SCYM Principles of Flow Cytometry & Instrumentation 5 — Questions and Answers
Question 1: What is the significance of the 'coefficient of variation' (CV) when evaluating flow cytometer performance?
- It measures the ratio of positive to negative cell populations
- It quantifies the spread (precision) of a fluorescent bead or cell population around its mean (Correct answer)
- It describes the efficiency of cell sorting recovery
- It indicates the percentage of cells that are viable in a sample
Correct answer: It quantifies the spread (precision) of a fluorescent bead or cell population around its mean
CV (standard deviation ÷ mean × 100%) measures how tightly a population clusters around its mean intensity; lower CV indicates better instrument precision and resolution.
Question 2: Which of the following best describes 'carryover' (also called cross-contamination) in a flow cytometer?
- Spectral spillover of one fluorochrome's emission into another detector
- Residual cells from a previous sample that appear in the next sample's data (Correct answer)
- Laser light that is not absorbed by the fluorochrome and passes to detectors
- Background autofluorescence that persists between samples
Correct answer: Residual cells from a previous sample that appear in the next sample's data
Carryover occurs when cells from a highly concentrated or strongly positive previous sample remain in the fluidic system and contaminate subsequent sample acquisitions.
Question 3: What is the role of the 'sample core' in hydrodynamic focusing?
- It provides the pressurized buffer that surrounds and aligns the sample stream
- It is the central narrow stream of sample fluid containing the cells being analyzed (Correct answer)
- It is the electronic signal pathway from the PMT to the data acquisition system
- It refers to the central wavelength of the laser beam used for excitation
Correct answer: It is the central narrow stream of sample fluid containing the cells being analyzed
The sample core is the inner fluid stream containing cells; it is focused to a narrow diameter by surrounding sheath fluid, ensuring cells pass through the laser one at a time.
Question 4: Why is it important to use antibody capture beads (rather than cells) as compensation controls when possible?
- Beads have no autofluorescence, giving a cleaner negative population than cells (Correct answer)
- Beads can be stained with any antibody regardless of species or isotype specificity
- Beads require no fixation and can be used with live-cell protocols only
- Beads display all antigens found on the target cell population
Correct answer: Beads have no autofluorescence, giving a cleaner negative population than cells
Antibody capture beads have minimal autofluorescence, providing a very clean negative population and a bright positive signal, which makes setting compensation values more accurate than using cells.
Question 5: In a cell sorter, what is the 'sort delay' (or 'drop delay') calibration used for?
- Adjusting the time the cell spends in the laser beam for optimal signal collection
- Determining the precise time between laser interrogation and droplet charging to ensure the correct droplet is deflected (Correct answer)
- Setting the interval between consecutive sort pulses to prevent double sorts
- Measuring the time required for cells to travel from the nozzle to the collection tube
Correct answer: Determining the precise time between laser interrogation and droplet charging to ensure the correct droplet is deflected
Sort delay calibration determines the exact time between when a cell is analyzed at the laser and when the droplet containing that cell breaks off and must be charged for deflection into the sort gate.
Question 6: Which parameter distinguishes a stream-in-cuvette instrument design from a jet-in-air design?
- Stream-in-cuvette systems can only use a single laser for excitation
- In stream-in-cuvette systems, cells pass through a closed quartz flow cell, reducing biohazard risk but limiting sort capability (Correct answer)
- Jet-in-air systems use hydrostatic pressure instead of pressurized sheath fluid
- Stream-in-cuvette systems require the use of conductive sheath fluid for cell sorting
Correct answer: In stream-in-cuvette systems, cells pass through a closed quartz flow cell, reducing biohazard risk but limiting sort capability
Stream-in-cuvette designs enclose the flow path in a quartz cuvette, reducing aerosol generation and biohazard risk, but the closed system limits compatibility with high-speed electrostatic sorting.
Question 7: What is the primary purpose of performing a daily quality control (QC) run with fluorescent beads on a flow cytometer?
- To calibrate absolute cell counts for clinical samples
- To monitor instrument stability, verify laser alignment, and ensure detector performance is within acceptable limits (Correct answer)
- To set compensation values for the day's multicolor experiment
- To verify that the sheath fluid is free of contaminating particles
Correct answer: To monitor instrument stability, verify laser alignment, and ensure detector performance is within acceptable limits
Daily QC with fluorescent beads tracks instrument performance over time by monitoring MFI values, CV, and peak position to detect instrument drift, alignment issues, or component failure.
SCYM Principles of Flow Cytometry & Instrumentation 5
What is the significance of the 'coefficient of variation' (CV) when evaluating flow cytometer performance?