Effects of swarm of particles in flow cytometers
When studying a subset of EV's with a flow cytometer, the results will be affected by the percentages of particles that are of non-interest1 . The effects are multiple, also when using fluorescence labeling.
When labeling EV's and using fluorescence triggering, it can happen that particles of not-interest are observed in scattering mode. This changes the results by moving the distribution towards higher values. But, overall, the number of detected events is related only to the labeled particles. Therefore, dilution shows a linear dependence with the events, but not with the scattered intensity.
In case there is a population of fluorescent particles, even if they are excited at different wavelengths, a signal may appear. This leads to the identification of false-positives.
Are the false positives of multiply labeled fluorescent beads due to energy transfer (à-la FRET)? The paper(Libregts 2018) attributes the effect to having many particles on the same spot
Diluting the samples is enough to overcome the problems, but the acquisition times become longer. Most of the problems are associated with swarming of particles, that can be overcome if the concentration of particles of non-interest is below a certain threshold, but it is not clear what threshold exactly.
Journal of Thrombosis and Haemostasis, 16, 1423-1436 (Libregts, ..., E.N.M. Nolte-'T Hoen, ...) ↩