Identification of eosinophils in lysed whole blood using side scatter and CD16 negativity

The identification of eosinophils in lysed whole blood by flow cytometry can be problematic, since these cells overlap significantly with the neutrophil cluster on forward scatter versus side scatter plots of whole blood samples. Current methods can be time-consuming when running multiple samples or may compromise yield in the interests of greater accuracy. The use of eosinophil purification techniques prior to FACS analysis or sorting as a way of ensuring purity may have unpredictable effects on eosinophil activation, leading to questionable data interpretation. Here we describe a simple, single-step method for definition of eosinophils utilizing their high side scatter and CD16 fluorescence negativity to differentiate them from neutrophils. The purity of the neutrophil and eosinophil populations sorted with this gate is close to 100% regardless of the peripheral blood eosinophil count, while the population obtained by sorting on a plot of FSC/SSC was a mixture of eosinophils and neutrophils. We suggest this method as a simple, reproducible, and accurate way of defining eosinophils by flow cytometry for analysis or sorting.