Although asthma is a common chronic airway inflammatory disease in children, the natural history of asthma remains unknown. It is difficult to diagnose asthma in children aged under 5 years because of the complexity of wheezing phenotypes at that age [
]. To date, no reliable biomarkers have been developed to distinguish asthma from non-asthmatic wheezing in very young children. The asthma prediction index (API) is a practical management tool for use on young patients experiencing recurrent wheezing in clinics [
], but the sensitivity and specificity of API are limited, especially for cases in which wheezing episodes are less frequent [
]. Therefore, it is urgent to explore potential effective biological markers for predicting asthma, especially in this age group.
Basophils are important immune cells that are involved in allergic diseases including asthma [
]. CD63-based basophil activation tests (BATs) are a useful tool for not only identifying specific allergens, but also monitoring immunologic homeostasis [
]. BATs are used for diagnosing food and drug allergies with high specificity and sensitivity [
]. Recent research has indicated that basophil allergen threshold sensitivity tests (CD-sens), which are based on BATs, have potential use for monitoring specific immunotherapy [
] and biological agent therapy [
]. Therefore, we investigated the potential clinical value of BAT results for predicting asthma in younger children in this pilot study.
The present prospective cohort study was conducted to explore the relationship between BAT results and final asthma outcomes in children with a recurrent wheeze. The clinical value of this approach for the early diagnosis of asthma in this age group is discussed.
API is a practical tool for predicting the risk of asthma in preschool children; however, its predictive values are limited when wheezing episodes are infrequent [
]. The discovery of a biological marker predictive of asthma development would be of great clinical value. To date, several biomarkers, such as the fractional concentration of exhaled nitric oxide (FeNO), blood eosinophils, sIgE, exhaled breath condensate, and periostin, have been evaluated with varying results, but none have been ideal [
In this pilot study, we examined the clinical value of the BAT for predicting asthma. We recruited children aged under 5 years who presented with wheezing or a history of wheezing episodes. We performed a BAT on patients’ samples at the time of their enrollment and followed the subjects for 2 years to see whether they were diagnosed with asthma during that time. We first analyzed the expression levels of the basophil activation marker CD63 at baseline and found no significant difference between children who were later diagnosed with asthma and those who were not. Because inhalants are closely related to asthma symptoms, we then performed a BAT on basophils stimulated with a mixture of inhalant allergens. The CD63 expression levels and positive rates of the CD63-based BAT were significantly higher in the children diagnosed with asthma than in those who were not diagnosed with asthma. Despite the low wheezing frequency in our subjects, the performance of the CD63-based BAT for predicting asthma diagnosis in our study was similar to that of API.
Basophils have been reported to play important roles in allergic diseases, such as asthma, AR, and AD, in animal disease models and in vitro studies [
]. Our group previously confirmed that basophils participate in Th2 immune responses and allergic airway inflammation as an important immune cell [
]. Despite ongoing debate about the importance of basophils in initiating Th2 immune responses and allergic inflammation [
], recently, increasingly more research has focused on the clinical utilization of BAT. This test not only has diagnostic value for the identification of specific allergen sensitization and diagnosis of food or drug allergy with high specificity and sensitivity [
], but it also has great clinical value in measuring a potential biomarker for monitoring allergen-specific immunotherapy [
] and biological therapies for allergic diseases [
]. Because of the many factors that influence wheezing and the complexity of the underlying mechanism of asthma, there are no known criteria for asthma diagnosis in younger children. The present study assessed a potential biomarker for the prediction and early diagnosis of asthma in young children.
In summary, our exploratory study revealed that the BAT has potential clinical value for predicting asthma diagnosis in children with recurrent wheezing episodes. However, this study had some limitations in its sample size and follow-up time; thus, the conclusions may have some limitations. Further studies with a larger sample size and prolonged follow-up time that additionally perform BAT when the subjects are free of symptoms and medication are needed to confirm our current findings.
Study design and population
This study was a prospective cohort observation trial conducted from July 2017 to March 2018 at Shanghai Xinhua Hospital and Shanghai Children’s Medical Center affiliated with Shanghai Jiao Tong University School of Medicine. Children aged under 5 years with a convincing history of wheezing episodes (confirmed by the researchers or documented as “expiratory wheeze” in previous medical records) were enrolled. The exclusion criteria were other coexisting chronic respiratory illnesses, e.g., bronchopulmonary dysplasia, tracheobronchial foreign bodies, congenital heart disease, or acute or chronic infectious diseases. Patients who had received immunomodulators 3 months before or during treatment were also excluded. The Ethics Committee at Xinhua Hospital approved the study. The children’s legal guardians provided written informed consent before the research began.
The children’s histories of allergic diseases (such as AD and AR as diagnosed by a specialist) and family histories of allergic diseases were obtained through detailed questionnaires completed by their parents.
Serum sIgE measurement
All enrolled subjects participated in sIgE testing. Serum samples were measured via the DX-Blot 45 Automatic Western Blotting instrument (Hangzhou Zhejiang University Dixun Biological genetic Engineering Co., Ltd. Hangzhou, Zhejiang, China) as per the manufacturer’s instructions.
Basophil activation test
Anticoagulated peripheral blood was collected and stored at 2–8 °C for up to 6 h. The Basophil Activation Test Set (Bühlmann Laboratories, Germany) was used for the BAT. In vitro stimulation was performed using an inhalant mix (BAG-IX1, Bühlmann Laboratories) that included common inhalation allergens (GX1, G12, T3, T4, W6, W9, M6, D1, D2, E1, and E2) as specific antigens. Cell stimulation buffer (100 μl) containing FITC-labeled anti-CD63 and PE-labeled anti-CCR3 monoclonal antibodies was added to three aliquots of 50 μl of blood. Every aliquot was combined with 50 μl of cell stimulation buffer containing 250 ng/ml inhalant mix as a test sample, 50 μl of cell stimulation buffer containing anti-FcεRI Ab as a positive control, or 50 μl of cellular stimulation buffer only as the baseline control. The samples were mixed and incubated at 37 °C in a water bath in the dark for 30 min. After the red blood cells were lysed, the cells were resuspended in phosphate-buffered saline and assessed with a CytoFLEX flow cytometer (Beckman Coulter, USA). Basophils were gated as CCR3
+ cells with low side scatter. The CD63
+ cells in this group were termed “activated basophils”. We acquired data for at least 500 basophils, and analyzed these data with CytExpert software (version 18.104.22.168). The test result was considered positive when the level of CD63 expression in stimulated cells was > 15% over the baseline level. All BATs were performed within 6 h after blood sampling.
The included children were followed up for 2 years in our clinic. Patients who required controller therapy visited the clinic monthly. Otherwise, the patients visited our clinic only when they had respiratory symptoms. All patients visited the clinic at least once a year, and an annual telephone interview was conducted with their parents through questionnaires regarding wheezing episode frequency, asthma diagnosis by a pediatric respiratory specialist, and medication for recurrent wheezing. Each asthma diagnosis was made by a pediatric respiratory specialist in accordance with guidelines for the diagnosis and management of asthma in children (2016), which were developed by the Chinese Pediatric Society, Chinese Medical Associatio n[
] and Global Strategy for Asthma Management and Prevention (2017) (Chapter 6, Asthma in children 5 years and younger). For children older than 5 years of age, the asthma diagnosis was based on typical respiratory symptoms, lung function test results, and FeNO levels. For children less than 5 years of age, the asthma diagnosis was evaluated at the end of the 2-year follow-up period on the basis of recurrent wheezing, response to bronchodilator treatment, history of allergic disease, allergen sensitization, history of asthma in first-degree relatives, and clinical improvement during 3 months of controller treatment (ICS or montelukast).
All patients were assessed by API on the basis of their parents’ responses to detailed questionnaires, medical record information obtained from annual outpatient services, serum sIgE, blood eosinophilic cell counts, and follow-up results by phone. Participants with one major or two minor risk factors were defined as API-positive [
]. Parental history of asthma, physician-diagnosed AD, and sensitization to no less than one inhalant allergen were defined as major risk factors. Minor risk factors included sensitization to food allergens, eosinophil levels in the peripheral blood of ≥4%, and wheezing unrelated to a cold. Children with no major factors and no more than one minor factor were defined as API-negative.
Participant demographic characteristics and wheezing features were summarized with amounts and percentages. Contingency tables were developed to compare the difference in qualitative variables between children diagnosed with asthma and those not diagnosed with asthma using a Chi-squared test. Continuous variables are represented by the median (interquartile range, IQR). A Mann-Whitney rank sum test was used to compare the non-normally distributed data. All statistical analyses were performed using SPSS v.22.0 (IBM SPSS Statistics, Armonk, NY, USA). A
p-value of < 0.05 based on a two-tailed test was considered statistically significant.
We thank Traci Raley, MS, ELS, and Katie Oakley, PhD, from Liwen Bianji, Edanz Editing China (
) for editing drafts of this manuscript. We thank the parents and children who participated in this study for their efforts.
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