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-bl ood flow cytometry, we measured intracellular antigen EG2 (a monoclona l antibody to eosinophil cationic protein) in 56 asthmatic patients (2 6 during an attack and 30 during an asymptomatic period) and 22 health y subjects to determine whether EG2 reflects the pathological stages o f allergy. Methods: In brief, preparations of the sample included the following procedures: (1) hemolyzation of heparinized or EDTA-mixed wh ole blood; (2) fixation of white blood cells with 0.4% parabenzoquinon e (PBQ) or paraformaldehyde (PFA); (3) permeabilizing the cell membran e with n-octyl-beta-D-glucopyranoside, and (4) staining of intracellul ar 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 tho se fixed with PFA. The number of EG2-positive eosinophils was signific antly 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 regardle ss of their current condition. Conclusion: Eosinophil intracellular EG 2 may indicate the pathological stage of asthma. This simple technique for analysing the properties of eosinophils using whole-blood flow cy tometry would save time and labor in laboratories.