Study subjects
The present study consecutively enrolled 32 patients with severe early-onset asthma (age of onset < 12 years) and 32 patients with severe late-onset asthma (age of onset > 12 years) at Shenzhen People’s Hospital (the First Affiliated Hospital of Southern University of Science and Technology, the Second Clinical Medical College of Jinan University) from July 2018 to January 2019. All participants were aged > 18 years. According to the Global Initiative for Asthma (GINA) 2017 classification, all subjects met the diagnostic criteria of severe persistent asthma (therapy step 4 or 5). All asthma patients had a history of asthma, and preformed sputum induction, spirometry test, fractional exhaled nitric oxide (FeNO), and venous blood sampling. Based on the age of onset, all subjects were divided into 2 groups: early-onset group and late-onset group.
Patients who met the following criteria were excluded: (1) acute heart failure, severe organ failure, malignant tumors; (2) other lung diseases such as pulmonary infection, pulmonary hypertension, bronchiectasis which seriously affect lung function, sputum cell classification and other test results; (3) pregnancy and lactation; (4) recent surgery history; and (5) anybody has been accepted biologic therapies recently.
This research protocol was approved by the Medical Ethics Committee of Shenzhen People’s Hospital, and all subjects have signed written informed consent before enrolment in the study.
Study design
On day 1 information of baseline characteristics and clinical scores of all subjects was collected, and the following determinations were also performed: FeNO, spirometry test, total Immunoglobulin E (IgE), antigen-specific IgE, IL-5, IL-17, HVEM, complete blood count (CBC), and differential leukocyte count (DLC). On day 3 induced sputum cell differential count from the subjects was detected.
FeNO measurements
Before spirometry analyses, FeNO was measured by the NIOX MINO device (Aerocrine AB, Solna, Sweden)at an exhalation flow rate of 0.05 L/s for 10 s according to the manufacturer’s instructions. The FeNO measurements were consistent with the American Thoracic Society (ATS) / European Respiratory Society (ERS) 2005 guidelines methods [12], and the normal exhaled NO value was set as 5–35 part per billion (ppb) for healthy adults.
Spirometry measurements
Spirometry test was performed using aV6200 spirometer (SensorMedics, USA) in accordance with the methods of the ATS/ERS task force to determine forced expiratory volume in 1 s (FEV1), FEV1 percentage of predicted (FEV1%pred), forced vital capacity (FVC) percentage of predicted (FVC%pred), FEV1/FVC ratio, maximal expiratory flow (MEF)25%, MEF50%, and maximal mid-expiratory flow (MMEF)75/25%. A predicted ratio for each parameter was calculated based on age, sex, height, and race.
Total IgE and specific IgE measurements
Total serum IgE levels were determined by an electrochemiluminescence immunossay (ECLIA) method from Roche (Mannheim, Germany). Antigen-specific IgE was measured by the enzyme-linked immunosorbent assay (ELISA) kits (HOB Biotech Group, Suzhou, China), and allergen-specific IgE levels were considered positive sensitization by a value ≥ 0.35 IU/L.
Serum cytokines and HVEM measurements
The serum levels of IL-5, IL-17 and HVEM were measured using ELISA kits according to the manufacturer’s instructions (Beiluo Biotechnology, Beijing, China).
CBC and DLC tests
CBC and DLC parameters including red blood cells (RBCs), platelets and leukocytes were measured by an automated Sysmex XN-3000 (Sysmex Corporation, Kobe, Japan), using impedance and optic scatter method.
Sputum induction and cell differential count
Sputum induction was performed with hypertonic saline inhalation, using a ultrasonic atomizer as previously described. The sample was considered qualified if it presented with cells viability ≥ 60% and ≤ 20% squamous cell. The cells were centrifuged and resuspended in saline, and sputum slides were prepared by cytocentrifugation for cytological examination. The slides were air-dried and stained with Giemsa. Differential cell count was measured by counting 400 nucleated cells per slide with high power (×400) magnification.
Statistical analysis
SPSS 13.0 (SPSS Inc., Chicago, USA) was used for statistical evaluations. Normally parametric and nonparametric data are presented as means (standard deviation, SD) and medians (quartile 1 − quartile 3). The independent-samples t test was applied for difference in parametric variables between two groups, and the Mann–Whitney U test for the nonparametric data between two groups. Categorical variables were compared using the chi-square test. Correlation between the asthmatic risks and parameters were assessed by multivariate logistic regression analysis. Receiver operating characteristic (ROC) curves were generated to evaluate the values of biomarkers for distinguishing between two groups. Linear correlation analysis was performed to assess the linear relationship between two variables. A P value of less than 0.05 (P < 0.05) was considered as a statistically significant difference.