SCYM Flow Cytometry Principles 5 — Questions and Answers
Question 1: Which gating hierarchy is standard for T-cell subset analysis in peripheral blood?
- CD3+ → Lymphocytes → CD4+/CD8+
- Lymphocytes → CD3+ → CD4+/CD8+ (Correct answer)
- CD4+/CD8+ → CD3+ → Lymphocytes
- CD45+ → CD4+/CD8+ → CD3+
Correct answer: Lymphocytes → CD3+ → CD4+/CD8+
Standard T-cell gating proceeds from scatter-based lymphocyte gate → CD3+ T cells → CD4+ helper or CD8+ cytotoxic subsets.
Question 2: What is autofluorescence in the context of flow cytometry, and how does it affect results?
- Fluorescence emitted by the sheath fluid that interferes with scatter signals
- Natural emission from cellular components that can mask dim fluorochrome signals (Correct answer)
- Cross-reactivity of secondary antibodies with multiple targets
- Laser-induced cell death that reduces positive events
Correct answer: Natural emission from cellular components that can mask dim fluorochrome signals
Autofluorescence from cellular components like flavins and NAD(P)H raises background fluorescence, reducing sensitivity for dim markers.
Question 3: In cell cycle analysis using propidium iodide (PI), cells in G2/M phase appear as:
- A peak at half the G1 fluorescence intensity
- A peak at twice the G1 fluorescence intensity (Correct answer)
- A broad distribution between G1 and G2/M peaks
- A peak with the same intensity as G1 but broader CV
Correct answer: A peak at twice the G1 fluorescence intensity
G2/M cells have doubled their DNA content compared to G1, so they incorporate twice as much PI and appear at double the fluorescence intensity.
Question 4: Which of the following correctly describes the role of Fc receptor blocking in flow cytometry?
- It prevents laser-induced photobleaching of fluorochromes
- It reduces non-specific antibody binding to Fc receptors on monocytes and NK cells (Correct answer)
- It enhances specific antibody binding to target antigens
- It blocks intracellular antigens from being stained by surface antibodies
Correct answer: It reduces non-specific antibody binding to Fc receptors on monocytes and NK cells
Fc receptor blocking agents (e.g., human IgG, FcR block) occupy Fc receptors on monocytes and NK cells to prevent non-specific antibody uptake.
Question 5: What is the advantage of using a violet laser (405 nm) in addition to a blue laser (488 nm)?
- It allows excitation of PE without spillover into FITC
- It enables use of violet-excited dyes like BV421, expanding the number of detectable parameters (Correct answer)
- It replaces the need for red laser excitation of APC
- It improves forward scatter resolution for small particles only
Correct answer: It enables use of violet-excited dyes like BV421, expanding the number of detectable parameters
A violet laser excites Brilliant Violet and Pacific Blue dyes that are not well excited at 488 nm, allowing additional parameters to be added to multicolor panels.
Question 6: When designing a multicolor flow cytometry panel, the brightest fluorochromes should be assigned to:
- The most abundantly expressed antigens
- The most dimly or rarely expressed antigens (Correct answer)
- The isotype control channels only
- The scatter parameters as a reference
Correct answer: The most dimly or rarely expressed antigens
Bright fluorochromes maximize detection of low-density antigens, while dim antigens would be missed if paired with dim fluorochromes.
Question 7: What does a bimodal distribution on a histogram suggest about an antigen's expression in a cell population?
- The antibody had poor binding affinity and produced nonspecific staining
- Two distinct subpopulations exist: one positive and one negative for the antigen (Correct answer)
- The instrument was misaligned, causing a split peak artifact
- Over-compensation resulted in false-positive and false-negative populations
Correct answer: Two distinct subpopulations exist: one positive and one negative for the antigen
A bimodal histogram indicates two distinct populations — one expressing the antigen (positive) and one not expressing it (negative), which is expected for many lineage markers.
SCYM Flow Cytometry Principles 5
Which gating hierarchy is standard for T-cell subset analysis in peripheral blood?