INTRODUCTION

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Several studies suggest that measured exhaled NO (ENO) might be useful as a marker of airway inflammation in asthma and other airway diseases (1). Both direct and indirect evidence supports the role of NO in the pathophysiology of asthma. Direct evidence comes from recent studies showing upregulation of inducible nitric oxide synthase expression in the airway epithelium of asthmatic patients, along with increased formation of nitrating species such as peroxynitrite and nitrotyrosine in lung tissue specimens (6, 7). On the other hand, ENO has been shown to correlate with other parameters of airway inflammation in asthma, such as eosinophils in induced sputum (8) and bronchial reactivity (9), and is therefore considered a more sensitive indicator of disease activity than other serum markers (10). In addition, persuasive evidence exists that ENO declines rapidly after treatment with antiinflammatory drugs such as corticosteroids (2, 9, 10, 11) and antileukotrienes (12) in asthmatic patients. For these reasons, ENO has been proposed as a surrogate marker of airway inflammation in asthma (2, 5), and the noninvasive nature of its measurement, together with the immediate availability of results of this, makes it ideally suited for the monitoring of childhood asthma.

Recently, great efforts have been made to standardize ENO measurement procedures (3). Basically, two methods exist for ENO measurement: online and offline sampling. Current thinking is that NO enters the airways with diffusion-limited (flow-based) kinetics after dissociation from other compounds or formation by nitric oxide synthase (4). For this reason, ENO levels are markedly flow-dependent, and a constant expiratory flow rate is required during measurement to obtain reproducible ENO results (4, 13). A constant expiratory flow can be achieved in different ways, but active and skilled cooperation of the patient is always required to maintain a constant flow for several seconds (4). In this regard, recent data indicate that 30% of children aged 7 to 16 yr are unable to perform a flow-controlled exhalation (14), and very few data are available about this for younger children (15).

To overcome this problem, we have developed a flow-driven (FD) method to measure ENO that does not require active cooperation in maintaining a constant expiratory flow. The aim of this study was to compare this modified online technique with the standardized (ST) online method (4), which is currently considered the method of choice for measuring ENO but is not easily performed by preadolescent children (4).

	METHODS

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Subjects

We enrolled 115 children, aged 4 to 16 yr, in the study. Of these children, 105 were asthmatic and 10 were healthy controls. The primary endpoint of the study was to validate and compare the FD method, which does not require active control of expiration, with the ST online single-breath method (4).

Asthmatic Subjects

The 105 children with asthma had clinically stable disease and ranged from 4 through 16 yr of age. They were consecutively recruited from the pulmonology/allergy outpatient clinic of the Department of Pediatrics of the University of Padua. The diagnosis of asthma was based on clinical history, examination, and pulmonary function parameters, according to international guidelines (16). Sixty-two of the children had been receiving continuous treatment with inhaled corticosteroids (beclomethasone propionate at 200 to 500 µg/d, or equivalent doses of fluticasone and budesonide) for at least 1 mo. Inhaled corticosteroids were delivered by a metered dose inhaler via a spacer device, or by a dry powder inhaler. Forty-three of the asthmatic children were steroid-naive, and were using only bronchodilators on demand.

Healthy Controls

Twenty-six children aged 4 through 13 yr and with negative skin prick tests and normal pulmonary function parameters were recruited. They had no history of allergy, respiratory disease, or recent respiratory tract infection. In 16 of these children ENO was measured only with the ST technique, whereas in the other 10 children it was measured with both the ST and the FD techniques.

Study Design

Children were randomized to begin ENO measurements with either the ST or the FD method. The same target exhalation flow rate was used with both methods. After description of the procedure and a practical demonstration to the subject, five attempts to perform the appropriate maneuvers were allowed for each method. Failure in five attempts was considered as inability to perform the maneuvers for a particular method.

ST Online Single-Breath Method

ENO was measured with an online method (4) by means of a computerized system (Exhaled Breath Analyzer; Aerocrine AB, Stockholm, Sweden) and chemiluminescence analyzer (CLD 77 AM; Eco Physics, Duernten, Switzerland). The analyzer has a response time of 100 ms and a detection limit of 0.1 ppb. Measurements of ENO were made in accordance with international recommendations (4). Without using a noseclip, subjects inserted a mouthpiece connected to a Y-piece with two one-way valves. They inhaled NO-free air through the mouth to TLC, and then exhaled immediately. The inhalation of NO-free air through the mouth was checked visually by monitoring for the opening of an inspiratory valve. Exhalation was performed through a heated pneumotachograph (Hans Rudolph Inc., Kansas City, MO) against a linear resistor with a resistance of 100 cm H₂O/L/s (Hans Rudolph) to increase oral pressure to 4.5 to 7 cm H₂O. A fraction of exhaled air was diverted into the NO analyzer at a flow rate of 110 ml/ min through a tube connected close to the mouthpiece (Figure 1). Expiratory flow was displayed in real time to each subject on a computer screen with a visual incentive developed for children (Aerocrine AB, Stockholm, Sweden). Children were encouraged to maintain a constant flow by being asked to maintain some party balloons through exhalation within a given flow range (45 to 55 ml/s), with a target flow of 50 ml/s. The upper and the lower limits of the flow range were represented, respectively, by images of the sky and water. Particular attention was used to avoid and detect gas leaks from the mouth. Children were asked to exhale for at least 6 to 7 s until at least a 2-s NO plateau was achieved. The NO concentration was calculated as the mean value from 50% to 90% of a whole breath, and the NO plateau was defined as occurring during a flow that varied by less than ± 10% with respect to the target flow. Exhaled NO was calculated as the mean of three NO plateau values that agreed to within 10% of the mean value, and suboptimal maneuvers were rejected. Results of the analyses (NO, flow, and pressure tracings) were recorded. At least 1 min was allowed to pass between each measurement. ENO concentrations are reported in parts per billion. The analyzer was calibrated regularly according to the manufacturer's guidelines, using certified NO gas (SIAD, Bergamo, Italy).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Breathing circuit used for online ENO measurement, with flow regulator for control of expiratory flow rate. A = pneumotachograph, B = mouthpiece, C = resistor, D = inspired NO-free air, E = NO sampling line, F = flow regulator. See text for explanation of techniques.

FD Online Single-Breath Method

To enable subjects to exhale with a constant flow rate without active cooperation, we developed a device allowing control of the subject's expiratory flow by the operator. This flow regulator consisted of a rubber tube (length 9 cm, diameter 0.9 cm) with an end restrictor of 2 mm. The flow regulator was placed in the exhalation circuit after the resistor, in the same apparatus used for the ST single-breath method (Figure 1). Bending of the flexible part of the regulator device immediately after the start of exhalation provided a variable resistance and allowed manual control of the expiratory flow rate with direct checking on the monitor by the operator. Expiratory flow was displayed in real time only to the operator, who maintained the flow rate at 45 to 55 ml/s, with a target flow of 50 ml/s. With this method, the subject was asked only to perform an exhalation from TLC, and was not required to self-regulate his or her expiration. As for the ST method, subjects were asked to exhale for at least 6 to 7 s, until a 2-s NO plateau was achieved. The NO concentration was calculated with the same parameters described earlier for the ST method.

Spirometry

Pulmonary function parameters (FEV₁, FVC, and forced expiratory flow from 25% to 75% of VC [FEF_25-75]) were measured by means of a 10-L bell spirometer (Biomedin, Padua, Italy), and the best of three maneuvers for each parameter was expressed as a percent of the predicted reference value according to Polgar and Promadhat (17). Spirometry was performed after ENO measurements.

Statistical Analysis

Data are expressed as mean ± SEM. The agreement between the FD and ST methods was assessed with the method of Bland and Altman (18), and the relationship between NO levels measured with the two techniques was evaluated by using Spearman's rank correlation analysis. Analysis of variance was used for statistical comparison of different groups. A computer package (Statistica; Microsoft Corp., Redmond, WA) was used for statistical analysis. Results were considered significant at a value of p < 0.05. The study was approved by the Institutional Review Board of the Department of Pediatrics of Padua, and all parents and children gave informed consent.

	RESULTS

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We enrolled 115 children, aged 4 to 16 yr, in the study. Of these, 110 children (96%) were able to perform the FD technique, whereas only 73 (63%) were able to perform the ST technique. The mean concentration of ENO of children who performed both techniques was similar, 36.1 ± 3.4 (mean ± SEM) ppb when measured with the ST technique and 33.8 ± 3.3 ppb when measured with the FD technique (p = NS). The mean flow rate with the ST method was 49.2 ± 0.1 ml/s, and with the FD method it was 49.6 ± 0.1 ml/s (p = NS). The mean mouthpiece pressure was 5.0 ± 0.1 cm H₂O with the ST technique and 13.5 ± 0.5 cm H₂O with the FD technique (p < 0.0001). In the 73 children who performed both methods, ENO levels with the two methods were highly correlated (r = 0.99, p < 0.0001) (Figure 2). The agreement between ENO levels measured with the ST and the FD methods was satisfactory, with no systematic error, and is shown graphically in Figure 3 in the manner proposed by Bland and Altman (18). For most measurements, the differences between the NO values obtained with the two methods were within 2 SD of one another, and the mean difference between the ST method and the FD method in ENO levels was 2.2 ± 0.4 ppb.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Relationship between ENO concentrations obtained with the ST and FD methods in 73 children who were able to perform the maneuvers for both techniques (r = 0.99, p < 0.0001). Each circle represents an individual subject.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Differences in ENO concentrations measured with the ST and FD techniques, reported graphically as proposed by Bland and Altman (18). The mean values of ENO concentrations measured with the ST and FD techniques for each subject are plotted against the difference between the ENO concentration measured with the FD and ST techniques. Good agreement was noted for results with the two methods. The solid line is the mean difference between ENO values obtained with the ST and FD methods, and the broken lines are the limits of agreement (mean ± 2 SD).

The full group of subjects was divided into two subgroups according to their age, with one group aged 4 to 8 yr (n = 74; mean age: 6.1 ± 0.1 yr) and the second group aged 9 to 16 yr (n = 41; mean age: 10.1 ± 0.2 yr). In the group aged 4 to 8 yr, 38 children (51%) were unable to perform the ST technique, whereas only five children (7%) failed to perform both the ST and FD techniques. In the group aged 9 to 16 yr, only 41 children (10%) were unable to perform the ST maneuver, and all four successfully performed the FD maneuver. The most frequent cause of failure in performing the ST maneuver was the inability to maintain a constant airflow during exhalation. Figure 4 shows the tracings of a successful test with the FD method and an unsuccessful test with the ST method, in which the subject was unable to maintain a constant expiratory flow. In the group aged 4 to 8 yr the mean value of ENO was 30.9 ± 5.0 ppb with the FD method and 33.1 ± 5.1 ppb with the ST method. In the group aged 9 to 16 yr the mean value of ENO was 41.3 ± 4.7 ppb with the FD method and 43.7 ± 5.0 ppb with the ST method. The difference between the younger group and the older group was not statistically significant (p = NS).

View larger version (21K): [in this window] [in a new window]   Figure 4.   ENO, flow, and pressure tracings for a 7-yr-old boy who correctly performed the FD maneuver (solid lines) achieving a good plateau, but who was unable to maintain a constant flow with the ST method (dotted lines).

In order to compare ENO levels in asthmatic and control children, we added 16 healthy children who had been asked to perform only the ST technique to the 10 who had been asked to perform both techniques. The mean level of ENO in the whole group of asthmatic children was 31.4 ± 2.5 ppb, which was significantly (p < 0.0001) higher than in the 26 healthy children (9.6 ± 0.7 ppb). Steroid-naive asthmatic children had significantly higher ENO values (40.7 ± 4.6 ppb) than did steroid-treated asthmatic children (25.1 ± 3.3 ppb, p < 0.01). Both steroid-treated and steroid-naive asthmatic children presented significantly higher ENO values than did healthy controls (p < 0.05 and p < 0.0001, respectively). No differences were found in spirometric parameters (FEV₁ and FEF_25-75) between steroid-naive and steroid-treated patients (FEV₁ = 92 ± 3% and 87 ± 3%; FEF_25-75 = 88 ± 5% and 78 ± 4%, respectively, p = NS).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, we investigated a modified restricted breath method for online measurement of ENO that overcomes the problem of actively maintaining a constant expiratory flow and may be easily performed by children from the ages of 4 to 5 yr onward. The only cooperative maneuver required of the child with this technique is an exhalation from TLC while the expiratory flow rate is kept constant by the operator, who manually modulates the flow regulator inserted in the expiratory breathing circuit (Figure 1).

Children found the FD technique very easy to perform, and only 7% in the group aged 4 to 8 yr were unable to perform it, whereas 51% were unable to perform the ST procedure. A satisfactory agreement and excellent correlation were found between ENO values measured with the FD technique and the ST technique, which is considered the method of choice for ENO measurement (4). The practical implication of these findings is that these two methods could be used interchangeably and NO measurement could be extended to young children and poorly cooperative subjects.

Confirming previous results, we found increased ENO values in asthmatic as compared with healthy children (19, 20), and among the asthmatic children, higher values in steroid- naive than in steroid-treated subjects. Also, steroid-treated subjects presented significantly higher ENO values than did healthy controls. These findings are in agreement with those of previous studies suggesting that low to moderate doses of inhaled steroids are insufficient to normalize ENO in asthmatic children (21).

Online measurement refers to ENO measured with a real-time display of ENO breath profiles, together with airway flow and pressure (4). A constant expiratory flow rate is the most critical factor for obtaining reproducible measurements with the online technique (3, 22). Even if ENO values are not pressure-dependent (13, 23, 24), the monitoring of mouthpiece pressure is important because it is known that an expiratory pressure  5 cm H₂O can ensure velum closure and exclude contamination with NO present in the nasal cavities (3, 4, 25).

The expiratory flow rate recommended for both children and adults by an official statement of the American Thoracic Society is 50 ml/s (4). Unfortunately, the online method requires active and skilled cooperation by the subject, and preadolescent children may have difficulty in maintaining a constant expiratory flow for several seconds (4, 14, 19). This was confirmed in our study, in which 51% of children under 8 yr of age failed to perform the ST online procedure. In this regard, a recent study by Jöbsis and colleagues (14) found that 30% of children aged 7 to 16 yr were unable to perform a 3-s flow-controlled exhalation, emphasizing the importance of developing alternative methods of measuring ENO for use in children. Similar data have been reported by Canady and coworkers (19). By contrast, 96% of children aged 4 to 16 yr were able to have ENO measurements with a constant flow with the FD method described in our study. The percentage of children able to maintain a constant expiratory flow with the ST method was low even with use of a visual incentive developed for children. It is therefore important to apply new technical solutions (mass flow controllers are under evaluation) together with age-appropriate visual biofeedback that, allowing passive flow restriction with a minimum of cooperation, will extend the application of NO measurements to young children. A potential limitation of our study that would be difficult to eliminate is a bias related to the influence of the operator in the head-to-head comparison of the FD and ST methods.

ENO values measured with the FD and ST methods were comparable and quantitatively similar. The mean expiratory flow was very similar with the two methods (49.6 ml/s and 49.2 ml/s, respectively, p = NS), whereas the expiratory mouth pressure was significantly higher with the FD than with the ST method (13.5 cm H₂O versus 5.0 cm H₂O, respectively, p < 0.0001). This is probably explained by the more physiologic and instinctive nature for children of blowing at higher pressures when not having to control the expiratory flow. The possible influence of pressure on ENO concentrations has been evaluated by several investigators, who concluded that in the presence of a constant expiratory flow rate, expiratory pressure does not affect NO measurement over a wide range of pressures (13, 23, 24).

An alternative technique for ENO measurement is the offline method, which has been shown to produce results that are well correlated with those obtained with online methods (14, 19, 26). The reservoir method offers the advantage of expirate collection when an NO analyzer is unavailable on site, and it is therefore particularly suited for epidemiologic studies (3, 4, 14, 19). We suggest that passive flow-controlling devices may also be usable in the offline method when it is applied to younger children, but specific studies are needed to evaluate this possibility.

In conclusion, we have described a modified online method for measuring ENO that is very simple and can be easily completed by most children from the age of 4 to 5 yr onward. There is a high correlation and satisfactory agreement between ENO values obtained with the FD method and the recommended ST method. We suggest that the FD method might be interchangeably used with the ST method, offering the possibility of extending ENO measurement to young children and poorly cooperative subjects.