Final Report: Off-Line Sampling of Exhaled Nitric Oxide in Respiratory Health Surveys

EPA Grant Number: R827352C011
Subproject: this is subproject number 011 , established and managed by the Center Director under grant R827352
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern California Particle Center and Supersite
Center Director: Froines, John R.
Title: Off-Line Sampling of Exhaled Nitric Oxide in Respiratory Health Surveys
Investigators: Gong, Henry , Linn, William S.
Institution: Rancho Los Amigos Medical Center , University of Southern California
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air


Topic C: Studies of the Effects of Varying Spatial and Temporal Patterns of Ambient Particulate Matter (PM) and Co-pollutants and Resulting Health Effects with Emphasis on the Role of Atmospheric Chemistry

Delayed offline measurement of exhaled nitric oxide (eNO) has important applications in environmental health screening studies where direct online measurement can be impractical. However, the use and application of delayed offline eNO has been limited by the instability of stored breath samples. Our goal was to develop a practical method for off-line measurement of eNO that can be applied in larger multiple-site epidemiologic surveys, with reliable preservation of exhaled breath samples for analysis at one central laboratory.

Summary/Accomplishments (Outputs/Outcomes):

Description of Research

Exploratory experiments indicated that breath could be collected satisfactorily in commercial eNO sampling bags (Sievers Instruments, Boulder, CO) or in structurally similar toy balloons made of aluminized polyethylene terephthalate film (Mylar or equivalent). We used both sampling bags and balloons with a Bag Collection and Sampling Kit (Sievers Instruments) that allowed a subject to take a vital-capacity inspiration of relatively NO-free air and immediately exhale it at a measured flow rate near 100 mL/sec. Exhaled air was allowed to escape for the first 3–6 sec to clear deadspace, then a sample of 0.5 to 1.5 L was collected. After preliminary experiments to optimize the procedure, we obtained 185 air samples, including:

  • breath from 38 nonsmoking volunteer subjects (10 adults, 22 high school students, and 6 children aged 9–13, including individuals with asthma or other chronic cardiorespiratory conditions),
  • ambient air over a range of NO pollution conditions,
  • air filtered by the breath collection apparatus (like the air inhaled by subjects during tests), and
  • commercial zero air free of NO.

Samples were collected in our laboratory and at 5 field locations (3 schools, 2 homes) in
metropolitan Los Angeles. Each sample was measured repeatedly with a chemiluminescent NO analyzer (Sievers NOA Model 280i) over a period of 1–7 days. A calibration check with zero and span gas was performed within 20 min of each sample measurement. Samples were stored at 42, 22, 6, or -14 degrees Celsius to evaluate effects of temperature on stability. Certain samples were stored in largely NO-free atmospheres, and compared with others stored in NO-polluted ambient air, to test effects of leaks or permeation.

Regression analyses were performed to evaluate samples’ changes in NO concentration over time and to identify factors that influenced those changes, including sample source, initial concentration and storage temperature.


We found both positive and negative changes in NO concentration during eNO sample storage, depending upon temperature, initial concentration and reaction with ambient air. For example, NO in breath samples stored at 22 degrees increased with time in approximately linear fashion; greater increase was noted for samples stored at 42 degrees. In contrast, storage at -14 degrees had a negative effect. Samples with initially high NO tended to show decreases over time, while samples initially low in NO showed very slow increases over time at 6 degrees. Variability within subjects was similar to that reported from previous studies of on and immediate offline measurements.

The findings of increased NO in samples with low or zero initial concentrations support an earlier paper in which it was suggested that aluminized Mylar containers can desorb NO into collected samples. In our hands, this desorption appears to be temperature sensitive.


Conclusions and Recommended Sampling Protocol

The study was successful in developing procedures suitable for studies that require large-scale collection of breath samples at field locations, and measurement of NO in a central laboratory. We found that numerous sources of variability may influence the delayed offline measurement of eNO. The resulting errors may differ in direction as well as magnitude, depending upon the collection and analysis system. Our system generally was associated with gain of NO at or above room temperature and loss of NO at lower temperatures. Given these findings, the key requirements for a successful study that were identified include refrigeration of Mylar sampling containers after collection and standardization of the time interval between collection and analysis. Based on our findings and information from published literature, we recommend the following for off-line eNO measurement in the Children’s Health Study and similar surveys:

  1. The Sievers Bag Collection and Sampling Kit and Sievers NOA 280i analyzer, or equivalent equipment, should be used. The manufacturer’s recommended procedures should be used, except that an abbreviated zero and span check should be performed at least every 20 min during analysis, and sample readings should be adjusted according to the zero/span check results closest in time.
  2. The subject should perform a vital-capacity inspiration through an NO-reducing filter, followed immediately by a flow-controlled vital capacity expiration. Expiratory flow should be standardized at 100 mL/sec, as read by the sampling kit’s expiratory pressure gauge with appropriate allowance for altitude. Deadspace gas must be discarded, so collection should begin between 3 and 6 seconds after the start of expiration.
  3. Two samples should be collected from each subject. One initial practice maneuver with no collection should be allowed, at least for younger children.
  4. The Sievers 1.5-L sample bag, washed 3 times with zero-NO gas and evacuated per manufacturer’s instructions, is the optimum sample container, reusable many times with careful handling. Bags must be individually identified, and the identity code must be recorded at each sample collection to facilitate quality review. Alternatively, aluminized Mylar self-sealing balloons may be used once and discarded.
  5. The same type of sample container should be used throughout a study.
  6. For each collected sample the technician should observe and code the expiration as satisfactory, slow, fast, or erratic.
  7. The time from collection to analysis, and the temperature during that time, must be similar for all samples. One possible strategy is to analyze samples 24 ± 4 hr after collection, keeping them refrigerated at approximately 5° C, or in heavily insulated containers surrounded by ice, in the interim.
  8. Insofar as practical, the analyst should be blind to the origin of each sample. If measurement is by the Sievers automated recording procedure, the point estimate for a sample should be the mean determined from a plateau of concentration with duration >15 sec. For manual measurement, at least three independent readings should be taken between 15 and 25 sec after starting the flow of sample air, and their median should be the point estimate for the sample. The mean reading from two samples should be the point estimate for a subject. If the two samples differ by >30% of the larger reading or by >3 ppb, whichever is greater, the data should be discarded as unreliable.

The work was published in:

Linn WS, Avila M, Gong H. Offline measurement of exhaled nitric oxide: sources of error. Archives of Environmental Health 2004;59(7):1-7.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 1 publications 1 publications in selected types All 1 journal articles
Other center views: All 150 publications 149 publications in selected types All 149 journal articles
Type Citation Sub Project Document Sources
Journal Article Linn WS, Avila M, Gong H Jr. Exhaled nitric oxide: sources of error in offline measurement. Archives of Environmental Health 2004;59(8):385-391. R827352 (Final)
R827352C011 (Final)
R827352C012 (Final)
R831861 (2005)
R831861 (Final)
R831861C001 (Final)
R831861C002 (Final)
R831861C003 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Abstract: AEH-Abstract
  • Supplemental Keywords:

    RFA, Health, Scientific Discipline, Air, Geographic Area, HUMAN HEALTH, particulate matter, Environmental Chemistry, State, Risk Assessments, Biochemistry, Health Effects, ambient aerosol, asthma, particulates, epidemiology, human health effects, toxicology, airway disease, allergic airway disease, air pollution, human exposure, toxicity, particulate exposure, indoor air quality, California (CA), allergens, breath samples, aerosols, atmospheric chemistry, dosimetry, human health risk, Nitric acid, genetic susceptibility, particle transport, particle concentrator

    Relevant Websites: Exit

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000
  • 2001
  • 2002 Progress Report
  • 2003
  • 2004

  • Main Center Abstract and Reports:

    R827352    Southern California Particle Center and Supersite

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827352C001 The Chemical Toxicology of Particulate Matter
    R827352C002 Pro-inflammatory and the Pro-oxidative Effects of Diesel Exhaust Particulate in Vivo and in Vitro
    R827352C003 Measurement of the “Effective” Surface Area of Ultrafine and Accumulation Mode PM (Pilot Project)
    R827352C004 Effect of Exposure to Freeways with Heavy Diesel Traffic and Gasoline Traffic on Asthma Mouse Model
    R827352C005 Effects of Exposure to Fine and Ultrafine Concentrated Ambient Particles near a Heavily Trafficked Freeway in Geriatric Rats (Pilot Project)
    R827352C006 Relationship Between Ultrafine Particle Size Distribution and Distance From Highways
    R827352C007 Exposure to Vehicular Pollutants and Respiratory Health
    R827352C008 Traffic Density and Human Reproductive Health
    R827352C009 The Role of Quinones, Aldehydes, Polycyclic Aromatic Hydrocarbons, and other Atmospheric Transformation Products on Chronic Health Effects in Children
    R827352C010 Novel Method for Measurement of Acrolein in Aerosols
    R827352C011 Off-Line Sampling of Exhaled Nitric Oxide in Respiratory Health Surveys
    R827352C012 Controlled Human Exposure Studies with Concentrated PM
    R827352C013 Particle Size Distributions of Polycyclic Aromatic Hydrocarbons in the LAB
    R827352C014 Physical and Chemical Characteristics of PM in the LAB (Source Receptor Study)
    R827352C015 Exposure Assessment and Airshed Modeling Applications in Support of SCPC and CHS Projects
    R827352C016 Particle Dosimetry
    R827352C017 Conduct Research and Monitoring That Contributes to a Better Understanding of the Measurement, Sources, Size Distribution, Chemical Composition, Physical State, Spatial and Temporal Variability, and Health Effects of Suspended PM in the Los Angeles Basin (LAB)