Airway Inflammation and Cellular Stress in Noneosinophilic Atopic Asthma: Study Design


Airway Inflammation and Cellular Stress in Noneosinophilic Atopic Asthma: Study DesignSpirometry and reversibility tests, skin-prick reaction tests, blood sampling for total serum IgE count, and radioallergosor-bent tests for a battery of common allergens were performed on the first visit. Spirometry was performed with a computerized system (MasterLab, 2.12; Jaeger; Wuerzburg, Germany) according to standardized guidelines. Subjects refrained from using long-acting and short-acting bronchodilators for 12 h and 6 h, respectively, prior to the measurements being made and did not smoke or drink tea or coffee during the morning of the investigation. Reversibility was assessed by the inhalation of 400 |j,g of salbutamol via a metered-dose inhaler.
Sputum induction was performed within 2 weeks after the first visit. According to sputum cytology findings, patients with NEA were identified and compared to EA and COPD patients. Using the data on the normal ranges of sputum cellular counts in the study by Belda et al, NEA was defined by the presence of eosinophils comprising < 2.2% of the total nonsquamous cell count.
Sputum was induced and processed as previously described. Based on previous reports that induction time significantly affects the cellular and fluid-phase measurements in induced sputum, we chose 20 min as the standard duration of sputum induction in all subjects. Total and differential cell counts were performed using standard methods.
Immunostaining procedures for HO-1, iNOS, and nitrotyrosine were performed as previously described. On immuno-stained slides, polymorphonuclear cells and macrophages were identified by morphologic analysis, and positively stained cells within 300 cells of each leukocyte population were counted using a light microscope. The results were expressed as the percentage of positive cells within each leukocyte population. All analyses were performed in a blind fashion by two investigators, and the results were averaged. Three replicate measurements were performed by each observer in 10 randomly selected slides. Both the intraobserver and interobserver coefficient of variation were < 10%.