Identification and analysis of eosinophils by flow cytometry using the depolarized side scatter-saponin method

Abstract

This study describes a method for flow cytometric (FCM) identification and analysis of eosinophlis (EOS) in mixed granulocyte populations without the need of further cell purification. Granulocytes were prepared from 10 ml blood samples obtained from 14 non-atopic healthy subjects and 16 hypereosinophilic subjects. The cells were fixed in paraformaldehyde (PFA) and permeabilized with either saponin or n-octyl-beta-D-glucopyranoside (OG) before FCM. Side scatter light (SS) and depolarized sideward light scatterings (dSS) were measured before and after granulocyte fixation and permeabilization. Based on autofluorescence of cells, EOS were depicted in forward scatter (FS)-SS and FS-dSS cytograms. FS-dSS analysis of PFA-saponin- treated cells provided the best discrimination between EOS (high dSS) and neutrophils (low dSS) in normal and hypereosinophilic blood samples (n = 22). Subsequent sorting and differential staining of the two populations further confirmed cell identity in high dSS (97.2 ± 3.0% EOS)and low dSS (99.6 ± 0.5% neutrophils) PFA-saponin-treated cells (n = 5). Similar FS-dSS profiles were obtained for hypodense (HBSS-1.082 g/ml interface) and normodense (1.082-1.100 g/ml interface) EOS separated by centrifugation on discontinuous Percoll gradients and treated with PFA-saponin. Using this method, we successfully detected the cytokines interleukin-2 (II-2), IL-4, IL-5, and interferon γ (IFNγ) in blood EOS of normal and hypereosinophilic subjects (n = 12). In conclusion, this method allows the analysis of EOS intracellular antigens in mixed granulocyte populations obtained from small blood samples. This analysis is not affected by the buoyant density of eosinophils, and can therefore be applied for the analysis of blood EOS in various types and severities of diseases.