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(Chest. 2002;122:955-959.)
© 2002 American College of Chest Physicians

Comparison of Sputum Induction Using High-Output and Low-Output Ultrasonic Nebulizers in Normal Subjects and Patients With COPD*

Martin G. Kelly, MB, BCH, BAO; Vanessa Brown, MSc; S. Lorraine Martin, PhD; Madeleine Ennis, PhD and J. Stuart Elborn, MD

* From the Department of Clinical Biochemistry (Dr. Ennis and Ms. Brown), Queen’s University of Belfast; Department of Respiratory Medicine (Drs. Kelly and Elborn), Belfast City Hospital; and Division of Biomedicinal Chemistry (Dr. Martin), School of Pharmacy, Queen’s University of Belfast, Belfast, Northern Ireland.

Correspondence to: Martin G. Kelly, MB, BCH, BAO, Specialist Registrar, Department of Respiratory Medicine, Level 11 Office, Belfast City Hospital, Lisburn Rd, Belfast BT9 7AB, Northern Ireland; e-mail: m.g.kelly{at}qub.ac.uk


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Induced sputum is used to investigate pulmonary diseases. Low-output ultrasonic nebulizers have become available and have potential advantages over high-output nebulizers. We hypothesized that a low-output nebulizer would give comparable results to a high-output nebulizer, with an acceptable safety profile.

Design: Randomized, crossover study.

Setting: University teaching hospital.

Participants: Ten normal subjects and 10 patients with COPD.

Interventions: Participants attended for sputum induction on two occasions in random order using low-output and high-output nebulizers.

Measurements and results: Lung function and oxygen saturation were measured during sputum induction, and tolerability of the procedure was assessed. Cell counts, interleukin 8, and neutrophil elastase were measured in sputum. Use of the high-output nebulizer resulted in a greater FEV1 (mean ± SEM, 0.29 ± 0.04 L vs 0.21 ± 0.04 L; p = 0.04) and percentage drop in FEV1 (25.8 ± 2.6% vs 19.5 ± 2.9%, respectively; p = 0.02) compared with the low-output nebulizer in patients with COPD. There was a shorter tolerated nebulization time with the high-output nebulizer compared with the low-output nebulizer: 12.7 ± 2.0 min vs 16.5 ± 1.8 min, respectively (p = 0.02). Modified Borg scores were lower with the low-output nebulizer than the high-output nebulizer in normal subjects: median, 0 (interquartile range [IQR], 0 to 1) vs median, 1.5 (IQR, 0 to 2), respectively (p = 0.05). There were no differences in cell counts and soluble markers of inflammation.

Conclusions: The low-output ultrasonic nebulizer is comparable to high-output nebulizer for cellular and soluble markers of inflammation, results in a smaller reduction in FEV1, is better tolerated, and is a suitable tool for investigating airway inflammation in patients with COPD.

Key Words: COPD • induced sputum • ultrasonic nebulizer


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COPD is an inflammatory disorder characterized by neutrophilic inflammation in airway secretions, with a predominance of macrophages and lymphocytes on biopsy.1 Bronchoscopic investigations are often not possible due to severity of disease. Since the 1980s, induced sputum has been used as a tool to investigate pulmonary diseases. It is less invasive and produces valid, repeatable results.2 3 4

Most studies use high-output ultrasonic nebulizers that deliver a large volume of hypertonic saline solution to the airways. More recently, low-output ultrasonic nebulizers have become available, which deliver a smaller volume over the same time. These have been used predominantly in asthma research.5 6 Few studies have reported the use of low-output nebulizers in patients with COPD, and no comparisons have been made with the high-output type.

We hypothesized that a low-output nebulizer would give comparable results to a high-output nebulizer, with an acceptable safety profile. The aim was to compare the two types of ultrasonic nebulizers, in normal subjects and patients with COPD, using the lowest concentration of saline solution (3%). This minimizes the load of saline solution delivered and is likely to be less irritant to the airways.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Participants
Normal Subjects:
Ten normal nonsmoking subjects (5 men) with no recent serious concomitant illnesses, history of atopy or asthma, or history of childhood chest disease were recruited: mean ± SEM age, 28 ± 1 years; FEV1, 3.65 ± 0.29 L; and FEV1 percent of predicted, 98.1 ± 3.2%.

Patients With COPD:
Ten patients (7 men) with stable COPD attending the Belfast City Hospital Respiratory Medicine clinics were also recruited: mean age, 63 ± 3 years; FEV1, 1.08 ± 0.14 L; FEV1 percent predicted, 40.7 ± 4.2%; pulse oximetric saturation, 91.4 ± 1.8%; and median cigarette consumption, 43.5 pack-years (interquartile range [IQR], 40 to 47.5 pack-years). The patients with COPD were selected based on a clinical history consistent with COPD as described in British Thoracic Society guidelines.7 We recruited patients > 40 years old with a >= 20 pack-year history of smoking, FEV1 < 70%, FEV1/FVC ratio < 75%, and < 15% reversibility in response to inhaled ß2-agonists. Patients were excluded if they were receiving long-term oral steroids, had any evidence of other inflammatory disorders, or had a history of respiratory tract infection in the last month. The Queen’s University of Belfast Faculty of Medicine Research Ethics Committee approved the study, and informed written consent was obtained from all patients

Sputum Induction
Spirometry was undertaken, and reversibility to salbutamol was tested in the patients with COPD. Fifteen minutes after inhalation of 200 µg of salbutamol, the patients rinsed their mouths with water. Spirometry was recorded to assess reversibility, and oxygen saturation and pulse rate were measured using a pulse oximeter (Sat-Trak Pulse Oximeter; SensorMedics; Bilthoven, the Netherlands). Subjects were randomized to either the high-output nebulizer (DeVilbiss Ultraneb 99; DeVilbiss Healthcare; Somerset, PA) or the low-output nebulizer (Sonix 2000; Clement Clarke International; Harlow, Essex, UK) initially, followed by the other nebulizer at least 1 week later. Randomization was achieved using random-number tables. The high-output nebulizer was set at approximately 3 mL/min (mean particle size < 4 µm), and the low-output nebulizer was set at approximately 0.4 mL/min (mean particle size of 4.9 µm).

After 5 min of nebulization with 3% saline solution (weight per unit volume), the subjects were asked to expectorate into a sterile sputum pot. Lung function and oxygen saturation were recorded; if there was a < 20% fall in FEV1, nebulization was recommenced. If there was a > 20% fall in FEV1, the procedure was stopped and inhaled salbutamol was administered if FEV1 was slow to return to baseline. The time taken for a > 20% fall in FEV1 was recorded. This procedure was repeated every 5 min for a total nebulization period of 20 min. Nebulization was then stopped. Patients were asked to score their symptoms using a modified Borg score (mBs)8 and a visual analog score (VAS) to assess chest tightness and the unpleasantness of the procedure.9 Changes in oxygen saturation and FEV1 were recorded.

Sputum Processing
Sputum was processed using the methods of Pavord et al.10 Sputum plugs were selected from the original known weight of induced sputum, to minimize salivary contamination. The plugs were weighed, and four times the selected sputum volume of freshly prepared 0.1% (weight per unit volume) dithiothreitol was added. After vortexing (15 s) and gentle mixing, samples were incubated in a shaking water bath at 37°C for 15 min. Subsequently, a further four volumes of phosphate-buffered saline solution were added and incubated for a further 5 min. The resultant suspension was filtered through 53-µm gauze (Lockertex; Warrington, England) and centrifuged at 200g for 10 min. The supernatant was removed and stored at - 80°C for interleukin (IL)-8 and neutrophil elastase (NE) analysis. For IL-8, 3.7 mM ethylene diamine tetra-acetic acid (inhibiting metalloproteinase-like activity) [Sigma Chemicals; Poole, England], 150 µM N-{alpha}-Tosyl-L-lysine chloromethyl ketone (inhibiting trypsin-like activity) [Sigma Chemicals], 20 µM Z-Phe-diphenylphosphonate (inhibiting chymotrypsin-like activity; supplied by the Division of Biomedicinal Chemistry, Queen’s University of Belfast), and 100 µM methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (Sigma Chemicals) were added (final concentrations shown). For NE, all protease inhibitors, except methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (NE inhibitor) were added in final concentrations as stated.

Slides were made using the glass coverslip method11 and stained with Diff-Quik (Hamilton-Thorne Research, Beverly, MA) for differential cell counting. Mast cells were enumerated after fixing in Carnoy solution (2.5 h) and staining with toluidine blue (0.5 h). Differential cell counts (at least 500 cells) and mast cell counts (at least 2,500 cells) were performed by one observer, who was blinded to the clinical history of the subjects.

IL-8 was measured using a commercially available enzyme-linked immunosorbent assay (R&D Systems; Abingdon, Oxon, England). Free NE activity was measured by a colorimetric assay12 using the elastase substrate Suc-Ala-Ala-Pro-Val-pNA (Bachem; Walden, Essex, England). Absorbance was measured at 405 nm on a microtiter plate reader (Spectramax 190; Molecular Devices; Sunnyvale, CA).

Statistical Analysis
Subject demographics, physiologic parameters, and time were expressed as mean (± SEM). Cigarette consumption, mBs, VAS, and all cellular data were expressed as median (IQR). Normal subjects and patients with COPD were compared as separate groups. The paired Student t test was used to compare parametric data, with other data being compared using Wilcoxon signed-rank test. Significance was assumed at p <= 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ten normal subjects and 10 patients with COPD were studied, and all successfully produced sputum. Nebulization was stopped due to a > 20% drop in FEV1 in three patients with COPD using the low-output nebulizer and seven patients with COPD using the high-output nebulizer. The high-output nebulizer produced a significantly larger drop in FEV1 (mean, 0.29 ± 0.04 L vs 0.21 ± 0.04 L, p = 0.04) and percentage drop in FEV1 (mean, 25.8 ± 2.6% vs 19.5 ± 2.9%, p = 0.02) compared with the low-output nebulizer in patients with COPD. There was a significantly shorter tolerated nebulization time with the high-output nebulizer compared with the low-output nebulizer: mean 12.7 ± 2.0 min vs 16.5 ± 1.8 min (p = 0.0156). There was no significant drop in oxygen saturation with either nebulizer, or any difference between the two nebulizers. Median mBs was lower with the low-output nebulizer than the high-output nebulizer in normal subjects: median, 0 (IQR, 0 to 1) vs median, 1.5 (IQR, 0 to 2) [p = 0.05], but there was no significant difference in patients with COPD (Table 1 ).


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  		Table 1. Physiologic and Safety Data*

 
Weight of selected sputum, cell viability, and cells per gram of sputum were similar in normal subjects and patients with COPD with both nebulizers. Differential cell counts were not statistically significantly different between the two nebulizers, in normal subjects or patients with COPD (Table 2 ).


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  		Table 2. Cellular and Fluid Phase Data*

 
IL-8 was detectable in 9 of 10 of the normal subjects, and in all patients with COPD. NE was detectable in all patients with COPD and in seven normal subjects. Both IL-8 and NE levels were significantly greater in patients with COPD compared to normal subjects. However, levels of both soluble markers were not significantly different between the low-output and high-output nebulizers (Table 2) . No order effect was noted for the two nebulizers with regard to cell counts or soluble markers.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that a low-output ultrasonic nebulizer can produce adequate and comparable samples to conventional, high-output ultrasonic nebulizers. It has a superior safety profile and better patient tolerance when compared to the high-output nebulizer.

The smaller fall in FEV1 and longer nebulization time suggests that the low-output nebulizer is more acceptable for use in patients with COPD. However, even with the low-output nebulizer, a > 20% fall in FEV1 occurred in three patients. This change, however, was only transient, and FEV1 subsequently returned to baseline rapidly in all patients. Bhowmik et al13 found statistically significant falls in FEV1 and oxygen saturation using a high-output nebulizer and a similar cohort of patients with COPD. Rytilä et al14 reported a better safety profile using a high-output nebulizer in patients with COPD. However, their study examined a group of patients with less severe COPD (mean FEV1, 53% of predicted). They also used 0.9% saline solution in patients with FEV1 < 1 L. This is likely to explain the better safety data. Brightling et al15 16 reported safety data on a cohort of patients with COPD using a low-output nebulizer; mean FEV1 percent predicted was slightly higher (44.3% vs 40.7%) than in our study, and preprocedure bronchodilatation was performed using a higher dose of salbutamol (2.5 mg nebulized). This may provide superior bronchoprotection during the sputum-induction procedure. The mBs was significantly lower in the low-output device in normal subjects, with positive trends in VAS score in normal subjects and patients with COPD, indicating better patient tolerance.

All samples were adequate for analysis, and there were no problems associated with sputum production in normal subjects, unlike previous studies.12 Differential cell counts were in agreement with published values.16 17 Median sputum IL-8 concentrations were similar to reported values in COPD,18 19 though higher than in some studies.3 16 The differences may be due to the presence or absence of protease inhibitors. The cocktail of inhibitors that we use leads to effective inhibition of protease activity.20

Our data indicate that low-output nebulization is satisfactory for analysis of lower airway secretions, in normal subjects and patients with COPD. The increased safety and tolerance enables the examination of patients with more severe COPD and those with exacerbations. There are some methodologic issues surrounding the use of induced sputum.12 13 21 Sputum induction may act as a proinflammatory stimulus, increasing airway neutrophils.22 This is present at 8 h23 and 24 h postnebulization,23 24 but absent at 6 days.12 This concurs with evidence showing IL-8–driven neutrophilic pulmonary inflammation after BAL.25 The reason for sputum induction causing neutrophilic inflammation is unclear, although it has been suggested that it is proportional to the load of saline solution delivered to the airways.22 A lower load of saline solution is desirable, as it may minimize the airway neutrophilia previously demonstrated with high-output nebulizers.22 Until now, the potential for this to obscure other changes has led to caution in the use of the technique for short-term serial assessment of lower airway markers of inflammation, particularly following specific interventions.22

In conclusion, cellular and soluble markers of inflammation are similar using a low-output ultrasonic nebulizer compared to a high-output nebulizer. The safety profile of the low-output device is superior, and it is well tolerated by subjects. It is a suitable tool with which to investigate airway inflammation in patients with COPD.


   Footnotes
 
Abbreviations: IL = interleukin; IQR = interquartile range; mBs = modified Borg score; NE = neutrophil elastase; VAS = visual analog score

Work performed at Department of Clinical Biochemistry, Queen’s University of Belfast and Department of Respiratory Medicine, Belfast City Hospital.

Research supported by a grant from the Northern Ireland Chest, Heart, and Stroke Association.

Received for publication October 1, 2001. Accepted for publication March 18, 2002.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rutgers, SR, Timens, W, Kaufmann, HH, et al (2000) Comparison of induced sputum with bronchial wash, bronchoalveolar lavage and bronchial biopsies in COPD. Eur Respir J 15,109-115[Abstract]
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  4. Claman, DM, Boushey, HA, Liu, J, et al Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects. J Allergy Clin Immunol 1994;94,861-869[CrossRef][ISI][Medline]
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  8. Killian, KJ, Summers, E, Watson, RM, et al Factors contributing to dyspnoea during bronchoprovocation and exercise in asthmatic subjects. Eur Respir J 1993;6,1004-1010[Abstract]
  9. Popov, TA, Pizzichini, MMM, Pizzichini, E, et al Some technical factors influencing the induction of sputum for cell analysis. Eur Respir J 1995;8,559-565[Abstract]
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  12. Castillo, MJ, Nakajima, K, Zimmerman, M, et al Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal Biochem 1979;99,53-64[CrossRef][ISI][Medline]
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  20. Martin, L, Lynas, JF, Walker, B, et al Improved protease inhibition for storage and analysis of CF sputum. Pediatr Pulmonol 1998;26(suppl 17),382-383
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