliers and skewed tails (Correct answer)
- MFI is the average intensity across all events; it captures total fluorescence accurately
- MFI is the geometric mean; it normalizes log-scale data automatically
Correct answer: MFI is the middle value of ranked events; it is resistant to outliers and skewed tails
MFI represents the 50th percentile of event intensities and is robust against outliers and non-normal distributions that are common in cytometry data.
Question 2: When setting up a multi-color panel, which tool is most useful for predicting and minimizing spectral spillover before running samples?
- Live-dead discrimination antibody titration
- Fluorochrome compatibility and spillover spreading calculators (e.g., Spectra Viewer) (Correct answer)
- Flow rate calibration with reference beads
- Side scatter threshold adjustment to exclude debris
Correct answer: Fluorochrome compatibility and spillover spreading calculators (e.g., Spectra Viewer)
Spectra viewers and spillover spreading calculators allow researchers to model expected spillover between fluorochrome combinations before committing to a panel, minimizing compensation burden.
Question 3: What is 'spreading error' in multicolor flow cytometry, and what is its primary consequence?
- Errors from high flow rate causing cell stream instability and overlapping events
- Increased variance in a negative population due to spillover from a highly expressed antigen (Correct answer)
- Systematic shift in FSC values caused by laser misalignment
- Reduction in absolute event counts from excessive threshold settings
Correct answer: Increased variance in a negative population due to spillover from a highly expressed antigen
Spreading error increases the variance (broadens the width) of a negative population when compensating for spillover from a brightly expressed antigen in an adjacent channel, reducing resolution.
Question 4: In Harmony batch correction for cytometry data, which type of variation does it specifically aim to remove?
- Biological variation between treatment conditions
- Technical batch effects due to sample processing or instrument differences (Correct answer)
- Compensation errors from improperly set spillover values
- Gating inconsistencies introduced by different operators
Correct answer: Technical batch effects due to sample processing or instrument differences
Harmony is an algorithm designed to align cells from different batches in a shared embedding space, removing technical variation while preserving true biological differences.
Question 5: Which cytometry analysis approach is best suited for identifying novel, previously undefined cell subsets without prior biological assumptions?
- Supervised gating based on established marker combinations
- Unsupervised clustering algorithms such as Phenograph or FlowSOM (Correct answer)
- Single-parameter threshold gating on individual markers
- Boolean combination gating using pre-defined positive/negative calls
Correct answer: Unsupervised clustering algorithms such as Phenograph or FlowSOM
Unsupervised clustering algorithms group cells based on their overall marker expression profiles without requiring predefined gate boundaries or assumptions about known populations.
Question 6: What does a bimodal distribution in a single-parameter histogram most commonly indicate in flow cytometry?
- Instrument detector saturation causing truncation of high-signal events
- Two distinct cell populations with different expression levels of the measured antigen (Correct answer)
- Inadequate compensation resulting in artificial peak splitting
- Sample aggregation causing doublets to appear as a separate peak
Correct answer: Two distinct cell populations with different expression levels of the measured antigen
A bimodal distribution with two peaks typically represents two cell populations expressing different levels (positive vs. negative, or high vs. low) of the measured antigen.
Question 7: In imaging flow cytometry analysis, which feature is calculated to help distinguish intact single cells from debris or cell fragments?
- Aspect ratio and area of the brightfield image (Correct answer)
- Median pixel intensity across all fluorescence channels
- Spectral spillover coefficient for each fluorochrome
- Time-of-flight value from the fluidic transit time
Correct answer: Aspect ratio and area of the brightfield image
Brightfield aspect ratio (height-to-width ratio) and cell area measurements allow discrimination of intact round cells from elongated debris or cell fragments.
SCYM Data Analysis 4
In cytometry, what is the definition of 'median fluorescence intensity' (MFI), and why is it preferred over mean in skewed distributions?