Data Analysis Flashcards

In flow cytometry, what does the term 'compensation' correct for?

Answer: Spectral spillover of fluorochromes into adjacent channels

Compensation mathematically removes spectral spillover, where emission from one fluorochrome bleeds into a detector channel assigned to another fluorochrome.

Which gating strategy is most appropriate for identifying rare cell populations comprising less than 0.01% of total events?

Answer: Sequential hierarchical gating with back-gating validation

Sequential hierarchical gating progressively enriches the population of interest, and back-gating confirms the identified population is correctly placed in earlier scatter plots.

What is the purpose of using a 'fluorescence minus one' (FMO) control?

Answer: To determine the boundary of positivity in the presence of spectral spillover

FMO controls contain all fluorochromes except the one being measured, revealing where the negative population boundary falls after compensation is applied.

When analyzing a bivariate dot plot, a 'cloud' of events forming a diagonal line from low-low to high-high typically indicates:

Answer: A positive correlation between the two measured parameters

A diagonal cluster from low-low to high-high in a bivariate plot indicates that the two parameters co-vary positively, suggesting co-expression or biological co-regulation.

In mass cytometry (CyTOF), which approach replaces traditional spectral compensation?

Answer: Minimal signal spillover due to narrow isotope mass windows

Mass cytometry uses heavy metal isotope tags with minimal mass overlap, so the need for spectral compensation is largely eliminated compared to fluorescence-based cytometry.

Which statistical method is most commonly used to assess whether two cell populations in a cytometry experiment are significantly different?

Answer: Student's t-test or non-parametric equivalent (e.g., Mann-Whitney U)

The t-test (or Mann-Whitney U for non-normal distributions) compares means of two groups, making it appropriate for evaluating differences between cell population frequencies or median fluorescence intensities.

What does a high coefficient of variation (CV) in a single-peak bead population indicate during quality control?

Answer: Broad peak width suggesting instrument performance issues

A high CV in a bead peak indicates broad distribution of events, which may reflect poor instrument alignment, suboptimal PMT voltage, or debris contamination.