METHODS
This was a cross-sectional observational study through the analysis of a spreadsheet containing data collected from asthma outpatient charts in a tertiary hospital. Stable asthmatics collected sputum from March 2018 to April 2020, once they met the sample viability criteria for analysis. The sociodemographic data, past and family history, skin allergy test (prick test), total serum IgE levels, blood eosinophil count, spirometry values and induced-sputum cytology of these patients were analyzed. The laboratory results used were those collected closest to the sputum collection date.
Asthma severity was classified according to the National Asthma Education and Prevention Program (NAEP) as mild, moderate, or severe5. Peripheral blood eosinophilia were considered when eosinophils count were above 400 × 10³/uL and total serum IgE was above 100 IU/mL6. Prick test was routinely performed in asthmatics. The tested allergens were Dermatophagoides pteronyssinus, Blomia tropicalis, Blatella germanica, Canis familiaris, Felis domesticus, Lolium multiflorum , in addition to a positive control (histamine 10mg/mL), and negative control (saline 0.9%). Tests were considered positive in the presence of a wheal with diameter ≥3 mm and a negative control (normal saline). Sputum collection and examination were performed as previously described by Pizzichini et al.7. Briefly, the sputum was separated from contaminating saliva. A mixture of sputum and dithiothreitol solution was vortexed for 15 min, and four volumes of Dulbecco’s PBS were added. The suspension was then filtered through 48-mm nylon gauze (Millipore Corporate Headquarters, Billerica, MA, USA) and centrifuged at 314×g for 10 min. Cell viability was determined by trypan blue exclusion. The cytospin slides prepared after cytocentrifugation were stained with
May–Grünwald–Giemsa to determine the differential cell count. Samples with up to 20% epithelial cells and more than 50% viability were considered adequate for analysis7. The differential counts in sputum were classified into one of the following standards: eosinophilic (eosinophils >2.5% and neutrophils ≤54%), neutrophilic (eosinophils ≤2.5% and neutrophils >54%), mixed (eosinophils >2.5% and neutrophils >54%), and pauci-granulocytic (eosinophils <2.5% and neutrophils <54%)8.
From spirometry results, forced expiratory volume value in the first second (FEV1) was considered normal if >80% predicted for age. In addition, patients with a ratio of forced expiratory volume in the first second to forced vital capacity (FEV1/FVC) <0.9 were diagnosed with obstructive ventilatory defect. The response to inhaled salbutamol (400mcg) was considered positive when FEV1 increased more than 12% of the baseline value9.
Patients responded to the asthma control test (ACT) questionnaire, and a score of ≥20 characterized controlled asthma10,11.
Continuous variables are expressed as means, standard deviation, median, and maximum and minimum values. The categorical variables (comparisons of cytological profiles with clinical, epidemiological, and functional variables) were analyzed using the Fisher’s exact test and considered significant when p-value <0.05.
The study protocol was approved by the Ethics and Research Committee of Hospital de Clínicas, Federal University of Paraná (CAAE: 29628220.4.0000.0096).