Whole blood flow cytometric analysis of eosinophil EG2 expression as a marker of the pathological conditions of asthma

Using a simple technique detecting the eosinophil fraction in whole-blood flow cytometry, we measured intracellular antigen EG2 (a monoclonal antibody to eosinophil cationic protein) in 56 asthmatic patients (26 during an attack and 30 during an asymptomatic period) and 22 healthy subjects to determine whether EG2 reflects the pathological stages of allergy. Methods: In brief, preparations of the sample included the following procedures: (1) hemolyzation of heparinized or EDTA-mixed whole blood; (2) fixation of white blood cells with 0.4% parabenzoquinone (PBQ) or paraformaldehyde (PFA); (3) permeabilizing the cell membrane with n-octyl-beta-D-glucopyranoside, and (4) staining of intracellular EG2 antigen with monoclonal EG2 antibody and FITC-labeled secondary antibody. Results: In PBQ-fixed samples, there was a clearer boundary of the eosinophil fraction with a higher yield and purity than in those fixed with PFA. The number of EG2-positive eosinophils was significantly greater in subjects during attacks than in asymptomatic patients. In addition, when compared with normal controls, asthmatic subjects had significantly greater numbers of EG2-positive eosinophils regardless of their current condition. Conclusion: Eosinophil intracellular EG2 may indicate the pathological stage of asthma. This simple technique for analysing the properties of eosinophils using whole-blood flow cytometry would save time and labor in laboratories.