Assay variation can be a major source of error, therefore it is critical to understand the statistics behind rare event analysis. The cytometry friendly Gaussian distribution cannot be applied to the analysis of such a low number of events (the term "rare" is given to events with a frequency of 0.01% or less) and the best approach is to use Poisson statistics (Fig.1). Good experimental practice suggests to keep Coefficient of Variation (CV) below 5%, and thus the number of events to acquire should be defined in order to maintain the lowest CV possible and in any case below 5%.
The number of events that satisfy a given criterion (e.g they are positive for a marker, P) is defined as
$R$=events that meet a given criterion
As with all statistical distribution,
the variance (V) is defined as
$V(R) = NP(1-P)$
The standard deviation (SD) is
and the coefficient of variation is
Figure 1. Poisson statistics defines the probability that a number of events will occur in a fixed interval of time/space/volume.