Identification of eosinophils by flow cytometry

A flow cytometric method to identify and characterize eosinophils in lysed whole blood samples was established. A gating protocol was applied that in the first step uses the high autofluorescence and the high sideward scatter of eosinophils. In the second step, eosinophils were differentiated from neutrophils by lack of CD16 expression or alternatively presence of CD49d expression. Eosinophils purified by density gradient centrifugation (purity: 93% eosinophils contaminated with 7% neutrophils) were used to evaluate the technique. We were able to identify eosinophils added back to lysed whole blood samples and to identify partial degranulated eosinophils after treatment with secretory IgA and anti-IgA. In addition we were able to show that due to a large overlap of sideward scatter, the technique is applicable to purified normodense as well as hypodense eosinophils. In addition, there was a good correlation (r = 0.921, P < 0.0001) between the percentage of eosinophils determined by flow cytometry and microscopic evaluation in 81 patients. In patients with atopic dermatitis there was a reasonable correlation between a severity score (SCORAD) and the number of eosinophils determined by flow cytometry (R = 0.6107, P = 0017). Since the technique proved to be able to identify activated eosinophils bearing the CD69 early activation antigen, the relation between serum creatinine and CD69 expression on peripheral blood eosinophils was analysed showing a positive correlation (r = 0.4344, P = 0.016).