Advancing Asthma

Management with
Exhaled Nitric Oxide
RESOURCE KIT

Summary
Exhaled nitric oxide (eNO) is an established marker of airway
inflammation that can be safely and accurately measured in
people with asthma. This resource kit provides comprehensive
information about the benefits and technology of eNO testing,
and highlights the medical necessity and the extent of acceptance
of eNO testing.
The Asthma and Airway Inflammation Dilemma
It is estimated that 22 million+ people in the U.S. suffer from asthma,
making it one of the most common and costly of all diseases. One
quarter of all emergency room visits are asthma related and asthma
is the one of the leading chronic childhood diseases.
Controlling inflammation of the airways has become the central
focus for managing asthma. Strong clinical evidence suggests
that asthma management and control can be significantly
improved by regularly monitoring airway inflammation. However,
current methods used to monitor and manage asthma, such as
lung function tests, do not measure airway inflammation.
Physicians have relied largely on correlating symptoms and disease
severity to assess their patients. Until now, the degree of airway
inflammation has not been measurable in a simple and practical way.
The eNO Solution
Exhaled nitric oxide has been established as a reliable marker of
airway inflammation in asthma for over 10 years. It has been the
central focus of studies, establishing its link to optimization of
medication and prediction of asthma exacerbations.
Measurement of eNO in the physician’s office is a much awaited
breakthrough in medical technology that provides physicians with
a reliable tool to measure airway inflammation as an adjunct to
the current diagnostic measures, such as lung function.
2
Table of Contents
Summary. . . . . . . . . . . . . . . . . . . . . 2
1. The Burden of Asthma . . . . . . . . . . . . . . 3
2. Exhaled Nitric Oxide (eNO). . . . . . . . . . . 4
3. Insight™ eNO System. . . . . . . . . . . . . . 6
4. Case Studies. . . . . . . . . . . . . . . . . . . 9
5. Exhaled Nitric Oxide Cost Model. . . . . . . . 12
6. Coding Information for Exhaled Nitric
Oxide (eNO)-CPT 95012. . . . . . . . . . . . . 12
Clinical Utility of eNO
Monitoring of eNO and adjusting medication accordingly could
significantly improve disease management, resulting in reduction
of the severity of symptoms, optimization of drug usage and
improvement of compliance in individual patients, and fewer
exacerbations. The end result is optimal use of healthcare
resources and improved quality of life for asthma patients.
Reimbursement for eNO Testing
In 2007, the CPT editorial committee added a specific code to
the CPT Coding Book for Nitric Oxide Expired Gas Determination
(CPT Code 95012©). With this action, the procedure should be
recognized as a standard service provided to patients. Exhaled
nitric oxide (eNO) determination is considered a service which is
consistent with contemporary medical practice for the evaluation
of patients with respiratory complaints.
Apieron and the Insight™ eNO System
Apieron, Inc. is a medical device company based in Menlo Park,
California. The company was founded in 2001 to develop the
first practical, office-based device for routine measurement of
exhaled nitric oxide. In March 2008, the company received FDA
510(k) clearance for the Insight™ eNO System, which is now
commercially available. Apieron’s goal is to offer a better way to
manage asthma and improve the standard of care for patients
who live with the disease.
The Insight system is a highly accurate device, expressly
designed for the physician’s office to measure nitric oxide in
expired human breath (eNO). It is non-invasive, safe, easy to
use, and provides results in less than a minute. Apieron’s unique
biosensor utilizes proprietary technology to detect trace amounts
of nitric oxide in a single human breath.
1. The Burden of Asthma
Asthma Overview
Asthma is a chronic disease characterized by inflammation of the
airways caused by allergens and other triggers. When airways
are inflamed, the inner walls of the airways swell making them
irregular. This causes the flow of air to become turbulent.
The events that lead to obstruction of airflow and thus to asthma
symptoms are complex and usually involve the following events:
• Bronchoconstriction, where the smooth muscle surrounding the
airways tightens in response to a trigger and narrows the airway.
• Inflammation, where inner walls of airways swell.
• Mucus formation within the airways that obstructs airflow.
Typical asthma symptoms include wheezing, coughing, chest
tightness (dyspnea) and shortness of breath.
The Role of Inflammation in Asthma
Asthma signs and symptoms evolve from three basic
characteristics that underlie the disease and its exacerbations:
airway obstruction, airway hyperresponsiveness and airway
inflammation. Airway obstruction and hyperresponsiveness
represent the classic physiology of asthma, and their contribution
to the disease process and symptomatology have been well
recognized for some time. Appreciation of the role of airway
inflammation in asthma has evolved more recently.
Today asthma experts consider airway inflammation a central
feature of asthma pathogenesis and its clinical manifestations.
In fact, airway inflammation likely plays a critical role in airway
obstruction and hyperresponsiveness. In recent years, clinical
and scientific knowledge of asthma has evolved from a model
of episodic constriction of bronchial smooth muscle to a model
which involves chronic airway inflammation.
Airway inflammation precedes symptoms. Evidence of
inflammation is present at the onset of symptoms in newly
diagnosed patients with asthma. Accordingly, treatment
algorithms for asthma have emphasized treatment of the
underlying inflammation, as well as the bronchoconstrictive
symptoms. By acquiring a better understanding and appreciation
of the inflammatory process, physicians can employ treatments
to inhibit specific steps in the process and improve control over
asthma and its symptoms.
The Cost of Asthma
Asthma affects 22 million Americans. A principal clinical
consequence of both acute and chronic inflammation is the
development of asthma exacerbations. Exacerbations of
asthma are not only an important clinical marker of inadequately
controlled or worsening asthma but are probably the most
important outcomes from both a humanistic and health
economics viewpoint. Severe asthma exacerbations lead to about
4,000 deaths and nearly 500,000 hospitalizations per year.
Healthcare costs for asthma include outpatient visits to physician
offices and hospital outpatient departments, visits to hospital
emergency departments (EDs) and hospitalizations. When
3
considering the prevalence of asthma and the frequency of such
visits, as outlined in the statistics below, the costs become
monumental.
• 34.1 million people have been diagnosed with asthma during
their lifetime.1
• 22.8 million people have asthma.1
• There were 14.1 million outpatient asthma visits to private
physician offices and hospital outpatient departments.2
• Children less than 18 years had 7 million physician office and
outpatient visits.3
Morbidity
• Asthma accounts for one quarter of all emergency room visits
in the U.S. each year, with 2 million emergency room visits.4
• Each year, asthma accounts for more than 1 million outpatient
visits4 and 500,000 hospitalizations.5
• The average length of stay (LOS) for asthma hospitalizations is
3 days.6
• Nearly half (41%) of asthma-related hospitalizations are for
children less than 19 years old.5
• Respiratory illnesses like asthma are the leading cause of
hospitalization for children. 6
Mortality
• There are about 4,000 deaths due to asthma each year, many
of which are avoidable with proper treatment and care.7
Social and Economic Costs
• The annual cost of asthma is estimated to be nearly $18 billion.
• Direct costs accounted for nearly $10 billion (hospitalizations
the single largest portion of direct cost) and indirect costs of $8
billion (lost earnings due to illness or death).8
• For adults, asthma is one of the leading causes of work
absenteeism and “presenteeism,” resulting in nearly 13 million
missed or lost (“less productive”) workdays each year.9
• Among children ages 5 to 17, asthma is the leading cause
of school absences from a chronic illness. It accounts for an
annual loss of more than 13 million school days per year.3 It is
estimated that children with asthma spend a nearly 8 million
days per year restricted to bed.8
1. “National Health Interview Survey,” National Center for Health Statistics, CDC, 2006.
2. CDC: http://www.cdc.gov/nchs/fastats/asthma.htm - accessed on Sep 2, 2008.
3. “State of childhood asthma in the United States,” CDC,1980-2005.
4. “National Hospital Ambulatory Medical Care Survey,” CDC, 2001-2004.
5. “National Hospital Discharge Survey,” National Center for Health Statistics, CDC,
2001-2004.
6. “National Health Statistics Survey,” National Center for Health Statistics, CDC, 2006.
7. “National Vital Statistics Reports,” Vol 56, Number 10, CDC, 2005.
8. “The Costs of Asthma,” Asthma and Allergy Foundation 1992 and 1998 Study,
2000 Update.
9. “Asthma Prevalence, Health Care Use and Mortality,” CDC, 2003-2005.
2. Exhaled Nitric Oxide (eNO)
Current Methods for Assessing Airway
Inflammation
Airway inflammation is shown to be an appropriate target for
improving asthma control10, and while it is recognized as playing
a key role in the pathophysiology of asthma, current methods to
evaluate a patient’s asthma status fall short because they do not
directly measure the degree of airway inflammation. Physicians
attempt to monitor asthma severity through clinical exam and
pulmonary function testing using spirometry or peak flow meters.
Unfortunately, these lung function tests do not directly measure
airway inflammation. Other markers of inflammation such as
bronchoalveolar lavage and induced sputum are invasive and
impractical to perform in a physician’s office. Therefore, when
prescribing and titrating medication, physicians have had to rely
on qualitative measures such as correlation of disease severity
and symptoms. Adding to this challenge is the fact that patients
with severe asthma may have a compromised perception of
airflow obstruction and dyspnea compared to normal individuals11
and, thus, underestimate the severity of their symptoms.
Without a convenient means to accurately and regularly assess
inflammation, it is difficult for physicians to manage asthma.
Inhaled corticosteroids (ICS) are the mainstay of treatment for
chronic asthma, and dosing should be adequate to control asthma
symptoms but also be as low as possible to avoid side effects.
Under-medication and over-medication are sub-optimal both
clinically and economically. If not managed properly, people with
asthma could experience permanent and irreversible damage
to their airways. Since the dose of medication required is highly
variable, both among patients and within individual patients,
physicians need an easy, effective and safe method to assist
them with titrating medication precisely.
The lack of knowing a patient’s degree of airway inflammation
creates a significant gap in asthma management today. With
direct markers of inflammation, this gap can be closed to improve
the overall treatment and management of asthma. Insight into a
patient’s airway inflammation could enable physicians to make
earlier interventions with appropriate levels of therapy, thereby
preventing emergency room visits and hospitalizations.
Exhaled Nitric Oxide (eNO) as a Biomarker of
Airway Inflammation
Nitric oxide (NO), a free radical, is produced by the body as part
of the inflammatory response and can be detected as exhaled
nitric oxide in expired human breath. Nitric oxide is produced
in the airway epithelial cells by a family of NO synthases called
inducible nitric oxide synthase. Inducible NO synthase expression
is sensitive to steroids and therefore shows a change in response
to treatment with anti-inflammatory medication like inhaled
steroids. Nitric oxide is a mediator of inflammation and it is an
10. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P,
Wardlaw AJ, Pavord ID. Asthma exacerbations and sputum eosinophil counts: A
randomised controlled trial. Lancet 2002;360:1715-1721.
11. Kikuchi Y, Okabe S, Tamura G, Hida W, Homma M, Shirato K, Takishima T.
Chemosensitivity and perception of dyspnea in patients with a history of near-
fatal asthma. N Engl J Med. 1994;330(19):1329–1334.
4
effective local vasodilator. It causes smooth muscle relaxation
thereby matching regional airflow and blood flow. Nitric oxide is
also important for ciliary action. Numerous studies over the past
decade have validated that exhaled nitric oxide (eNO) can serve as
a biomarker of airway inflammation in asthma.
In 1991, NO in exhaled breath was first reported.12 Studies have
shown that eNO is correlated with other markers of inflammation.
For example, exhaled nitric oxide correlates with eosonophilic
airway inflammation measured in induced sputum13 and in
bronchoscopy (lavage and biopsy), most commonly seen in
asthma but also in any condition where eosonophils are present,
such as allergic rhinitis, eosonophilic bronchitis14 and chronic
obstructive pulmonary disease (COPD).15
It has also been shown that eNO levels are elevated in steroid-
naive asthma,16 and eNO levels fall rapidly with anti-inflammatory
medication (e.g. inhaled corticosteroids)17, oral steroids, anti-
leukotrienes (e.g. montelukast)18 and anti-IgE (e.g. Xolair).19 Exhaled
nitric oxide also correlates with non-specific bronchial reactivity
to methacholine (typically assessed in methacholine challenge) in
steroid-naïve patients. Increased reactivity is reflected by higher
eNO levels and is an indirect marker of airway inflammation.
It is now well established that the concentration of nitric oxide
in exhaled breath (eNO) is a reliable indicator of the degree of
inflammation in the airways. Exhaled nitric oxide levels increase as
inflammation increases and decrease as inflammation decreases.
Clinical Recommendations for eNO
Clinical recommendations exist for use of eNO measurements in
asthma management, and such recommendations are currently
being reviewed and updated:
• The American Thoracic Society and European Respiratory
Society (ATS/ERS) have published recommendations for the
standardized measurement of eNO.20
12. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous
nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans.
Biochem Biophys Res Commun. 1991;181(2):852-857.
13. Jatakanon A, Lim S, Kharitonov SA, Chung KF, Barnes PJ. Correlation between
exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in
patients with mild asthma. Thorax 1998;53:91-95.
14. Berlyne GS, Parameswaran K, Kamada D, Efthimiadis A, Hargreave FE. A
comparison of exhaled nitric oxide and induced sputum as markers of airway
inflammation. J Allergy Clin Immunol 2000;106:638-644.
15. Fabbri LM, Romagnoli M, Corbetta L, et al. Differences in airway inflammation
in patients with fixed airflow obstruction due to asthma or chronic obstructive
pulmonary disease. Am J Respir Crit Care Med 2003;167:418-424.
16. Alving K, Weitzberg E, and Lundberg JM. Increased amount of nitric oxide in
exhaled air of asthmatics. Eur Respir J. 1993;6(9):1368-1370.
17. Yates DH, Kharitonov SA, Robbins RA, Thomas PS, Barnes PJ. Effect of a nitric
oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am J
Respir Crit Care Med. 1995;152(3):892-896.
18. Bratton DL, Lanz MJ, Miyazawa N, White CW, Silkoff PE. Exhaled nitric oxide
before and after montelukast sodium therapy in school-age children with chronic
asthma: A preliminary study. Pediatr Pulmonol 1999;28:402-407.
19. Silkoff PE, Romero FA, Gupta N, Townley RG, Milgrom H. Exhaled nitric oxide
in children with asthma receiving xolair (omalizumab), a monoclonal anti-
immunoglobulin e antibody. Pediatrics 2004;113:e308-312.
20 Recommendations for standardization procedures for the online and offline
measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in
adults and in children. Am J Respir Crit Care Med. 2005;171:913-930.
Separate recommendations have also been developed for children. • Monitoring patient compliance
An elevated eNO level is suggestive of an inadequate anti-
Clinical Utility of eNO inflammatory regimen, which may be due to poor compliance
Exhaled nitric oxide measurement offers new benefits for or under-medication.24 A study by Beck-Ripp and his colleagues
asthma management. showed that there was a strong correlation between reduction
in eNO values and patient compliance (measured as percentage
• Predicting steroid response of prescribed medication taken). In this study, the researchers
An elevated eNO level is highly predictive of a positive response followed 54 patients between the ages of 6 and 16 over 16
to ICS.21 Smith et al showed that eNO levels are very good weeks. The results showed that there was a 50% reduction in
indicators of response to steroid in people with undiagnosed eNO values with higher compliance.
respiratory symptoms. Exhaled nitric oxide was shown to be
• Predicting exacerbations
more accurate than spirometry, peak flow meters, bronchodilator
response and airway hyper-responsiveness (AHR). This study An elevated eNO level at a clinic visit has been shown to be
followed 52 subjects in a single-blind, fixed sequence, placebo- associated with an increased risk for an exacerbation in the
controlled trial of inhaled fluticasone over 4 weeks. Similar results following two weeks.25 In this study, moderate and severe-
were found in another study 22 (Figure 1.1 below) involving 73 persistent asthma patients were evaluated during a routine clinic
steroid-naïve subjects with uncontrolled asthma which showed visit and then noted whether they had an exacerbation within
about 50% reduction in eNO levels following ICS therapy over 2 weeks of the initial appointment. Those with an exacerbation
2 weeks. The study also showed that eNO was a significantly had a higher mean eNO (29.67 ppb ± 14.48) compared to those
better predictor of response to steroid therapy than conventional who did not (12.92 ppb ± 5.17), p = 0.002. A nominal logistic
lung function tests. Additionally, a 35% improvement in asthma regression model to determine those variables that predict
symptom scores were seen following the 2-week therapy. asthma exacerbation found that eNO was the only significant
predictor, p = 0.03.
TAS = Total Asthma In another study26 with 31 subjects (19 adults and 12 children)
Normalized measure of ability to predict steroid use.

eNO is a more reliable Score

predictor of steroid eNO = Exhaled Nitric
using the Insight system, it was shown that by regularly
response than lung Oxide monitoring the diurnal changes in eNO levels, it is possible to
function
FEV1 = Forced predict asthma exacerbations up to 1 week in advance. In this
Expiratory
Volume in
study, spirometry measurements showed no indication of an
1 Second impending exacerbation. During this study, subjects measured
Rev = FEV1 and recorded eNO levels and peak flow values twice daily
Reversibility
(morning and evening). Once a week they also performed a
4-Var = Regression
Model Using spirometry measurement at the study site. The subjects also
All 4 Variables maintained a diary of asthma symptoms. The analysis showed
4-var TAS eNO Rev FEV that a change in the week-to-week pattern of diurnal change in
eNO values predicted an exacerbation. It was also clear that
morning eNO values are better predictors than evening eNO
Figure 1.1 values. See Figure 2.1.

• Improving titration of medication

Study Day Relative
Smith et al showed that using eNO to guide ICS dose in addition 80
eNO (ppb), PEF ( % of Personal Best)

to Exacerbation
to clinical management compared with clinical management alone 70
Evening eNO
significantly reduced the dose of inhaled steroid while showing a 60 Morning eNO
numerical reduction in exacerbations.23 In a single-blind, placebo- 50 Overnight
controlled trial, the researchers randomly assigned 97 patients 40 Change
Evening PEF
with asthma who had been regularly receiving treatment with 30
Morning PEF
inhaled corticosteroids, to have their corticosteroid dose adjusted, 20
No. of subjects
in a stepwise fashion, on the basis of either eNO measurements 10
observed
or an algorithm based on conventional guidelines. After the 0
-14 -7 0 7
optimal dose was determined (phase 1), patients were followed
up for 12 months (phase 2). They found that a 40% reduction in
the dose of ICS can be achieved without loss of asthma control. Figure 2.1

21 Smith AD, Cowan JO, Brassett KP, et al. Exhaled nitric oxide: a predictor of steroid
response. Am J Respir Crit Care Med. 2005;172(4):453-459.
22 Dreon DM, Berger WE, Hutchins EE, and Parikh BR. Exhaled Nitric Oxide Predicts
Use of Controller Medication. J Allergy Clin Immunol 2008;121(2):S157 Scientific
poster at AAAAI 2008 (NOT PUBLISHED).
23 Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric
oxide measurements to guide treatment in chronic asthma. N Engl J Med.
2005.;352(21):2163-2173.
24 Beck-Ripp J, Griese M, Arenz S, Koring C, Pasqualoni B, Bufler P. Changes of
exhaled nitric oxide during steroid treatment of childhood asthma. Eur Respir J
2002;19(6):1015-1019.
25 Harkins MS, Fiato KL, Iwamoto GK. Exhaled nitric oxide predicts asthma
exacerbation. J Asthma. 2004;41(4):471-476.
26 Wolfe JD, Dreon DM, Hutchins EE, and Parikh BR. Relationship of Exhaled
Nitric Oxide and Peak Expiratory Flow to Loss of Asthma Control. J Allergy Clin
Immunol 2008;121(2):S159 Scientific poster at AAAAI 2008 (NOT PUBLISHED).

5
Performing eNO Testing is Simple and steadily through a disposable breath tube, and the results appear
Straightforward on the display within one minute. Patient data can be stored and
maintained in patient cards for trend analysis or downloaded to a
Exhaled nitric oxide testing is an accurate and reliable measure of printer for patients’ files.
airway inflammation that is:
With an accurate measure of their patients’ airway inflammation
• Non-invasive and customized reports for each patient, physicians can
• Patient-friendly significantly improve the precision of prescribed therapy.
• Fast and suitable for routine clinical analysis
The eNO test is simple to administer compared to some of the
conventional lung function testing methods or breath analyses.
The patient has to be seated and is required to exhale steadily for
11 seconds after taking in a deep breath. The device measures the
eNO level and displays a number on the screen. The interpretation
is based on this single numerical value rather than on a complex
tracing or profile. Historical values of eNO can be added to gain
better understanding of the patient’s disease progression.
Important aspects of any system that measures exhaled nitric
oxide include:
Figure 3.1. Insight Monitor
• Detection of trace amounts of the gas in parts per billion (ppb)
• Constant expiratory flow rate due to flow dependence of Insight Sensor Technology
exhaled nitric oxide
Apieron is the first company to successfully implement a
• Exclusion of nasal nitric oxide technique utilizing sol-gel technology to make a commercial
biosensor for the detection of exhaled nitric oxide. The Insight
3. Insight™ eNO System sensor is a disposable, plastic cartridge that contains a path for
exhaled breath to flow through and a sol-gel based biosensor for
Indications for Use detecting nitric oxide. The gas flow path contains desiccant to
The intended use of the Apieron Insight™ eNO System is to control the environment and condition the gas inside the sensor.
quantitatively measure exhaled nitric oxide (eNO) in expired The biosensor consists of a sol-gel matrix (a glass-based three-
human breath as a marker of inflammation in persons with dimensional matrix) with a porous structure that envelopes
asthma. Measurement of eNO in expired human breath by protein molecules which are sensitive to nitric oxide. The
the Apieron Insight eNO System is a non-invasive, simple and biosensor matrix changes its light absorption characteristics when
safe method to measure a decrease in eNO in asthma patients exposed to nitric oxide (NO). This optical change can be measured
that often occurs after treatment with anti-inflammatory and correlated to the concentration of nitric oxide.
pharmacological therapy as an indication of the therapeutic
effects in patients with elevated eNO levels. The Apieron Insight
eNO System is suitable for use in children 8 to 17 years of
age, and in adults 18 years of age and older. As an adjunct to
established clinical assessments, such as spirometry and physical
examination, eNO measurements give the physician an objective
marker to evaluate the patient’s response to anti-inflammatory
therapy. The Apieron Insight eNO System can be used by
trained operators in a physician’s office or laboratory setting. The
Apieron Insight eNO System should not be used in critical care,
emergency care or in anesthesiology.
Prescription device: Federal law restricts this device to sale by or
on the order of a physician.

A Convenient Way to Measure eNO Levels

The Insight eNO System was developed to provide a practical
and accurate means for measuring eNO levels via a simple breath
test. Designed for routine use in physician offices and clinics, the
Insight system enables physicians to monitor and manage asthma
on a regular basis. The system includes a small desktop monitor
with a large color display. It employs an eNO sensor to measure
nitric oxide from the breath sample. The patient breathes out
Figure 3.2. Insight Sensor
How Is eNO Measured?
The biosensor encapsulates a heme protein that has a high
affinity for nitric oxide. The heme protein has a metal active
site that is specific to nitric oxide (Figure 3.3). As exhaled nitric

6
oxide molecules from the breath sample flow into the sensor,
they diffuse into the porous matrix and react with the protein
molecules. This results in a change in the optical transmission
properties of the matrix in a consistent and measurable way
when light of a certain wavelength (412 nm) is passed through
the matrix (see Figure 3.4).
The optical signal generated is proportional to the concentration of
nitric oxide and enables the Insight system to accurately detect trace
amounts of nitric oxide molecules in a single breath sample. Nitric
oxide measurement results are reported in parts per billion (ppb).
Optically transparent
porous glass matrix
• A carbon dioxide filter to remove CO2 from the breath sample.
• A preconditioned desiccant to control the concentration of
water vapor surrounding the biosensor and to condition the
incoming breath sample.
• Temperature controllers to control the temperature of the
sensor and gas and to prevent temperature-induced shifts in
optical absorbance.
Insight Monitor Technology
The Insight monitor consists of three subsystems controlled
by a central microprocessor: an eNO measurement system, a
pneumatic system and an user interface.
• Measurement system—contains the hardware required to
stabilize the Insight sensor to its operating temperature and
measure the optical absorbance of the biosensor material
when exposed to the breath sample.
• Pneumatic system—controls the exhalation flow rate as outlined
in the American Thoracic Society 2005 recommendations.
• User-friendly interface—guides the operator through the test
sequence and guides the patient through the breath sampling
maneuver.

Embedded heme-protein Accuracy of the System

A study was conducted on 82 non-randomized asthmatic subjects
at a single site comparing eNO measurements taken with both
Figure 3.3. Biosensor the Insight eNO System and the Aerocrine NIOX® System. The
demographic data for this study is presented below. A total of 58
adults (ages ≥18 years) and 20 children (ages <18 years) completed
the study resulting in 78 subjects eligible for analysis. Each subject
2.5 performed two breath maneuvers on each system, and the results
ranged from 10 ppb to 197 ppb on the Insight eNO System. The
testing was performed by three trained technicians. Results were
2
analyzed to evaluate the performance of the Insight eNO System
as it compares to the NIOX System in a clinical setting.
1.5
Optical
Absorbance

measurement
1 band
200
Insight eNO System Result (ppb)

0.5
150

0
395 405 415 425 435 100
Wavelength (nm) NIOX Result ( ppb)
eNO
50 Line of Identity
Figure 3.4. Absorption Spectra (dotted line) Standard
Regression
0
Overcoming Technical Challenges 0 50 100 150 200
NIOX Results (PPB)
Although the heme protein is highly selective for NO, certain
factors will affect the optical absorbance properties of the Figure 4.1. Correlation to reference
biosensor. Detrimental factors include exposure to CO2 from the
breath sample, changes in humidity and changes in temperature.
The Insight system incorporates specific features to counteract
these factors:

7

Parameter Analysis Results [95% CI]
Ordinary Least Squares Regression
Agreement Slope 0.95 [0.90 to 1.00]
Intercept 1.3 ppb [-1.9 to 4.5]
Bias Mean Difference -1.14 ppb [-3.08 to 0.80]
Precision Mean Absolute Difference
Insight
Within-Subject Standard Deviation 3.96 ppb (2.95 to 4.98)
Repeatability
Clinical Agreement Positive Percent Agreement
@ 30 ppb Negative Percent Agreement
Table 4.1. Performance of the Insight eNO System
Agreement: Regression analysis performed on the first
measurement from each system demonstrated agreement,
with an R2 of 0.95, (y=0.95x+1.3 ppb) between the Insight eNO
System and the NIOX System.
Bias: Mean difference between the Insight eNO System and
the NIOX System was -1.14 ppb, demonstrating that there is no
consistent bias between the two systems in clinical use.
Precision: The mean absolute difference between the Insight
eNO System and the NIOX System was 5.86 ppb.
Repeatability: The first and second measurements taken on the
Insight eNO System were compared, and the average standard
deviation between measurements was 3.96 ppb for the Insight
eNO System.
Clinical Agreement: Using a clinically relevant decision point
(30 ppb), an analysis was performed that compared the results
of measurements taken on the Insight eNO System as they
compared to the NIOX System results from the same subject.
The 51 subjects identified as elevated by the NIOX System were
also identified as elevated by the Insight eNO System (100%
positive agreement). Twenty-seven subjects were identified as
normal by the NIOX System and 25 of these were identified as
normal by the Insight eNO System (93% negative agreement).
This demonstrates that both the NIOX System and the Insight
eNO System are highly consistent in detecting subjects with both
normal and elevated eNO measurements.
Interference
Two sets of endogenous interference experiments were conducted.
In the first set of experiments, interference testing was
performed on the Insight eNO System for the following common
constituents of exhaled breath:
• 200 ppm H2 (hydrogen)
• 3 ppm CO (carbon monoxide)
• 100% Relative Humidity (water vapor)
• 0.5% CO2 (carbon dioxide)
• 0 ppb NO (nitric oxide)
Twenty-four samples containing all of the above potential
interferents (balance air) and 24 samples containing only air were
R2 = 0.95 prepared and tested on the Insight eNO System. The mean
difference in response was less than 0.05 ppb and was not
statistically significant between the samples containing potential
interferents and those containing only air.
5.86 ppb
In the second set of experiments seven additional compounds
(isoprene, ethanol, acetone, ammonia, acetaldehyde,
methanethiol and methane) were tested at physiologically
relevant concentrations by comparing the response of the Insight
100% eNO System to a test gas. The test gas contained 0 ppb NO
93% (nitric oxide). A compound was deemed non-interfering if, at
the tested concentration, the response was within ±5 ppb NO
equivalent when compared to the nitrogen control.
None of the tested compounds exhibited interference greater
than ±5 ppb equivalent NO when compared to a nitrogen control.
Clinical interference testing (exogenous compounds):
The influence of mouthwash containing alcohol, alcohol-
free mouthwash, toothpaste, breath mints, throat lozenges,
carbonated beverage with caffeine and caffeine-free carbonated
beverage on eNO was assessed in a clinical study. Twelve
healthy adult subjects (9 males and 3 females; mean age 36 ±
11.8 yrs; range 20-62 yrs) participated in the clinical study. The
eNO measurements for adults ranged from 5 ppb to 99 ppb.
The endpoint was the difference in eNO before as compared to
one hour after exposure to each compound. None of the tested
exogenous compounds were found to interfere (at ± 5 ppb) with
the eNO measurement at one hour post-exposure.
In pediatric subjects, the influence of alcohol-free mouthwash,
toothpaste, breath mints, and caffeine-free carbonated beverage
on eNO was assessed in a clinical study. Seven healthy children
(6 males and 1 female; ages 5-17 yrs) participated in the clinical
study. The eNO measurements for children ranged from 3 ppb
to 27 ppb. The endpoint was the difference in eNO before as
compared to one hour after exposure to each compound. None of
the exogenous compounds tested were found to interfere (at ± 5
ppb) with the eNO measurement at one hour post-exposure.
These studies show that the Insight eNO System can
accurately and reliably measure exhaled nitric oxide
8
 Case Study 1
4. Case Studies

“Let’s go fishing dad!” – Maya, 10, Pensacola FL

Maya is 10 years old and loves
to go fishing with her dad on
the weekends. She has
moderate persistent asthma.
She is very compliant and
often corrects her dad when it
comes to managing her
asthma. She gets on very well
with her doctor, who has been
seeing her for 3 years now.
Maya has been on Pulmicort®
DPI 200 mcg QD. Based
on her record of asthma
control, her doctor wanted
to optimize Maya’s therapy. But he had no objective measure
of her airway inflammation to monitor her response. Once he
had access to exhaled nitric oxide (eNO) measurement with
the Insight system, he incorporated exhaled nitric oxide (eNO)
testing into her treatment plan. When he first measured Maya’s
eNO, it was 44 ppb. He felt it was a little high for her age, but
he wanted to establish her usual eNO levels over time before
changing her medication. Over the next 8 weeks, he found that
her eNO values settled at around 30 ppb after initially fluctuating.
He increased her Pulmicort dosage to 200 BID. Over the next 8
weeks, Maya’s eNO values decreased to the low 20’s. Her doctor
was very pleased with the results but the next time he measured
her, she was at 40 ppb. An almost two-fold increase in her eNO
bothered him but further investigation with Maya and her father
revealed that she had missed taking her medication recently.
Her eNO values dropped back down to around 20 ppb once she
resumed her treatment. By continuing to monitor Maya’s eNO
levels regularly, her doctor was confident that he could fine-tune
her medication.

Maya’s history and medical information Titrate medication and monitor compliance with precise information
• Age:10-year-old female
• Classification:Moderate persistent 70 6 weeks
4 months
• Medication:Pulmicort 60

• Dosage:200 mcg QD 50
Missed medication
eNO in ppb

shows up as abnormally
• FEV1:80% high eNO values
40
• eNO:44 ppb
• Other information: 30

- Non-allergic rhinitis 20
200 QD
- Non-allergic conjunctivitis Pulmicort 200 BID Pulmicort
10
- Cold sores
- Occasional headaches Time

Benefits of eNO:

Treatment gap revealed by eNO measurement with the Insight™ eNO System
• Objective validation for a sound clinical decision
• Insight into airway inflammation not manifesting as symptoms
• Timely identification of suboptimal compliance to medication and therapy

Patient benefits from the new treatment paradigm

• Better titrated dosage enables patient to maintain control

9
 Case Study 2

“I like to win.” – Scott, 23, Fresno CA

Scott is a talented young racer
who loves to drive his all-wheel
drive car on wet and slippery
dirt roads. He competes in
many local and regional events.
He has a keen eye for those
fast corners and usually leaves
the rest of the racers in the
dust.
Scott spends a lot of time
outdoors and has complained
about worsening asthma
symptoms with increased
activities. His doctor, Jim
Brewster, knows him too well to ask him to reduce his need
for speed. Dr. Brewster decided to assess Scott’s airway
inflammation via exhaled nitric oxide (eNO) values as an aid to
titrating his controller medications. Scott was on Advair® 500 BID.
He suspected that Scott was not compliant and Dr. Brewster
knew that Scott had some asthma attacks in the past that landed
him in emergency care.
Scott’s eNO was 32 ppb at his first visit, but Dr. Brewster
planned to measure his eNO values over several visits and look
for patterns before making any treatment changes. Consistent
with Scott’s irregular compliance, his eNO values varied
widely. However, these variations did not coincide with Scott’s
symptoms. Most of the time, Scott’s symptoms were normal
and he felt fine, but his eNO levels were high. Dr. Brewster then
shared the eNO values with Scott and explained the significance,
hoping to convince Scott to take his medications seriously and
regularly. Scott liked having a number that showed him the status
of the inflammation of his airways, and started to improve his
compliance which resulted in a reduction and stability of his eNO
levels. Subsequently Dr. Brewster changed Scott’s medication
from Advair 500 BID (a combination of the 500 mcg of the inhaled
steroid fluticasone and salmeterol, a long acting bronchodilator)
to fluticasone 220 BID alone. Scott’s eNO values are now in the
low 20’s and do not fluctuate like before. Scott has not had any
exacerbations since he started monitoring his eNO values, and
Dr. Brewster has been able to reduce the daily dose of inhaled
steroids by 50% and stop salmeterol.

Improve patient compliance and confidence

Scott’s history and medical information
Prednisone Prednisone & Augmentin
• Age:23-year-old male 80
• Classification:Severe persistent Improved compliance
70
• Medication:Advair
60
• Dosage:500 BID Advair 500 BID to
eNO in ppb

• FEV1:117% 50
Flovent 220/2inh BID
• eNO:32 ppb 40
• Other information 30
- Allergic rhinitis
20
- Severe asthma attacks Advair 500 BID
10
- Hospitalized /intubated in last 18 months
- Occasional headaches 0
1 2 3 4 5 6 7
Time (months)

Benefits of eNO testing

Treatment gap revealed by eNO testing

• Simple and compelling evidence to highlight to the patient the need to follow treatment plan and medication
• Confirm suboptimal compliance with medication and treatment plan
• Provide objective validation for clinical treatment decisions

Patient benefits from the new treatment paradigm

• More confidence from a precisely tailored therapy that fits lifestyle requirements
• Greater awareness and motivation to improve compliance

10
 Case Study 3

“It feels good to be in control.” - Lynn, 36, Long Island NY

Lynn works in
emergency care at the
city hospital as a nurse
and loves her job. She
lives with her husband
and two children. Her
life is demanding to
say the least. She has
been living with asthma
for almost 14 years
now and feels that she
can’t quite get it under
control. Her health has
been a big concern for
her whole family. She
has persistent asthma with symptoms most of the time. She has
missed many days at work due to her asthma.
While on Advair® 250/50 BID and QVAR® 80mcg BID (i.e.
2 inhaled steroids), her doctor decided to monitor Lynn’s
inflammation using eNO testing. Her eNO levels were very high
(80-128 ppb where normal is about 20 ppb) and Lynn continued
to have almost daily symptoms. Her doctor doubled her QVAR
dosage to 160 BID. Over time, this brought her eNO down to the
45-50 ppb range but these levels were still elevated. Around that
time, Lynn moved to a new house in Long Island (a surprise family
inheritance) and after the move, there was a further drop in eNO
to around 20 ppb. Lynn also reported that her symptoms were
getting much better. Lynn and her doctor figured that her older
house, which had a moldy basement, might have triggered her
symptoms. Her doctor then reduced her dosage of QVAR back
to just 1 inhalation. He felt confident that he could further drop or
even eliminate her dosage of QVAR and significantly reduce her
medication by following her eNO levels regularly. Lynn continues
to work in ER and is happy to be able to focus more on her work
and family.

Track disease progression and see impact of environment

Lynn’s history and medical information
• Age: 36-year-old female 140
• Classification:Severe persistent 120
• Medication: Advair, QVAR
100
• Dosage:Advair 250/50 BID and QVAR 80 BID QVAR 80 2inh BID
eNO in ppb

• FEV1:51% 80
• eNO: 85 ppb Moved to a
60
• Other information: new house
40 Decreased to
- Asthma x 14 years QVAR 80
- Regularly symptomatic 1 inh BID
20
- Allergic Rhinitis
0
- Ragweed, mold, dander 1 2 3 4 5 6 7 8
- Intermittent psoriasis Time (months)

Benefits of eNO testing

Treatment gap revealed by eNO testing

• Despite 2 controller medications, eNO indicated that airway inflammation was uncontrolled
• Exhaled nitric oxide (eNO) showed that airway inflammation was decreasing after a change in environment.
• Exhaled nitric oxide (eNO) allowed the tapering of medication in the better environment.

Patient benefits from the new treatment paradigm

• Titration of therapy to optimize asthma control
• Improved quality of life

11
5. Exhaled Nitric Oxide Cost Model
This cost model provides an insurance payer with the
reimbursement costs of eNO patient testing with the Insight
system and the expected savings in medical resources resulting
from improved asthma control.The cost model:
• Estimates the number of patients that are likely to undergo
eNO testing over a three year period.
• Estimates total eNO testing reimbursement costs to the
insurance payer.
• Estimates the number of asthma exacerbation events likely to
be avoided by patients monitored with eNO.
• Estimates cost savings in medical resources (hospitalization,
ER visits, office visits, drug use) due to avoidance of asthma
exacerbation events.
• Estimates the net cost to the insurance payer of eNO testing
reimbursement minus medical resource savings.
Please contact us at care@apieron.com if you would like to see
how the model can be applied to your plan.
Managing asthma is a breath away™
12
6. Coding Information for Exhaled Nitric
Oxide (eNO)-CPT 95012
In 2007, the CPT editorial committee added a specific code to the
CPT Coding Book for Nitric Oxide Expired Gas Determination.
With this action, the procedure should be recognized as a
standard service provided to patients. The committee felt it
appropriate to add this code to the Category I codes instead
of placing it in the Category III codes as a temporary or new
services code. The Category I codes are considered consistent
with contemporary medical practice and are performed by
providers across the country. Determination of exhaled nitric
oxide (eNO) is considered a service which is consistent with
contemporary medical practice for the evaluation of patients with
respiratory complaints.
Centers for Medical Services (Medicare) also determined that
the service should be recognized as a payable service for all
Medicare beneficiaries. In doing this, the RUC committee
(Relative Value Update Committee) assigned an RVU value of
0.50. This value does not have a physician work component RVU,
only an overhead and malpractice RVU. RVU or relative value unit
determines what the reimbursement will be for Medicare as well
as many other carriers. Medicare has a conversion factor which
may be higher or lower than the third party payers to determine
the final reimbursement for the code.
The code assigned for the Nitric Oxide Expired Gas Determination
is 95012©. According to the CPT subsection heading for 2008,
the interpretation and report for the test is included in the testing
code. If a “significant and separately identifiable” evaluation
and management service is provided in addition to the testing,
then an evaluation and management code may be coded in
addition to the testing code. The ‘25 modifier’ would need to be
added to the evaluation and management code (E/M) in order for
both the test, and the evaluation and management code to be
appropriately reimbursed.
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