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The measurement of nitric oxide (NO) in exhaled breath has given us the ability to learn about and monitor the inflammatory status of the airway through a non-invasive method that is easy to perform and repeat. This has been most useful in the diagnosis and management of asthma and has promised a seemingly unlimited potential for evaluating the airways and how clinical decisions are made (Grob N M and Dweik R A 2008 Chest 133 837–9). The exhaled NO field was initially limited, however, due to the absence of standardized methodology. The ATS and ERS jointly released recommendations for standardized methods of measuring and reporting exhaled NO in 1999 that were revised in 2005 (1999 Am. J. Respir. Crit. Care. Med. 160 2104–17; 2005 Am. J. Respir. Crit. Care. Med. 171 912–30). In this paper, we summarize the literature that followed this standardization. We searched the literature for all papers that included the term ‘exhaled nitric oxide’ and selected those that followed ATS guidelines for online measurement for further review. We also reviewed cut-off values suggested by groups studying exhaled nitric oxide. We found a wide range of NO values reported for normal and asthma populations. The geometric mean for FENO ranged from 10 ppb to 33 ppb in healthy adult control populations. For asthma, the FENO geometric mean ranged from 6 ppb to 98 ppb. This considerable variation likely reflects the different clinical settings and purposes of measurement. Exhaled NO has been used for a multitude of reasons that range from screening, to diagnosis, to monitoring the effect of therapy. The field of exhaled NO has made undeniable progress since the standardization of the measurement methods. Our challenge now is to have guidelines to interpret exhaled NO levels in the appropriate context. As the utility of exhaled NO continues to evolve, it can serve as a good example of the crucial role of the standardization of collection and measurement methods to propel any new test in the right direction as it makes its way from a research tool to a clinically useful test.
The nitric oxide (NO) field has come a long way since the identification of NO as what been known as ‘endothelial-derived relaxing factor’ in the late 1980s [4, 5]. Soon after that in the early 1990s, the ability to measure NO in exhaled breath by chemiluminescence opened a new window in the ability to study lung physiology and disease . While the field of exhaled breath research had already made significant strides by then, rapid development and standardization of exhaled NO measurements brought this method to the forefront of breath analysis as a non-invasive tool in diagnosing and monitoring disease. Nearly every year brings more discoveries in exhaled NO than the year before (figure 1). Asthma is one of the diseases most studied by this technique and will be the primary focus of this paper.
NO is endogenously synthesized by a group of enzymes, nitric oxide synthases (NOS), that help regulate the tone of blood vessels and airways, provide signals for neurotransmission in the bronchial wall, and control ciliated beating of epithelial cells [7–11]. One form of NOS, inducible NOS (iNOS), is thought to be the source of increases in NO during inflammation: its expression is induced by inflammatory mediators (TNF-α,IL-1β,IFN- α) and inhibited by corticosteroids [12, 13]. In asthma, a disease characterized by airway inflammation, exhaled NO levels have been found to correlate with eosinophilic airway inflammation [14–17]. The ability to learn the inflammatory status of an airway through a non-invasive measurement has promised a seemingly unlimited potential for evaluating the airways and how clinical decisions are made .
Exhaled NO can be measured by two main techniques: online and offline measurements. The online measurement refers to the fractional concentrations of orally exhaled nitric oxide (FENO) testing with a real-time display of NO breath profiles; the offline testing refers to a collection of exhalate for later analysis . FENO levels measured may vary depending on the technique . Similarly, the flow rate at which FENO is measured is another important component of variability. At higher flow rates, the transit time for NO to diffuse into the airway is limited: as the flow rate increases, FENO decreases [20–22].
Many promising trends were discovered early in the investigations of FENO. Smoking was found to reduce FENO [19, 23–25], while asthmatic smokers still had elevated FENO . Age also had an effect on FENO:FENO increased with age in children [27–29]. The ingestion of caffeine and nitrates influenced the FENO measurement [30, 31]. Respiratory track infections also elevated FENO in asthmatics [32, 33]. This mileu of trends (table 1) suggested the need for a task force to outline guidelines to standardize this new measurement technique to reduce the confounding effects of many of these newly discovered variables on future NO measurements.
The ATS and ERS jointly released recommendations for standardized methods of measuring FENO in 1999 that were revised in 2005 [2, 3]. One of the main reasons that stimulated the development of the guidelines was the seminal observation that the exhaled NO levels were dependent on the expiratory flow rate . The recommendations provided standards for online and offline procedures including background information regarding the choice between the two techniques. The documents included descriptions of NO terminology and units, factors influencing FENO values, techniques for exhaled NO measurement, recommendations for expiratory flow rate and interpretations of NO single breath values. The recommendations also included information for optimizing NO measurement in special populations, including children.
Since the guidelines have been released, FENO has emerged as a highly reproducible technique for non-invasively examining the airways. Before the guidelines were released, research in this field focused on studying the different methods and determining whether NO changes could be found in specific conditions. Since the guidelines were published, however, the field has moved much further: NO levels have been determined in several conditions such as asthma, chronic obstructive pulmonary disease, bronchiectasis, cystic fibrosis, sarcoidosis, upper respiratory infections, primary ciliary dyskinesia, pulmonary hypertension, lung transplantation rejection and others. In asthma, one of the diseases most examined by this technique, FENO values have been used to predict exacerbations [34–36], predict the outcome of inhaled corticosteroid (ICS) withdrawal , monitor adherence to treatment  and to aid in the management of asthma . There has also been significant work in establishing normal FENO values [40–45].
We have searched the literature for all papers that included the term ‘exhaled nitric oxide’ and selected those that followed ATS guidelines for online measurement for further review (table 2). Despite the new guidelines, we found a wide range of NO values reported for normal (table 3) and asthma (table 4). The geometric mean for FENO ranged from 10 ppb to 33 ppb in healthy adult control populations; in healthy children FENO ranged from 5 ppb to 14 ppb. For asthma, the FENO geometric mean ranged from 6 ppb to 98 ppb; in asthmatic children, this range was from 11 ppb to 44 ppb. Groups such as Travers et al used multivariate modeling to suggest that reference NO values should be grouped by gender, atopy status and smoking status; this study was one of the first to publish the expected reference values for exhaled nitric oxide based on the presence of these variables . Sex, atopy and smoking status significantly impacted normal values for exhaled nitric oxide; the presence of multiple factors had a multiplicative effect on reference ranges determined by multivariate modeling .
We also reviewed cut-off values suggested by groups studying exhaled nitric oxide. They include the following suggestions for adults:
Similar recommendations were made for children and nitric oxide:
Combined with the fact that there is considerable overlap in FENO between healthy individuals and asthmatics, defining different cut points for different clinical settings may be more clinically useful than normative values. Once the clinical setting is taken into consideration, certain patterns begin to emerge. The FENO levels above 45–50 ppb may predict steroid responsiveness while the levels below 35 ppb can suggest optimal asthma control in an asthmatic on therapy. The FENO levels above 20–25 ppb suggest the presence of asthma in a steroid naive individual with symptoms while the lower levels are not likely to be associated with airway inflammation . What needed now are interpretation guidelines to make the FENO levels more clinically useful to practitioners . These are currently under development by the American Thoracic Society (ATS).
Although the field has made undeniable progress, many important questions remain before FENO and its clinical implications can be fully understood. The pathobiologic role of NO, as fundamental as it is, remains incompletely understood. Some groups believe NO is involved in pro-inflammatory conditions that trigger immunomodulatory reactions , while others consider its effect on smooth muscle relaxation a protector against inflammatory reactions such as airway hyperresponsiveness [56, 57]. A fundamental understanding of the role of NO in physiology and how that function is altered in pathology is key to being able to interpret FENO values.
In addition to learning more about NO, several steps must be taken in order to improve the integration of FENO into the clinical setting. First, more work needs to be done in order to better define a normal healthy reference population. Many studies thus far with normal healthy controls report ‘normal’ FENO without addressing the effect of potential confounders in this control population (i.e., atopy, smoking status, gender, etc). Other studies that focus on establishing reference values for the healthy population have been too small and the ranges of accepted values have been too great to draw conclusions from. There is a need to establish a large normal healthy reference population, taking note of the presence of these known confounders, in order to obtain a better understanding of what a normal FENO really is. The recent inclusion of FENO measurement in the National Health and Nutrition Examination Survey NHANES is expected to provide more useful population-based normative values.
Next, once normal values are better understood, relationships between FENO and confounders need to be determined. For example, a clinician should be able to adjust the expected FENO for a healthy patient after a change in the smoking status. The ultimate goal of being able to predict a FENO measurement in a patient based on knowledge of their smoking status, age, gender, height and other factors is known to impact the FENO measurements. Until such predicted values are available for FENO, certain cut-off points may be helpful in deciding how to use a particular FENO value in a particular individual. However, in addition to the possible confounding variables, the clinical context, the reason for the test and the pretest probability of the outcome may all need to be taken into consideration. This requires the development of guidelines for the interpretation of FENO, a need that is currently being addressed by the ATS which has already taken on this task of developing interpretation guidelines for FENO.
As the utility of exhaled NO continues to evolve, it can serve as a good example of the crucial role of the standardization of collection and measurement methods to propel any new test in the right direction as it makes its way from a research tool to a clinically useful test.