ISAC Data Analysis and Interpretation 2 — Questions and Answers
Question 1: In flow cytometry, what does the term 'spread' refer to when evaluating instrument performance?
- The width of a population cluster on a dot plot
- The difference between the highest and lowest MFI values
- The CV of a bead population measured on a single parameter (Correct answer)
- The distance between positive and negative populations
Correct answer: The CV of a bead population measured on a single parameter
Spread refers to the coefficient of variation (CV) of a bead population, indicating how tightly the instrument measures a single parameter.
Question 2: Which gating strategy is BEST suited for identifying rare populations that overlap with more abundant cells?
- Rectangular gates on a dot plot
- Hierarchical sequential gating with Boolean combinations (Correct answer)
- Threshold gating on a single parameter histogram
- Polygon gates applied directly to the total population
Correct answer: Hierarchical sequential gating with Boolean combinations
Hierarchical sequential gating combined with Boolean logic progressively enriches for rare populations by eliminating unwanted cells step-by-step.
Question 3: What is the primary purpose of performing a fluorescence minus one (FMO) control?
- To determine the spillover coefficient for compensation
- To set the gate boundary for a specific marker by showing background spread (Correct answer)
- To verify that the laser is calibrated to the correct wavelength
- To confirm that all antibodies are at saturating concentrations
Correct answer: To set the gate boundary for a specific marker by showing background spread
FMO controls reveal the spread of fluorescence into a detector from all other fluorochromes except the one being gated, defining the true boundary of negative populations.
Question 4: When using viSNE (t-SNE based visualization) for high-dimensional cytometry data, each point on the map represents:
- A cluster of cells averaged together
- An individual cell from the original dataset (Correct answer)
- A single fluorescence parameter
- A time point from a kinetic experiment
Correct answer: An individual cell from the original dataset
In viSNE, each point represents a single cell, with proximity on the map reflecting similarity in high-dimensional marker expression.
Question 5: A researcher notices that their CD4 and CD8 staining produces a population with double-positive cells that should not exist physiologically in peripheral blood. What is the MOST likely cause?
- Instrument PMT voltage set too high
- Insufficient compensation for spectral overlap between the fluorochromes used (Correct answer)
- Incorrect threshold setting on the forward scatter parameter
- The antibody clones are recognizing the same epitope
Correct answer: Insufficient compensation for spectral overlap between the fluorochromes used
Inadequate compensation causes spillover fluorescence to falsely elevate signal in adjacent channels, creating artifactual double-positive populations.
Question 6: In mass cytometry (CyTOF), what replaces fluorescent dyes as reporters?
- Quantum dots of varying sizes
- Heavy metal isotope-conjugated antibodies (Correct answer)
- Radioactive isotope-labeled antibodies
- Enzyme-linked polymers detected by mass spectrometry
Correct answer: Heavy metal isotope-conjugated antibodies
CyTOF uses antibodies conjugated to heavy metal isotopes (e.g., lanthanides), which are detected by time-of-flight mass spectrometry rather than fluorescence.
Question 7: What does a high coefficient of variation (CV) in a peak separation experiment indicate?
- Excellent instrument resolution and alignment
- Poor precision in measuring that fluorescence parameter (Correct answer)
- High signal intensity from the fluorochrome used
- Successful compensation of spectral overlap
Correct answer: Poor precision in measuring that fluorescence parameter
A high CV indicates that measurements of a single population are widely spread, reflecting poor instrument precision or suboptimal setup.
ISAC Data Analysis and Interpretation 2
In flow cytometry, what does the term 'spread' refer to when evaluating instrument performance?