SCYM Compensation Techniques 2

Question 1

In multicolor flow cytometry, what is the consequence of under-compensation for spectral spillover?

Accepted Answer

False-positive signal appears in the spillover channel

Answer

Under-compensation leaves residual donor signal in the acceptor channel, creating a false-positive shift.

Question 2

Which mathematical relationship defines the spillover coefficient used in compensation matrices?

Accepted Answer

Ratio of signal in off-target channel to signal in primary channel for a single-stained control

Answer

The spillover coefficient is calculated as the off-target channel signal divided by the primary channel signal from a single-color control.

Question 3

When using AutoMACS beads for compensation controls, what is the critical requirement for accuracy?

Accepted Answer

Bead signal must be at least as bright as the brightest cell population

Answer

Compensation beads must generate a positive signal at least as bright as experimental cells to correctly determine spillover.

Question 4

In a compensation matrix for an 8-color panel, how many spillover values must be determined?

Accepted Answer

56

Answer

For n colors, there are n×(n−1) = 8×7 = 56 off-diagonal spillover values in the compensation matrix.

Question 5

What effect does increasing PMT voltage have on the measured spillover coefficient?

Accepted Answer

It has no effect if the detector is operating in the linear range

Answer

Spillover coefficients are ratios and remain constant across PMT voltages as long as detectors remain in their linear range.

(SCYM) Specialist in Cytometry Practice Test