Lung clearance index is elevated in young children with symptom‐controlled asthma

Abstract Background Pulmonary function testing has been recommended as an adjunct to symptom monitoring for assessment of asthma control. Lung clearance index (LCI) measures ventilation inhomogeneity and is thought to represent changes in the small airways. It has been proposed as a useful early marker of airway disease in asthmatic subjects, and determining it is feasible in preschool children. This study aims to assess whether LCI remains elevated in symptomatically controlled asthmatic children with a history of severe asthma, compared with healthy controls. A secondary aim was to determine whether the results were consistent across the preschool and school‐aged populations. Methods Using a case‐control design, we compared 33 children with currently well‐controlled symptoms who had a history of severe asthma, to 45 healthy controls (age 3‐15 years) matched by age, height, and sex. We performed multiple breath washout tests using sulfur hexafluoride as a tracer gas, to determine their LCI and Scond values. Results In the overall study, LCI z‐score values were on average 0.86 units (95% confidence interval: 0.24‐1.47, P = 0.01, t‐test) higher in children with a history of severe asthma with current well‐controlled symptoms compared with healthy controls. In addition, within the subgroup of preschool children (age ≤ 6), the asthmatic had significantly higher LCI z‐score values than their healthy controls peers (mean (SD), 0.57 (2.18) vs −1.10 (1.00), P = 0.03, t‐test). Twenty‐seven percent (27%; 9/33) of subjects had an LCI value greater than the upper limit of our healthy controls despite being symptom controlled. Amongst preschool children, 5 (42%; 5/12) of the asthmatic children had abnormal LCI at the individual level. Conclusions LCI is elevated in children with asthma, which may be driven by differences in the preschool population. LCI may be useful in defining preschool asthma endotypes with persistent ventilation inhomogeneity despite symptomatic control.


| INTRODUCTION
Asthma is characterized by airway inflammation and airway hyperreactivity, leading to variable airflow obstruction and associated respiratory symptoms. 1 Clinical evaluation and spirometry are the current standards for the diagnosis and monitoring of asthma. 1 Many longitudinal cohorts report decrements in spirometry in children with asthma (compared with healthy children) that appear by school-age and persist through to adulthood, [2][3][4] yet, in practice, spirometry is often within the normal range in pediatric asthma patients and is challenging to perform in preschool asthmatics, where burden of disease is highest. 5 Although asthma is a disease of both central and peripheral airways, 6-8 FEV 1 , a spirometry-based test normally required for the objective assessment of asthma severity, measures mainly central airways obstruction. The small airways have been called the "quiet zone", 8,9 alluding to the challenges in its evaluation using conventional spirometric maneuvers. Therefore, interest in the development of technologies tailored to recognize early peripheral airway changes in asthma, feasible for all ages, has been a priority.
Lung clearance index (LCI), attained during the multiple breath washout (MBW) test, measures ventilation inhomogeneity. Because MBW depends on tidal breathing measures, it is feasible to measure in very young children. It has been suggested in the literature that LCI represents changes in the peripheral airways. 10 Airway biopsy studies suggest that inflammation persists in the small peripheral airways despite therapy, and in the presence of normal range spirometry. 11 Very little data exists in the literature about LCI in children with asthma and whether it may offer insight into endotypes of pediatric asthma. A study by Sonnappa et al 12 suggested that MBW indices were elevated in children with multi-trigger wheeze compared with healthy controls.
This study included symptomatic children and did not include an assessment of symptom control. Another study in older children also documented elevated LCI values compared with healthy controls, but again, no assessment of symptom control was made. 13 Magnetic Resonance Imaging data suggest persistent ventilation defects are present in children with preschool asthma, the size of which correlates with disease severity. 14 It is not clear if LCI, which provides a global estimate of lung ventilation heterogeneity, may provide insight into lung physiologic changes in asthmatic children who despite a history of severe exacerbations, are currently under symptomatic control.
The aim of this study was to determine whether LCI remained elevated in a cross-sectional cohort of asthmatic children with a history of severe exacerbations who currently had well-controlled symptoms of asthma compared with healthy controls. A secondary aim was to explore age-related differences in LCI amongst the asthmatic children. We hypothesized that LCI would be higher in asthmatic children compared with healthy controls despite their symptoms being well controlled.

| Study design and participants
We designed a case-control study in which children with asthma (cases) were compared with healthy children (controls).
Children (3-18 years old) with asthma were recruited from the severe Asthma outpatient clinics at the Hospital for Sick Children between December 2009 and May 2015. All children who were approached for this study had specialist physician-confirmed asthma that included the following: a history of physician-documented wheeze on >2 occasions, and either a clinical response to a bronchodilator or a bronchodilator response of greater than 12% change in FEV 1 . All children had a history of at least 1 hospitalization or >2 courses of oral corticosteroids within the past year for uncontrolled asthma symptoms or a history of uncontrolled asthma symptoms despite adequate treatment (Step 3-5 NHLBI treatment). For inclusion into this study, children with physician-diagnosed asthma had to have evidence of current symptom control, defined as: use of a short-acting bronchodilator <3 times per week; daytime asthma symptoms <3 times per week, and nocturnal awakenings due to their asthma ≤1 per week. Children ≥6 years of age able to perform spirometry had to have an FEV 1 in the normal range (> 80% after race-correction based on the reference equations from Global Lung Initiative (GLI) 15 ). Exclusion criteria included prematurity or low birth weight, a history of congenital heart disease, neuromuscular disorder or bone disease, a history of chronic lung disease other than asthma, respiratory infection or change in medication within the past 3 weeks, and history of smoking. Symptoms and medical history were obtained by questionnaire and chart review.
Healthy children were recruited from the general population to serve as the control group. Exclusion criteria included prematurity or low birth weight, a history of congenital heart disease, neuromuscular disorder or bone disease, history of chronic productive cough or recurrent wheezing or shortness of breath within the last 12 months, a history of any chronic lung disease including a diagnosis of asthma or reactive airway disease, any previous hospital admission for a respiratory condition, and history of smoking.
This study received approval from the Hospital for Sick Children research ethics board (REB # 1000013927), and consent was obtained from all parents or participants, where applicable.

| Pulmonary function tests
Pulmonary function was performed in a standardized order: MBW followed by spirometry. In children able to perform acceptable and reproducible spirometry testing, as per our clinical protocol, bronchodilator (Salbutamol 400 μg) was administered using a holding chamber.
Post-bronchodilator spirometry and MBW testing were repeated 15 minutes after bronchodilator administration. was used to measure LCI, using an AMIS 2000 mass spectrometer (Innovision A/S, Odense, Denmark). Washout testing was completed when the test gas concentration fell below 1/40 th of the initial concentration. All MBW trials were recorded and analyzed using a custom data acquisition and offline analysis program (TestPoint, Measurement Computing, Norton, MA, USA). Quality control was assessed posttesting as per the ATS/ERS statement 16 and included a stable washin with little fluctuation of the SF 6 signal, a clean disconnect below 0.1% SF 6 , a clean, regular, and representative functional residual capacity (FRC) breath (before disconnect), and finally, a stable and steady breathing pattern. Pre-gas sampling point and post-gas sampling point dead space was set to 0 and 0.0154 L, respectively. LCI, calculated as the number of lung turnovers required to reach a concentration in the tracer gas equal to 1/40 th of the starting concentration, is derived from the TestPoint software, as are S cond values, as previously described. 17 Quality control for the breath-by-breath analysis was performed first, before an S cond value was generated. As per previously described, 18 we excluded any breaths in each trial with >25% deviation from the mean tidal volume and any trials that had <66% of the breaths remaining after exclusion.

| Spirometry
Spirometry was performed using the Vmax Encore system (CareFusion, San Diego, CA, USA) and reported as per the ATS/ERS guidelines. 19,20 Spirometry z-scores and percent predicted values were calculated using the GLI equations. 15 Participants were grouped into self-reported ethnic groups, and parameters were race-corrected, as per GLI equations.

| Atopy
Aeroallergen sensitization was defined as a wheal size ≥2 mm larger than the saline control, to a standard panel of 14 aeroallergens. These aeroallergens included house dust mites (Dermatophagoides farine and pteronyssinus), cockroach, cat, dog, mouse, horse, feathers, tree mix, grass mix, ragweed, alternaria, hormodendrum, aspergillus mix. Eczema and allergic rhinitis history was based on parental report of a past diagnosis or typical symptoms. Atopic status was defined as any of the following: aeroallergen sensitization, a history of eczema, or atopic rhinitis. Atopy sensitization in controls was not assessed.

| Statistical analysis
Height, weight, and body mass index (BMI) centiles were calculated using WHO growth charts (WHO 2006). 21 Healthy controls and asthma children were matched by age, height, and sex. Baseline population characteristics were presented as median (interquartile range) or frequency (percentage), where appropriate. Comparisons between healthy and asthma groups for baseline population characteristics were performed using Student t-test or Mann-Whitney U test, where appropriate, for continuous variables, and chi-square test, for categorical variables.
The primary statistical analysis was to determine whether LCI remained elevated in asthmatic children with a history of severe exacerbations who currently had well-controlled symptoms of asthma, compared with healthy controls. Given our sample size of 45 healthy and 30 asthmatic children, we were able to detect a 0.7 z-scores difference and 0.8 z-scores difference between the 2 groups, with 80% and 90% power, respectively, at 0.05 significance level. Lung function parameters were presented as mean (standard deviation, SD) and analyzed both as raw scales and z-scores, calculated using the published reference equations for MBW 22 and spirometry. 15 To further investigate MBW (eg, LCI) and spirometry parameters (eg, FEV 1 ) in children with asthma compared with healthy controls, linear regression models were used, and difference and its associated 95% confidence interval (CI) between the 2 groups were estimated. The analyses were adjusted for age and height values when raw scale values were used, whereas no adjustment were performed when z-score or percent predicted values were used.
To investigate the secondary aim of age-related differences in LCI, we included an interaction term between age and group in the linear regression models to test its significance. In addition, we performed a subgroup analysis by dividing the children into preschool-age children (age ≤ 6) and school-age children (age > 6). Comparison between asthmatic and healthy controls within each age group was performed using 2-sample t test or Mann-Whitney U test, where appropriate.

Correlations between lung function parameters and clinical factors
were assessed using a Spearman's correlation. According to ATS/ERS recommendation, 23 the lower limit of normal and upper limit of normal (ULN) of lung function parameters were defined as 5 th and 95 th percentile of our healthy controls, respectively. Comparisons of lung function parameters between pre and post BD were performed using paired t-test. Statistical analyses were performed using SAS version 9.4 (SAS Statistical Software, Cary, NC, USA). Statistical tests were 2 sided, and significance level was set at P < 0.05.

| Study participants
The current study included 33 asthmatic and 45 healthy controls children matched for age, height, and sex. Demographic comparisons between the 2 groups are presented for the overall age (Table 1), and by preschool-age and school-age (   (Table 4). We repeated the analysis by removing 2 subjects with extreme LCI values, and our results remained robust, that is LCI is still elevated in asthmatic children compared with healthy controls in the overall population (P = 0.03, t-test). In addition, we found that the age group (preschool vs school-age) had a significant interaction effect on LCI z-score (P = 0.04, t-test of coefficient from linear regression), indicating that there was an age-related different in LCI z-score between the 2 groups. This was confirmed with the analysis within subgroups of preschool-age children (age ≤ 6) and school-age children (age > 6) ( Figure 1). The preschool asthmatic children had significantly higher LCI z-score values than that in their healthy controls peers     Table 5).
At the individual level, 9 children with asthma (27%; 9/33) had an LCI value that fell above the ULN of our healthy control population (> 6.94, ie, 95 th percentile of our healthy controls) despite being under symptomatic control. Amongst preschool children, 5 (42%; 5/12) of the asthmatic children had abnormal LCI at the individual level. None of the clinical factors (past hospitalizations, use of oral steroids or emergency visits, type of controller therapy, treatment level or dosage) were significantly associated with abnormal LCI. There was also no correlation between any of the spirometry parameters and LCI.
In the asthmatic group, the mean (SD) changes between post and pre bronchodilator in LCI raw scale and z-score were 0.05 (0.58) and 0.02 (1.16), respectively, neither of which was significant (Table 6).
Finally, although post-BD FEV 1 (% predicted) was statistically significantly higher compared with pre-BD, the change of 3.9 (2), P < 0.001 (paired t-test), which was less than the cut-off 12% as recommended by ATS/ERS, 24 was not considered clinically significant.    in monitoring asthma control. By contrast, we noted that almost half of our preschool asthmatic population (42%; 5/12) had an LCI value above the ULN (> 6.94, ie, 95 th percentile of our healthy control range). Our population was recruited from a specialty tertiary care asthma clinic and may represent a more severe phenotype. Of note, while most (>90%) of the asthmatic children were on regular controller therapy, 9/12 (75%) preschool asthmatic children were on monotherapy whereas only 2/12 (17%) were on multiple controllers ( Table 3).

| DISCUSSION
The use of additional therapy in the older age group could account for better control and, therefore, lower LCI values. In addition, LCI is calculated as the ratio of cumulative expired volume divided by FRC; therefore, small decreases in FRC would be magnified in the overall LCI calculation. Although the differences in FRC noted between the asthmatic and healthy children were quite similar in the overall population, amongst preschool children, the FRC measured by MBW was lower numerically in asthmatic children compared with healthy controls. This may have accounted for the elevated LCI values in this FIGURE 1 LCI z-score plotted against height by age groups. Left panel: LCI z-score vs height for preschool children (age ≤ 6). Right panel: LCI zscore vs height for school-age children (age > 6)

FIGURE 2
Comparison of LCI z-score between asthmatics (N = 12) and healthy controls (N = 13) in preschool children (age ≤ 6) and between asthmatics (N = 21) and healthy controls (N = 32) in schoolage children (age > 6). The lines and whiskers in the boxplot denote median and ranges, respectively. P values were calculated using t-test  Variables are presented as mean (SD).
*P values were calculated using paired t-test.
assessments of disease severity. The final limitation pertains to the use of SF 6 as a tracer gas for MBW. This technique is being replaced by nitrogen washout, and comparison of results from studies using a different gas must be interpreted with caution.
In summary, we showed that LCI is elevated in children with asthma despite physician-assessed symptom control. This difference may be driven by the preschool population. The implications of an abnormal LCI require further investigation, including imaging and assessment of inflammatory phenotype. MBW testing may prove to be a useful monitoring tool in the assessment of asthma in young children. Future studies should assess the utility of different preschool lung function tests such as MBW and spirometry in assessing disease severity in preschool children with asthma.

FUNDING
Don and Debbie Morrison and SickKids Foundation.