SCYM Data Acquisition & Analysis 5

Question 1

In mass cytometry (CyTOF), heavy metal isotopes replace fluorochromes. Which statement best describes why spillover is minimized in CyTOF?

Accepted Answer

Atomic mass channels are nearly discrete with minimal spectral overlap, unlike broad fluorochrome emission spectra

Answer

Mass-to-charge detection separates isotopes by atomic mass, which have much narrower 'overlap' than optical fluorochrome emission spectra, allowing 40+ parameters with minimal crosstalk.

Question 2

After acquiring data, a researcher finds the negative population in the FITC channel is shifted higher than expected. The MOST likely cause is:

Accepted Answer

Autofluorescence from the cells contributing to the FITC channel signal

Answer

Cellular autofluorescence is most prominent in the FITC (green) channel and raises the apparent fluorescence of unstained cells, mimicking dim positive staining.

Question 3

Which parameter is most commonly used as the trigger (threshold) channel in standard flow cytometry of whole blood samples?

Accepted Answer

FSC (forward scatter)

Answer

FSC-based triggering is standard for most applications because cell size (forward scatter) effectively distinguishes cellular events from electronic noise.

Question 4

A researcher is comparing two experiments run on different days and finds the MFI of a common marker differs by 30%. The best strategy to enable cross-experiment comparison is:

Accepted Answer

Use standardized reference beads to normalize instrument settings and MFI values across runs

Answer

Calibration beads with known fluorochrome intensities enable instrument-independent normalization, allowing MFI comparisons across different days and instruments.

Question 5

In Boolean gating analysis, what does the gate 'CD3+ AND CD4+ AND NOT CD8+' identify?

Accepted Answer

Helper T cells (CD4 single-positive)

Answer

CD3+CD4+CD8− defines conventional CD4 helper T cells, which express CD3 and CD4 but not CD8.

(SCYM) Specialist in Cytometry Practice Test

(SCYM) Specialist in Cytometry Practice Test

SCYM Data Acquisition & Analysis 5