INTRODUCTION

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The assessment of airway inflammation in asthma involves either invasive techniques such as fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) and biopsies, or noninvasive techniques such as sputum induction. Sputum production is a common symptom of asthma and airway disease. It can be used in patients recovering from an acute exacerbation (1), but spontaneous sputum production is often inadequate for many patients with asthma (2). Sputum production induced by hypertonic saline was used in subjects who were unable to produce sufficient sputum normally (3). An aerosol of hypertonic saline (3, 4, and 5%) was generated using a nebulizer. A bronchodilator (salbutamol 200 µg) was administered before the procedure to prevent possible bronchoconstriction induced by hypertonic saline (15). Sputum production under these conditions was found to be safe and effective (2). However, most of the patients tested in previous studies had mild asthma, and the safety aspect has not been assessed except in one abstract (16). Hypertonic saline has been used as a bronchoconstrictor to assess nonspecific bronchial hyperreactivity (8, 17). Thus, inhalation of hypertonic saline by patients with uncontrolled asthma may lead to serious side effects.

The aim of this study was to assess the safety of hypertonic saline-induced sputum using the method of Pin and colleagues (3, 24) after premedication with 200 µg of salbutamol administered by a metered-dose inhaler (MDI). Safety was assessed clinically using a symptom score and FEV₁ measurements. Spirometry was performed just before sputum induction and 5 min after the end of each period of inhaling hypertonic saline. Nine control subjects and 64 asthmatic patients with variable disease severity (FEV₁ ranging from 40 to 126% of predicted values) were tested. Of these asthmatic subjects, 21 had uncontrolled asthma and 43 were considered to have a severe form of the disease.

	METHODS

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Patient population. In a preliminary study we enrolled eight severe asthmatics with FEV₁ < 1 L and found that seven of eight presented major side effects, with the occurrence of two severe asthma attacks requiring intensive treatment (see Table 1). We decided to exclude from the present study severe patients with such low lung function, based on these preliminary findings and previous reports (24).

Sixty-four asthmatic patients ranging in age between 19 and 74 yr (median, 54 yr) were studied. Asthma was defined using Pin and colleagues' criteria (24). All patients had a reversible airway obstruction characterized by an increase of at least 12% in the baseline FEV₁ value, and an absolute value of 200 ml, after inhalation of 200 µg salbutamol. Patients were excluded from the study if they were current smokers or if they had smoked over 10 pack-years, if they had had a respiratory infection within the month preceding the study, or if the FEV₁ value was < 1 L. All patients were true asthmatic patients: nonsmokers whose symptoms began before the age of 40 yr, with recurrent symptoms of chest tightness and wheezy dyspnea. They displayed a variable peak expiratory flow rate, which is not a common feature of chronic obstructive pulmonary disease. All had displayed an improvement in FEV₁ of more than 12% in the past year.

The study was approved by the Ethics Committee of our Institution, and the patients gave their informed consent.

Characteristics of asthma. The clinical severity of asthma was assessed according to the scoring system of Aas (25), which ranges from 1 to 5. Although this scoring system may not be the most recent, it has been validated in many studies. All patients were treated according to recent guidelines (26) and monitored for 15 d by symptom-medication scores and peak flow rates before sputum induction. The treatment regimen of the patients was kept constant for the 2 wk preceding the study. Twenty-one patients were considered to have uncontrolled asthma according to their symptoms; specifically, all 21 had nocturnal symptoms and a diurnal variation in peak flow > 20% (26).

Protocol for sputum induction. Sputum was induced according to the method of Pin and colleagues (3). Before sputum induction, all subjects underwent spirometry and then received a dose of 200 µg salbutamol by MDI. The sputum induction procedure was performed after a second spirometric investigation, and spirometry was repeated at 5-min intervals throughout the procedure and immediately after sputum induction was completed. The patients were asked to rinse their mouths out with water before induction to avoid salivary contamination of induced sputum samples as much as possible.

The hypertonic saline (3, 4, and 5% saline) was nebulized with a DP100 (Syst'Am, Paris, France) ultrasonic nebulizer and inhaled in 5-min periods for up to 30 min. This nebulizer generates particles with a mean mass aerodynamic diameter of 4.5 µm and has an output of 2.4 ml/min. The saline concentration was increased every 10 min. Patients were encouraged to cough throughout the procedure, and they regularly stopped to expectorate sputum into a clean plastic container. If troublesome symptoms occurred (i.e., wheezing, severe cough, or dyspnea), nebulization was discontinued. The nebulization was stopped when a sputum sample was obtained according to the criteria of Pin and colleagues (3).

Parameters for evaluating safety. Safety was assessed by both objective parameters (% drop in FEV₁) and the clinical tolerance of the patient to the procedure. Sputum induction was stopped if one of the following parameters were observed:

(1) Drop in post-salbutamol FEV₁ of > 20%: If FEV₁ dropped > 10% during hypertonic saline nebulization, the next concentration of saline was not administered.

(2) Severe dyspnea, cough, or wheezing: A symptom score was used to assess dyspnea and cough more precisely. These symptoms were marked by the investigator as absent, mild, moderate, or severe. The wheezing was scored either present or absent.

A total symptom score of 7 was the highest possible.

Statistical analysis. Results are given as medians and 25-75% percentiles. We used the Wilcoxon (W) test for paired data and the Mann-Whitney (U) test for unpaired data to perform statistical analysis. Correlation coefficients were calculated by the Spearman-Rank test ().

	RESULTS

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Characteristics of the patients. The characteristics of the patients are shown in Table 2. Twenty-one patients had mild to moderate asthma (Aas score of 1, 2, or 3) and 43 had severe asthma (Aas score of 4 and 5). Their FEV₁ scores ranged from 40 to 126% of predicted values (median 81.5%). Twenty-one patients had uncontrolled asthma as defined above. The medications used for asthma treatment were 2 bronchodilator as required for mild asthmatics; inhaled steroids for 31-50% of those with mild and 100% of those with severe asthma; of the patients with severe asthma, 62-93% were treated with oral corticosteroid and 77-85% received long-acting 2 agonists (Table 2). These treatments explain the low reversibility of their lung function obstruction to short-acting 2 agonists observed on the day of the procedure.

Tolerance of sputum induction. In control subjects, there were no clinical sign of intolerance to the procedure and the median FEV₁ reduction was small (Table 3). However, some patients with uncontrolled severe asthma were unable to perform the whole protocol. The fall in FEV₁ values was usually small and never exceeded 20% from the baseline value measured after inhalation of salbutamol. There was no significant correlation between FEV₁ drop and the baseline FEV₁ (Spearman rank correlation test). The reduction of FEV₁ values for the control subjects and patients with mild controlled asthma (n = 13) was < 10%. However, 2 of 8 patients with mild, uncontrolled asthma; 7 of 30 patients with severe, controlled asthma; and 2 of 13 patients with severe, uncontrolled asthma showed an FEV₁ reduction > 10%. The time course of the drop in FEV₁ is shown on Figure 1. It was usually progressive and easily reversed by a nebulization of 10 mg of salbutamol. We assessed the symptoms and compared them with the observed changes in lung function. There were no differences among the symptoms reported by the patients within the three groups. We failed to find any correlation between the drops in lung function and the occurrence of symptoms. The drop in FEV₁ was significantly greater in the patients with severe asthma compared with mild asthma (p < 0.02, Mann-Whitney U test). The control of the disease (i.e., nocturnal asthma, peak expiratory flow rate, diurnal variability > 15%) was not associated with any difference in the occurrence of symptoms or, more importantly, with a drop in FEV₁.

View larger version (37K): [in this window] [in a new window]   Figure 1.   Time course of FEV1 measurements before, during, and after the procedure in 20 patients who presented a fall in their lung function during the procedure. Horizontal axis represents progress of sputum induction: post 2, after 200 µg of salbutamol; T1, after 3% hypertonic saline nebulization for 5 min; T2, after 3% hypertonic for 5 min; T3, after 4% hypertonic for 5 min; T4, after 4% hypertonic for 5 min; T5, after 5% hypertonic for 5 min; T6, after 5% hypertonic for 5 min; postNeb, after 10 mg salbutamol nebulization for 10 min.

Sputum production. Sputum was obtained in 76% of asthmatic patients and in about 50% of the control subjects (Table 3). The definition of sputum efficiency is derived from Pin and colleagues (24). We did not find any significant correlation between the occurrence of symptoms and the production of sputum or with a fall in lung function.

	DISCUSSION

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In this study, we assessed the safety of sputum induction in a large group of asthmatic patients with variable disease severity. Using a validated method of sputum induction (24), we observed that the procedure is safe and that sputum can be obtained from > 75% of asthmatic patients. The occurrence of symptoms was not related to the changes in lung function. The changes in FEV₁ were significantly more prominent in severe patients, but the control of the disease was not associated with less tolerance of the procedure. However, the most severe asthmatic group was excluded from the study because a preliminary study showed an unacceptable rate of side effects, including severe asthma exacerbation.

Few studies have investigated the safety of sputum induction using hypertonic saline. Pin and Fahy and their colleagues were the first to report saline-induced sputum in asthmatic patients and to study the adverse reactions occurring with the technique (3, 16, 24). Few adverse reactions were reported, but the exact numbers were not given. A fall of FEV₁ varying from 1.3 to 5.3% from the basal value was reported in these studies. In the first report (3), 1 of 17 patients presented an FEV₁ reduction of > 20%, compared to 2 of 12 in the second study (24). These asthmatic reactions were rapidly reversed by administration of an inhaled bronchodilator. Fahy and colleagues (5, 16) reported the safety of this technique in a more heterogeneous population of asthmatic subjects, with FEV₁ varying from 26 to 123%. However, few patients had severe asthma in this study and the mean fall in FEV₁ was 7.4%; one patient had a 47% fall, although a detailed description of this patient was not given. Other adverse reactions were reported as salty taste, increased salivation, sore throat, mild nausea, cough, and bronchorrhea. All these studies used ultrasonic nebulization of hypertonic saline (3-5%) and premedication with 2 nebulized bronchodilator. Other studies did not specifically report side effects.

In this study, we aimed to investigate the safety of the procedure and therefore tested patients with severe and/or uncontrolled asthma. However, we excluded the most severe cases, who had baseline FEV₁ values of less than 1 L, because in a preliminary study using a different technique we observed that these patients may present a severe risk of acute bronchoconstriction (Table 1). Moreover, this cutoff limit has been proposed previously (24). In our study, many of the patients with severe asthma received treatment with oral steroids and they were placed into two categories depending on the control of their asthma by Global Initiative for Asthma (GINA) guidelines (26). We found that even in patients with severe uncontrolled asthma, the procedure did not induce a severe reaction, even though it had to be stopped in 23% of cases. A drop of FEV₁ of > 10% from baseline values was rarely observed, even in the most severe cases. However, patients with mild uncontrolled asthma appear to be at greater risk than those with mild controlled disease. Changes in lung function were rapidly reversed by a nebulized solution of bronchodilator with no need for other therapeutical interventions. Many hypotheses could be advanced to explain the poorer tolerance of patients with severe asthma to sputum induction. However, the most likely is related to the bronchoconstrictive activity of hypertonic saline, which is commonly used to assess nonspecific bronchial hyperreactivity (17).

In conclusion, we found that hypertonic saline-induced sputum is a safe technique when using a controlled increase in the hypertonic saline concentration. It is safe even for patients with severe asthma, although they need very careful observation. We could not discriminate in the present study whether symptoms or measures of lung function better relate to the occurrence of side effects. We did not find any predictive factor except the overall severity of the disease, which can be determined before the procedure begins.