Identification and validation of nebulized aerosol devices for sputum induction

Omron NE-U17 (Omron, Japan) and EASYneb II (Flaem Nuova, Italy) systems were found to have similar measurements of aerosol size. There was no significant difference in induced sputum cell results between the AeroNeb Solo and EASYneb II devices. DISCUSSION: There is a need for rigorous, scientific evaluation of nebu-lizer devices for clinical applications, including sputum induction, for measurement of cell counts. CONCLUSION: The present study was the most comprehensive analysis of different nebulizer devices for sputum induction to measure cell counts, and provides a framework for appropriate evaluation of nebulizer devices for induced sputum testing.


Identification and validation of nebulized aerosol devices for sputum induction
Warren J Davidson MD 1 , John Dennis PhD Telephone 403-943-8864, fax 403-943-8666, e-mail wdavidso@ucalgary.caI nduced sputum cell count measurements are a noninvasive, reliable method for evaluating the presence, type and degree of airway inflammation in patients with asthma and various other airway diseases (1)(2)(3).Sputum cell counts can be used clinically to assist with patient management, including characterization of asthma phenotypes that may benefit from emerging biological therapies (4-7), and titration of inhaled corticosteroid therapy in patients with asthma (8).Tailoring asthma management based on sputum eosinophil counts has been shown to be effective in reducing asthma exacerbations (9)(10)(11)(12) and is now considered to be standard of care for the management of patients with moderate-severe asthma in the Canadian asthma guidelines (13).
A wide range of commercially available nebulizer devices are available for clinical and research use.Traditional jet nebulizers are available in several different formats: constant output, breath-enhanced and dosimetric output.Unlike jet nebulizers, ultrasonic devices do not use compressed airflow and have traditionally been used for sputum induction.Vibrating mesh nebulizers are designed to improve the versatility, portability and efficiency of particle delivery (14).Established guidelines have stressed the need for performing rigorous, scientific testing of nebulizer devices for clinical applications (15).However, there remains a relative paucity of literature testing various nebulizer designs for use in sputum induction for the purposes of measuring sputum cell counts.
The desirable aerosol characteristics for sputum induction include a relatively low output and a large particle size (2).The range of nebulized aerosol sizes required for therapeutic respiratory deposition (including sputum induction) is 0.5 μm to 5 μm in a patient breathing at tidal volume (16).However, given that the aim of the present study was to determine nebulizer aerosol output characteristics (output and size) and identify a replacement nebulizer system with similar aerosol output properties, our study focused on comparing two nebulizers with a similar aerosol size and output with a well-established nebulizer device whose performance range is known to support sputum induction.
In Calgary (Alberta), induced sputum collection and cell count analysis began in 2005 as part of the Department of Medicine Innovation Funding Program.The clinical use of induced sputum testing continues to increase and, to date, we have analyzed approximately 3100 clinical samples within the Calgary region and surrounding referral areas.Relatively low-output ultrasonic nebulizers, of which the Universal III ultrasonic nebulizer (Flaem Nuova, Italy) is a prototype, have been widely used throughout Canada, Europe and Australia for sputum induction.Recently, the manufacturer (Flaem Nuova, Italy) replaced this device with the EASYneb II ultrasonic nebulizer.However, both units are labelled as single-patient devices by the manufacturer and cannot be used on multiple patients as per infection prevention and control requirements.Therefore, there is a need for alternative nebulizer devices that are either disposable or labelled for multipatient use.The aim of the present study was to apply validated rigorous, scientific testing methods to identify and validate nebulizer devices appropriate for use in a clinical sputum induction program.Specifically, we aimed to identify nebulizer devices with aerosol characteristics comparable with the current 'gold standard' EASYneb II and would be appropriate for use in a large clinical sputum induction program.

METHODS
The present study was approved by the institutional research ethics board and written informed consent was obtained from all subjects.

Study design
The primary objectives were: to define the in vitro nebulized aerosol characteristics (aerosol output and aerosol size) of the Universal III and EASYneb II ultrasonic nebulizers; to screen a range of candidate alternative nebulizers for aerosol output and aerosol size using rigorous in vitro criteria; and to compare induced sputum cell counts in healthy adult subjects obtained using the EASYneb II ultrasonic nebulizer with a selected nebulizer device that was either disposable or labelled for multipatient use.

Aerosol testing
In vitro tests applied methods embodied in the European Standard EN13544-1 (17)  Testing of aerosol output incorporated sinusoidal breath simulation.Inhaled aerosol was collected on low-resistance filters (Filtrete, 3M, USA) and salt residue was subsequently assayed using ion-specific electrochemistry.Following initial testing of seven potentially suitable nebulizers for aerosol output, two specific nebulizers were then selected for aerosol size measurement, based on the following a priori established criteria: met current health authority requirements for infection control; in vitro characteristics of aerosol output; calculated time to complete patient sputum induction collection using the Universal III device as the baseline; ease of use by staff; and equipment costs, including purchase price, maintenance, cleaning and storage.Aerosol size measurement incorporated Next Generation Impactor (NGI) cascade impaction of saline aerosols and subsequent desorption of collected size fractions by ion-specific electrochemistry.

Study subjects
Healthy nonsmoking adult study participants were eligible provided they had normal spirometry (forced expiratory volume in 1 s [FEV 1 ] >80% predicted and FEV 1 to forced vital capacity [FVC] ratio [FEV 1 / FVC] >0.75), with no significant bronchodilator response (FEV 1 and FVC <200 mL increase and <12% increase following short-acting bronchodilator administration).Potential study participants were excluded from the study if they had any of the following: a positive methacholine challenge test (defined here as a provocative concentration of methacholine required to yield a 20% reduction in baseline FEV 1 [PC 20 ] <16 mg/mL); a smoking history ≥5 pack years or had smoked within the past 12 months; a clinical history of atopy or allergic rhinitis; a diagnosed upper respiratory tract infection four weeks before study participation; or a history of any inflammatory condition considered by the investigators to be clinically relevant, including any underlying autoimmune disorder, infectious disease, immunodeficiency, malignancy or clinically relevant comorbidity.Women who were pregnant, or who were planning to become pregnant during the study period, and women who were breastfeeding were also excluded.Finally, only participants who provided written informed consent were included in the study.
Study subjects attended the laboratory on three separate occasions.During the initial screening visit, the subject's medical history was reviewed to determine whether they met inclusion/exclusion criteria.Suitable candidates were randomly assigned to undergo initial induced sputum collection with either the EASYneb II or the selected nebulizer device.At the second visit, subjects performed the first sputum induction.Forty-eight hours later (visit 3), they returned for the second sputum induction using the alternate nebulizer.The randomization sequence was computer-generated and stored in opaque, sealed envelopes held by an individual not involved in the study.

Protocol
Aerosol testing: For aerosol output testing, the test nebulizer containing 0.9% sodium chloride was fitted to a breath simulation device (Pari Compas Breathing Simulator, Pari GmbH, Germany), which simulated tidal breathing (sinus flow of 500 mL tidal volume at 15 breaths/min).Test nebulizers were operated according to the manufacturers' recommended flow rate.A low-resistance electrostatic filter fitted to the nebulizer mouthpiece collected all 'inhaled' nebulized aerosol.Aerosol residue was desorbed from collection filters and chloride was desorbed and quantified electrochemically, using a chloride ion-specific electrode (Jenway pH/ion model 3040 meter, Bibby Scientific Limited, United Kingdom) against known standards.The collected volume of deposited aerosol was then calculated and presented as μL/min output rate, as well as total μL dose.Total nebulization time was also recorded.
For aerosol size testing, the test nebulizer containing 0.9% sodium chloride was adapted to fit a cascade impactor (M170 NGI, MSP Inc, USA).Test nebulizers were operated according to manufacturers' recommended flow rate (where appropriate).Emitted nebulized aerosol was drawn into the NGI at 15 L/min and aerosol droplets were separated into different size fractions within the impactor over a timed 3 min collection period.Aerosol residues in each collection plate were quantitatively desorbed and the amount of chloride determined electrochemically.The amount of aerosol residue deposited on each stage was used to generate cumulative and normative size distributions, from which the mass median aerodynamic diameter (MMAD) and geometric SD of MMAD (GSD) were determined.The MMAD was defined as the diameter at which 50% of the particles by mass were larger and 50% were smaller.The GSD is a measure of the spread of an aerodynamic particle size distribution and typically calculated as follows: GSD = (d84/d16)1/2, in which d84 and d16 represent the diameters at which 84% and 16% of the aerosol mass are contained Figure 1.Sputum induction and processing: Sputum induction was performed using escalating doses of hypertonic saline as per standardized guidelines (18).Before nebulized saline administration, spirometry was performed, pre-and postbronchodilator, according to American Thoracic Society criteria (19).For the EASYneb II ultrasonic nebulizer, 9 mL of 4% sterile saline were placed into the well.The subject inhaled the nebulized mist for 7 min at tidal volume.At 7 min, the subject was instructed to blow their nose, rinse their mouth with water, then cough and expectorate sputum into a specimen container.Spirometry was repeated to ensure the change in FEV 1 was <10% of baseline.These steps were repeated with 9 mL of 4% and 9 mL of 5% sterile saline.For the AeroNeb Solo device, the same procedure was used except 6 mL of the respective saline solutions were placed into the well, as per manufacturer's instructions.
After induction, samples were placed on ice and processed for differential cell counts using dithiothreitol within 2 h of collection (20).Total cell counts were obtained manually using a Bright-line Neubauer hemacytometer (Hausser Scientific, USA); the viability of each sample was assessed using the trypan blue exclusion method (21).Cells were diluted in phosphate-buffered saline to 1×10 6 /mL and cytospin slides were prepared before being stained with Congo red for differential sputum cell counts.Samples were considered to be adequate for analysis if there was <20% squamous cell contamination and a 400 nonsquamous cell differential cell count could be obtained.Measurements were compared with reported normal values (22).Cell counts were performed by an experienced cytotechnologist blinded to subject characteristics and nebulizer device used.

Analysis
Aerosol results were compared using the Kruskal-Wallis test.Where a significant effect was detected, appropriate post hoc testing was used to assess where that difference existed.Values from the different sputum collections were compared using the paired t test for normally distributed data and the Wilcoxon signed-rank test for nonparametric data; P<0.05 was considered to be statistically significant.

Aerosol output testing
Table 1 summarizes the results of the aerosol output testing.Given that the Universal III device has historically been viewed the gold standard (13) nebulizer for sputum induction in Canada, six of these  devices were tested to establish baseline data with which the other nebulizer devices were then compared.The results in Table 1 show that the aerosol output at 1 min for the Universal III device was different than the Respigard II and Omron NE-U17 devices; it was similar for all the other devices.Total nebulization time was similar among all devices except the Omron NE-U17.

Aerosol size testing
After the initial aerosol output testing was completed, two nebulizer devices were then chosen for measurement of aerosol size and comparison with the Universal III.Criteria for selection of the nebulizers included: met current health authority requirements for infection control; in vitro characteristics of aerosol output; calculated time to complete patient sputum induction collection using the Universal III device as the baseline; ease of use by staff; and equipment costs, including purchase price, maintenance, cleaning and storage.The AeroNeb Solo was chosen because it is a readily available, small, relatively inexpensive, single-use disposable unit that had aerosol output similar to the Universal III.Furthermore, the calculated time for induced sputum collection was not greater than current practice and the pulmonary laboratory staff performing sputum induction found it easy to use.The Omron NE-U17 was also chosen because it is labelled for multipatient use, and is easy to use and could reduce the time needed for the induced sputum procedure.The ability to calibrate aerosol output with the Omron NE-U17 was believed to be beneficial when testing a broad clinical spectrum of patients (ie, the nebulizer output can be adjusted depending on how the patient tolerates the sputum induction procedure).
The results of aerosol size testing are summarized in Table 2.All three devices had similar MMAD and GSD.The Universal III had a marginally larger fine particle fraction of aerosol mass <5 μm (fine particle fraction % <5 μm).

Induced sputum cell counts
At the time the present study was conducted, the Omron NE-U17 had not yet received CSA approval for clinical use in Canada.As a result, we chose to proceed with the AeroNeb Solo device.Given that the original Universal III model had been replaced by the manufacturer (FlaemN, Italy) with the the EASYneb II, the induced sputum cell counts obtained using the AeroNeb Solo device were compared with those obtained using the EASYneb II.Fourteen healthy adult subjects were tested, with 10 providing adequate sputum samples; two inadequate sputum samples were obtained from each type of nebulizer.Subject characteristics at baseline are listed in Table 3.The majority of the subjects were Caucasian (n=8) and female (n=9).The study population had normal spirometry and airway responsiveness (PC 20 >16 mg/mL).None of the subjects were taking regular medications at the time of enrollment.The sputum cell count results are summarized in Table 4.There were no significant differences in cell viability, and total and differential sputum cell counts between the two devices.No subjects experienced symptomatic bronchospasm or a >10% decrease in FEV 1 following administration of hypertonic saline.

DISCUSSION
Currently, standard nebulizer devices used for sputum induction are labelled as single-patient devices by the manufacturer and cannot be used on multiple patients as per infection prevention and control requirements.As such, these devices cannot feasibly be used in a large clinical sputum induction program.Therefore, there is a need for alternative nebulizer devices that are either disposable or labelled for multipatient use.To date, the present study was the most comprehensive analysis objectively assessing different nebulizer devices for use in performing sputum induction for the purposes of measuring sputum cell counts.When compared with the gold standard Universal III ultrasonic nebulizer, the different devices had variable aerosol outputs.Aerosol size testing showed that both the AeroNeb Solo and Omron NE-U17 systems had slightly larger aerosol size than the EASYneb II, but were considered to be equivalent in terms of the aerosol deposition associated with tidal-flow breathing patterns and with nebulized aerosol administration.There were no differences in the induced sputum cell counts obtained using either the EasyNeb II or the AeroNeb devices.Our study provides a framework for the appropriate evaluation of nebulizer devices for induced sputum testing.This framework adheres to the strict European Respiratory Society guidelines for nebulizer testing and selection for clinical applications (15).
In an earlier study assessing the use of induced sputum cell counts, Pin et al (23) used the Fisoneb ultrasonic nebulizer (Canadian Medical Products Ltd, Canada), which was then used in other sputum induction studies (2,24).The Fisoneb nebulizer became the precursor of the Medix ultrasonic nebulizer (Clement Clarke International Ltd, United Kingdom), which was then used in various applications of sputum induction (25,26).Both the Fisoneb and Medix ultrasonic nebulizers have highly similar aerosol characteristics.Belda et al (27) compared the relatively low output, large particle size Medix ultrasonic nebulizer with the higher output smaller particle size DeVilbiss   Ultraneb 99 nebulizer (DeVilbiss Co, USA).There were no significant differences observed with regard to sputum cell counts between the two nebulizers, although the higher output nebulizer did result in lower eosinophilic cationic protein and interleukin-8 levels within the sputum fluid-phase.More recently, Loh et al ( 28) compared a Taiwanese-made compressed air nebulizer (BestNeb, Apex Medical Corp, Taiwan) with an ultrasonic nebulizer, Medix Sonix 2000 (Clement Clarke International Ltd, United Kingdom).Sixteen healthy adult subjects underwent sputum induction with the different devices with either hypertonic saline or isotonic saline.The ultrasonic nebulizer and hypertonic saline resulted in higher total sputum cell counts and improved cell viability.There was no difference in the differential cell counts.There were several limitations of this particular study including the assessment of only two nebulizer devices and the lack of aerosol testing.The order of nebulizer use was not randomized.Furthermore, the sputum induction procedures were performed over four weeks, increasing the chance of external factors affecting the sputum cell count.The current study extends our knowledge in the area of sputum induction by performing rigorous aerosol testing on several commercially available nebulizer devices and controlling for the potential limitations identified in earlier studies.
When comparing aerosol output among different designs, the most useful metric is the aerosol output rate (ie, the volume of aerosol 'inhaled' by a patient in 1 min [μL/min]) (29).This metric enables comparison of various nebulizer designs that may require different volume fills of test saline.With a focus on aerosol output rate, a suitable alternative nebulizer was deemed to be one that would provide a similar rate to the Universal III.The EASYneb II provided a similar output rate but was not a suitable alternative due to the fact that it is labelled as a single-patient device.The Respigard II had a very low aerosol output rate of 11 μL/min.The MistyMax 10 and the PARI LC Sprint, both jet nebulizers, along with the AeroNeb Solo, a vibrating mesh nebulizer, had an aerosol output rate on the lower end of the Universal III.When tested at maximum settings (airflow and nebulization rate), the Omron NE-U17 had an aerosol output rate higher than the Universal III.The overall aerosol output for the Omron unit appeared to be lower than other nebulizers due to a large dead volume (the minimum volume of saline required by the unit to produce an aerosol).The MMAD, as a single unit description of aerosol size, is the most useful metric for evaluating different systems (30).The AeroNeb Solo and the Omron NE-U17 were found to have similar MMADs compared with the EasyNeb II device.
Determining whether induced sputum cell counts differ among different nebulizer devices is an important clinical question.Sputum cell counts can be used clinically to assist with management of airway inflammation, including titration of inhaled corticosteroid therapy in asthma management (9).Given that we wanted to assess changes in sputum cell counts only related to the type of nebulizer device, and not related to underlying airway disease, we chose to study healthy, nonsmoking subjects.In an effort to reduce systematic bias, we randomized the type of nebulizer device used for the first induced sputum collection.Although the respiratory therapist performing the induced sputum test could not be blinded to the nebulizer device, the cytotechnologist preparing the samples and performing the cell counts was blinded to the type of nebulizer.We scheduled the second induced sputum collection 48 h after the first collection because repeated induced sputum procedures within 48 h have been shown to alter the cellular composition of sputum (31)(32)(33).
There were a few limitations to the present study.First, the choice of nebulizer devices was limited to those that were CSA approved and commercially available and supported in Canada.Second, we did not assess whether sputum fluid-phase mediators, such as sputum cytokines, differed among the nebulizer devices.Third, testing was not performed on patients with asthma.However, given that the principle aim of the present study was to assess changes in sputum cell counts related only to the type of nebulizer device, we did not include subjects with asthma.Having identified the AeroNeb Solo as being the best candidate among seven potential nebulizers in the present study among healthy subjects, future studies will now focus on comparing the AeroNeb Solo with the EASYneb II nebulizer in patients with asthma and other inflammatory airway diseases.Fourth, we did not perform a formal cost analysis.However, compared with the cost of the single-patient device EASYneb II ($325), the AeroNeb Solo was substantially less expensive ($47) in Canada.

CONCLUSION
The present analysis was the most comprehensive study formally assessing different nebulizer devices for use in performing sputum induction for the purposes of measuring sputum cell counts.The various nebulizers we tested had variable aerosol outputs.The AeroNeb Solo, Omron NE-U17 and EASYneb II systems were found to have similar measurements of aerosol size.We found no significant difference in the induced sputum cell counts obtained with the AeroNeb Solo and the EASYneb II devices.Consequently, we have formally adopted the AeroNeb Solo device as the nebulizer of choice in our clinical sputum induction program at the University of Calgary.Our study provides a framework for the rigorous, scientific evaluation of nebulizer devices for induced sputum testing using accepted guidelines.

Figure 1 )
Figure 1) The plots show two expressions of a typical nebulized aerosol size distribution.The x-axis defines nebulized aerosol droplet (Dp) size between 0.1 μm and 100 μm in a log-normal scale.The cumulative size distribution (triangles) plots the cumulative mass of size fractions obtained from a nextgenration cascade impactor (15 L/min flow) and relates to the left y-axis (cumulative undersize %).From the cumulative size plot, the mass median aerodynamic diameter can be determined as the size intercepting the 50% cumulative mass, which is 5.9 μm in this example.The circles represent a normative plot of the same size distribution whose shape indicates a near symmetrical log-normal distribution typical of many nebulized aerosol size distributions.AeroNeb (Aerogen Ltd, Ireland); conc Concentration 1,2 , Stephanie The BSc 1 , Belinda Litoski RRT 1 , Cora Pieron PhD 1,2 , Richard Leigh MD PhD 1 1 Department of Medicine, University of Calgary; 2 SolAero Ltd, Calgary, Alberta Correspondence: Dr Warren J Davidson, Department of Medicine, University of Calgary, 7007-14th Street Southwest, Calgary, Alberta T2V 1P9.