Pediatric Pulmonology 48:981–995 (2013)

Fraction of Exhaled Nitric Oxide (FeNO) Norms inHealthy North African Children 5–16 Years Old

Sonia Rouatbi, MD, PhD,1,2* Ashraf Alqodwa, MD,1 Samia Ben Mdella, MD,1 andHelmi Ben Saad, MD, PhD

1,2

Summary. Aims: (i) To identify factors that influence the FeNO values in healthy North African,

Arab children aged 6–16 years; (ii) to test the applicability and reliability of the previously pub-

lished FeNO norms; and (iii) if needed, to establish FeNO norms in this population, and to pro-

spectively assess its reliability. Population and Methods: This was a cross-sectional analytical

study. A convenience sample of healthy Tunisian children, aged 6–16 years was recruited. First

subjects have responded to two questionnaires, and then FeNO levels were measured by an

online method with electrochemical analyzer (Medisoft, Sorinnes [Dinant], Belgium). Anthropo-

metric and spirometric data were collected. Simple and a multiple linear regressions were

determined. The 95% confidence interval (95% CI) and upper limit of normal (ULN) were de-

fined. Results: Two hundred eleven children (107 boys) were retained. Anthropometric data,

gender, socioeconomic level, obesity or puberty status, and sports activity were not indepen-

dent influencing variables. Total sample FeNO data appeared to be influenced only by maximum

mid expiratory flow (l sec�1; r2 ¼ 0.0236, P ¼ 0.0516). For boys, only 1st second forced expi-

ratory volume (l) explains a slight (r2 ¼ 0.0451) but significant FeNO variability (P ¼ 0.0281).

For girls, FeNO was not significantly correlated with any children determined data. For North

African/Arab children, FeNO values were significantly lower than in other populations and

the available published FeNO norms did not reliably predict FeNO in our population. The

mean � SD (95% CI ULN, minimum–maximum) of FeNO (ppb) for the total sample was

5.0 � 2.9 (5.4, 1.0–17.0). For North African, Arab children of any age, any FeNO value greater

than 17.0 ppb may be considered abnormal. Finally, in an additional group of children prospec-

tively assessed, we found no child with a FeNO higher than 17.0 ppb. Conclusion: Our FeNO

norms enrich the global repository of FeNO norms the pediatrician can use to choose the most

appropriate norms based on children’s location or ethnicity. Pediatr Pulmonol. 2013; 48:981–

995. � 2012 Wiley Periodicals, Inc.

Key words: exhaled nitric oxide; norms; interpretation; child; Tunisia.

Funding source: none reported.

INTRODUCTION

Nitric oxide (NO) is produced by a wide variety ofcell types including airway nerves, epithelial, inflamma-tory (macrophages, neutrophils, mast cells), and vascu-lar endothelial cells. It is generated via a five-electronoxidation of a terminal guanidinium nitrogen on theamino acid L-arginine; this reaction is catalyzed by NOsynthase.1–3

Since the demonstration of the presence of NO in theexhaled air by Gustafsson et al.4 in 1991, there havebeen several publications showing that the fraction ofexhaled NO (FeNO) is elevated in many respiratory dis-eases, especially bronchial asthma in children.5,6 NOcan be detected in exhaled air by several methods suchas chemiluminesence, spectroscopy, electrochemicalportable, and other methods currently under develop-ment.1,7 Cheaper and easy to use,8 FeNO analyzers arenow readily available and increasingly used not onlyfor the diagnosis of eosinophilic airway inflammation

Additional supporting information may be found in the online version of

this article.

1Service of Physiology and Functional Explorations, Farhat HACHED

Hospital, Sousse, Tunisia.

2Laboratory of Physiology, Faculty of Medicine of Sousse, University of

Sousse, Sousse, Tunisia.

Conflict of interest: None.

*Correspondence to: Prof. Sonia Rouatbi, MD, PhD, Faculty of Medicine

of Sousse, «Ibn El Jazzar», Street Mohamed Karoui, Sousse 4000, Tunisia.

E-mail: sonia.rouatbi@rns.tn

Received 20 May 2012; Accepted 3 October 2012.

DOI 10.1002/ppul.22721

Published online 20 November 2012 in Wiley Online Library

(wileyonlinelibrary.com).

� 2012 Wiley Periodicals, Inc.

which is seen mainly in asthma,9 but also for its assess-ment.10,11 In addition, the American Thoracic Societyand European Respiratory Society (ATS/ERS) havejointly demonstrated that some factors (i.e., age, atopy,and gender) may affect the FeNO values’.12

Interpretation of FeNO data relies upon comparison ofmeasured values with predicted ones available frompublished norms (reference equations or normal valuestables). However, to our knowledge, FeNO norms haveonly been established in four children populations.13–16

However, neither of these studies provided prospectiveverification for their studied populations, nor proposeda method of interpreting the measured FeNO (e.g., usingan upper limit of normal (ULN) or a fixed percentageabove which measured FeNO values would be consid-ered as abnormal). The need for normal values specificto North African populations has been demonstrated forseveral physiological parameters.17–21 The applicabilityand the reliability of published FeNO norms13–16 shouldbe assessed as regards to North African children, in or-der to avoid erroneous clinical interpretation of FeNO

data in this population.Moreover, the ATS/ERS have encouraged investiga-

tors to publish physiological norms for healthy popula-tions of various racial backgrounds, to enable individualsubject results to be compared with data from a raciallysimilar population.12 The use of the same kind ofassessment equipment and procedure is also recom-mended.12 Therefore, the present study aimed:

(1) To identify factors that influence the FeNO values’of healthy North African, Arab children aged 6–16 years.

(2) To test the applicability and reliability of the pre-viously published FeNO reference equations13–15

or normal values16 in this population (the null

hypothesis is that there will be no difference be-tween measured and predicted FeNO mean values).

(3) If needed, to establish FeNO norms in this popula-tion, and to prospectively assess its reliability.

METHODS

Study Design

Our study is a cross-sectional analytical study spreadover 7 months (June 2011 to December 2011). It wasconducted in the Department of Physiology and Func-tional Explorations in the Farhat HACHED Hospital inSousse (Tunisia; altitude <100 m).

Study design consists of a sample of healthy Tuni-sian children (Arab race) in the region of Sousse,aged 6–16 years. Subjects were recruited from thechildren of the hospital workers, and from public andprivate schools. Information letters, clarifying theaims of the study, were put up in the Medicine Facul-ty and in the local different schools. When a childwas interested, an appointment for medical question-naires and exploration was fixed. Data from each vol-unteer child included: gender, age, height, weight,birth height and weight, smoking history (child orparents), medication use, medical history, physical ex-amination, pubertal stage, sports activity, FeNO, andspirometry data. All children received a copy of theirexploration, and when an unknown dysfunction wasdiscovered, they were sent to a specialist. Study ap-proval was obtained from the hospital Ethics commit-tee, and written informed consent was obtained fromall children and/or their parents.

Sample Size

The sample size is calculated according to the fol-lowing predictive equation22: n ¼ (Z2 � p � q)/D2,where ‘‘n’’ was the number of required children, ‘‘Z’’was the 95% confidence level (¼1.96), ‘‘q’’ was equalto ‘‘1 � p,’’ ‘‘D’’ was the precision (¼7%), and ‘‘p’’was the estimation of children aged 5–16 years with anormal FeNO value. According to Buchvald et al.,16

among the 721 recruited children, only 405 children(P ¼ 0.56) without outliers and atopics were retained(Supplementary E. Table 1). Plugging this relevant val-ue into the predictive equation, the sample size wasthus 193 children. Therefore, to determine the influenc-ing factors and to establish FeNO norms, we recruitedan initial group of 211 children (104 girls; 107 boys).

To verify the reliability of our norms, we prospec-tively measured the FeNO in a second group of 24 addi-tional healthy children (12 boys) meeting the inclusioncriteria of the present study but having not participatedto the first part.

ABBREVIATIONS:

ATS American thoracic society

BMI Body mass index

BSA Body surface area

CI Confidence interval

ERS European respiratory society

FEF25–75% Forced expiratory flow from 25% to 75% of FVC

FeNO Fraction of exhaled NO

FEV1 1st second forced expiratory volume

FVC Forced vital capacity

IgE Immunoglobulin E

LOA Limits of agreement

NO Nitric oxide

ppb Parts per billion

r Correlation coefficient

r2 Determination coefficient

SD Standard deviation

SEL Socioeconomic level

ULN Upper limit of normal

982 Rouatbi et al.

Pediatric Pulmonology

Subjects

Volunteer children aged from 6 to 16 years wereincluded. The following non-inclusion criteria wereapplied: chronic illnesses; a history of pulmonary dis-eases or related respiratory symptoms [history of asthmaor medication asthma use, current or past symptomsof wheeze, chronic cough; abnormal lung function(obstructive ventilatory defect or abnormal spirometrydata23)]; oto-rhino-laryngologic diseases or symptoms[allergic rhinitis, rhinitis; symptoms and signs of acuteupper respiratory infection during 2 weeks prior toassessment; recent upper airway infection (cold, flu,sore throat within the last 7 days)]; atopic dermatitis oreczema; regular medication use (especially steroidsor b-agonist, leukotriene receptor agonist, etc.); heartdisease; premature birth (i.e., birth before 36 weeksgestational age); active smoking; inability to performproperly FeNO measurement and imperfect realizationof required respiratory maneuvers or inability to complywith the study procedure. The FeNO measure was per-formed after the non-inclusion criteria had been verified.

Medical Questionnaires

Two medical questionnaires recommended for epide-miological research were combined and used to assessseveral children characteristics.24,25 Questionnaireswere written in Arabic and were composed of ques-tions, mainly closed response and usually dichotomous.Questions were asked by an examiner with whom chil-dren or children-parents were not familiar. Children orparents of the subjects answered questionnaires regard-ing demographic data, general health information (espe-cially birth weight, height, and gestational age notedfrom the personal health records), questions on clinicalsymptoms and diagnosis of allergic diseases.

Two subgroups of children were formed accordingto sports activity (non-active; active) based on the re-sponse to the following question18: do you practice anysports activities outside of school?

Two socioeconomic levels (SEL) were distinguishedbased on a socioeconomic score: ratio between thenumber of inhabited rooms and household size (<1.5:unfavorable; �1.5: favorable).26

Physical Examination

Anthropometric data were verified, measured, or cal-culated: age, height, weight, body mass index (BMI),and body surface area (BSA). The decimal age (accuracyto 0.1 years) was calculated from the date of measure-ment and the date of birth. Due to the failure of softwareto compute decimal age as the difference between testdate and birth date, age was taken as the number of com-plete years from birth to the date of the study. Height

(cm) was measured with height gauge, in children shoestaken off; heels joined and back well straight. Weight(�1 kg) was measured in children without heavy clothes.BMI (weight/height2, kg m�2) was calculated. Over-weight and obesity were defined according to the criteriaof Cole et al.27 depending on age and BMI. Two groupsof children were identified (normal weight, overweight,or obesity). BSA was calculated28: BSA ðm2Þ ¼0:007184�Weight0:425

kg � Height0:725cm

.Pubertal status was evaluated by two methods. Quali-

tative score of Tanner, based on the five-point ratingscale of morphologic breast changes for girls and pubichair changes for boys (T1–T5).29 The qualitative scoreof Tanner was converted into a quantitative variable[pubertal stage by Tanner scale]: T1: 0; T2: 0; T3: 1;T4: 2; and T5: 3.

FeNO Measurement

The FeNO (expressed in parts per billion, ppb) wasmeasured by Medisoft HypAir FeNO method using anelectrochemical analyzer (Medisoft). The ATS/ERS rec-ommendations were respected.12 The instrument wascalibrated and used according to the manufacturer’sinstructions, and work in conjunction with a personalcomputer. The software supplied by either manufacturerprovided both audio and visual feedback allowing theparticipant to maintain a constant exhaled breath flowrate.

The online method, with constant flow rate, which isconsidered the method of choice, was used. The pediat-ric FeNO task force recommends expiratory flow ratesof 50 ml/sec for online collection.30 After a full un-forced exhalation outside the mouthpiece, a maximalinspiration was performed through an absorber to en-sure NO-free air. The child then performed a controlledexhalation using flow control at an exhalation pressureof 4–10 cmH2O for at least 6 sec, during whichtime sample collection and gas analysis was performed.Nasal contamination is presented by closure of the ve-lum by using 5 cmH2O oral back-pressure. A nose clipwas not used. To encourage the child to exhale at afixed flow rate of 50 ml/sec, the flow indicator on thedevice was replaced by a cartoon of a dolphin movingthrough hoops. Children were asked to not eat, drink,or participate in strenuous activity for 1 hr prior to thetest.

Three acceptable measurements were taken at a flowrate of 50 ml/sec within a 15-min period according toATS/ERS guidelines.12 The mean of the three valueswas used.13–16

Spirometry Function Test

Spiromety was carried, according to the recent inter-national recommendations,23 out in the sitting position,

FeNO Norms of Healthy North African Children 983

Pediatric Pulmonology

and a nose clip was applied. All tests, performed bythe same investigator using a spirometer (ZAN 100,Messgerate GmbH, Ober-thulba, Germany), were doneafter the FeNO measurement.12 The flow sensor of thespirometer was calibrated daily with a 3-L syringe.

The following parameters were measured/calculated:forced vital capacity (FVC, l); 1st second forced expira-tory volume (FEV1, l); forced expiratory flow from25% to 75% of FVC (FEF25–75%, l sec�1) and FEV1/FVC ratio (absolute value). The results were comparedwith local age- and gender-matched reference values.21

An obstructive ventilator defect was retained whenthe FEV1/FVC ratio was lower than the lower limit ofnormal.23 FEV1 and FVC were considered as abnormalwhen they were lower than the lower limit of normal.23

Statistical Analysis

Data Analysis

For each child, the mean of the three FeNO valueswas used for statistical analysis. Preliminary descriptiveanalysis included frequencies for categorical variablesand mean � SD for continuous ones. The dependentvariable (FeNO) was normally distributed and FeNO

results were presented as mean � SD and as upper95% confidence interval (95% CI).

All analyses were performed without outliers, definedas FeNO values above arithmetic mean þ 3 SD.16

Univariate and Multiple Regression Analysis:Influencing Factors

t-Tests were used to evaluate the associations be-tween FeNO and the categorical variables (gender, SEL,sports activity, and obesity status). Pearson’s product-moment correlation coefficients (r) evaluated the associ-ations between FeNO and the continuous measures (age,height, weight, birth height and weight, socioeconomicscore, gestational age, BMI, BSA, qualitative score ofTanner, and pubertal stage by Tanner scale, FEV1 (l,%), FVC (l, %), FEV1/FVC (absolute value), FEF25–75%

(l sec�1, %)).The linearity of association between FeNO and the

continuous measures was checked graphically by plot-ting each regressor against the FeNO. Only significantlyand linearly associated variables were entered into themodel. A linear regression model was used to evaluatethe independent variables explaining the variance inFeNO. Candidate variables were stepped into the modelwith a stepwise selection method. To determine entryand removal from the model, significance levels of 0.15and 0.05 were used, respectively. No colinearity be-tween predictors was detected with variance inflationfactors. The linearity was evaluated by correlation (r)and determination (r2) coefficients and the standard

error. The 95% CI (¼1.64 � residual standard devia-tion) was calculated.22

Comparison With Published Norms

Comparison was made by two ways:

(i) Individually measured FeNO were compared withthe predicted FeNO from the published norms forthe same age or height ranges as in the correspond-ing study, using paired t-tests and scatter plots13–15

or non-parametric tests and histograms.16

(ii) As proposed by Bland and Altman,31 comparisonsbetween measured and predicted FeNO

13–16 wereperformed by means of the limits of agreement(LOA), where individual differences (measuredminus predicted FeNO) were plotted against thecorresponding mean value. From these data, LOAwere then calculated.31 The reference equationthat provides the LOA closest to zero will be themost appropriate for our population.

It is well known that FeNO values obtained with dif-ferent devices are not directly comparable.32 As theAerocrine devices are much more commonly used andmost of the other devices give pretty similar results32

and as measurements on the HypAir FeNO are 1.6 timeshigher than those obtained with the Aerocrine NIOX33

and for a better interpretation of our data, we have ad-justed our results in accordance with Brooks et al.33 Forthat reason all FeNO predicted values from the publishednorms13–16 were divided by 1.6 and individually mea-sured FeNO were compared with the predicted/adjustedFeNO from the published norms as described above.

FeNO Normal Values

A table for each age range and for the total sample,presenting FeNO mean � SD, 95% CI ULN, and mini-mum–maximum is provided. Three ways are proposedto interpret a measured FeNO value:

(i) Use of the total sample 95% CI ULN as a thresh-old. In this case each child aged 5–16 years FeNO

value higher than the total sample FeNO 95% CIULN, will be considered as abnormal.

(ii) For more accuracy, a specific threshold (95% CIULN) for each age range will be evaluated. Forexample, for a given child aged 12.0–12.9 years,each FeNO value higher than the 12.0–12.9-yearage group FeNO 95% CI ULN, will be consideredas abnormal.

(iii) Use of the total sample maximum FeNO valueas a threshold. In this case each child aged 5–16 years FeNO value higher than the total samplemaximum FeNO value, will be considered asabnormal.

984 Rouatbi et al.

Pediatric Pulmonology

Reliability of the North African FeNO Norms

The reliability of our norms was evaluated in the sec-ond group of 24 healthy children. The number of chil-dren having a measured FeNO values higher than 95% CIULN predicted normal values for each age range or forthe total sample is determined. FeNO predicted normalvalues will be considered as reliable when no child fromthe second group will have a measured abnormal value.

Analyses were carried out using Statistica (StatisticaKernel version 6, StatSoft, Maisons-Alfort, France).Significance was set at the 0.05 level.

RESULTS

Children Data

An initial sample of 354 voluntary children Arabrace was examined. Non-inclusion criteria, presentedin detail in the Supplementary Data, were found in119 children. Two hundred eleven healthy children(107 boys) were included to establish FeNO norms.

The SEL, sports activity, obesity, and puberty statusof the 211 children are shown in Table 1. The two mainconclusions from this table are: (i) Significantly fewergirls had a favorable SEL, or, were categorized as beingactive, than boys. (ii) Compared to boys, there are asignificantly higher numbers of girls having a T5 of thequalitative score of Tanner or having level 3 of thepubertal stage by Tanner scale.

The number of children in each age group, the gen-der distribution, the anthropometric, spirometric, andFeNO data are given in Table 2. Among included chil-dren, the birth weight, birth height, and FVC (% pre-dicted) were significantly higher among boys than girls,and the former’s FEV1/FVC was significantly lower.

Supplementary E. Figure 1 shows the distribution ofthe 211 healthy children according to gender and agerange. The two main conclusions from this figure are:(i) Compared to boys, there are a significantly lowernumber of girls aged 13.0–13.9 years. (ii) Compared toboys, there are a significantly higher number of girlsaged 14.0–14.9 years.

The FeNO data of the 211 children are shown inFigure 1, according to age, height, and weight ranges(Fig. 1A–C, respectively). A significant FeNO differenceis found between children at the age of 9.0–9.9 and10.0–10.9 years.

Univariate Analysis

In the total sample, gender (Table 2), SEL, obesitystatus, and sports activity (Table 3) did not significantlyaffect the FeNO value.

The correlation coefficient (r) between FeNO and thecontinuous children’s data are shown in Table 3. Forthe total sample, FeNO was significantly correlated(P < 0.05) with FEF25–75% (l sec�1). For boys, FeNO

was significantly correlated (P < 0.05) with age andFEV1 (l). For girls, FeNO was not significantly correlat-ed with any of the other variables.

Multivariate Analysis: FeNO Influencing Factors

Table 4 presents the cumulative r2 of the independentinfluencing factors included in the FeNO forward linearstepwise multiple regressions.

For the included boys, only FEV1 (l) explains a slight(r2 ¼ 0.0451) but significant FeNO variability (FeNO

(ppb) ¼ 3.17682 þ 0.75009 � FEV1 (l)). After thepredicted FeNO value for a given boy was computedfrom this equation, the ULN for the child could beobtained by adding 4.8 ppb.

For the total sample, only FEF25–75% (l sec�1) explainsa slight (r2 ¼ 0.0236) but significant FeNO variability(FeNO (ppb) ¼ 3.91283 þ 0.46666 � FEF25–75% (l sec�1)).After the predicted FeNO value for an individual childwas computed from this equation, the ULN for thechild could be obtained by adding 4.6 ppb.

Comparison With Published Norms

Comparison Without Values Adjustment Accordingto Brooks et al.33

Figure 2 shows individually measured FeNO plottedagainst the corresponding predicted value for the same

TABLE 1— Socioeconomic Level, Sports Activity, Obesity,and Puberty Status

Boys

(n ¼ 107)

Girls

(n ¼ 104)

Total sample

(n ¼ 211)

Socioeconomic level

Unfavorable 85 95 180

Favorable 22 9� 31

Sports activity

Active 59 38� 97

Non-active 48 66 114

Obesity status

Normal weight 80 80 160

Overweight or obesity 24 24 48

Puberty status: qualitative

score of Tanner

T1 40 41 81

T2 26 25 51

T3 17 12 29

T4 19 15 34

T5 5 11� 16

Puberty status: pubertal

stage by Tanner scale

0 66 66 132

1 17 12 29

2 19 15 34

3 5 11� 16

Data are number. �P < 0.05: Boys versus Girls. Bold values:

statistically significant difference.

FeNO Norms of Healthy North African Children 985

Pediatric Pulmonology

age range, using Kovesi et al.13 (Fig. 2A), Yao et al.15

(Fig. 2B), and Malmberg et al.14 (Fig. 2C) referenceequations, respectively. As can be seen, the data showedwide disparity compared with the identity line.Figure 2D shows the measured FeNO of different agegroups compared with those measured in the multi-centre study (Europe and USA).16 In all instances, ourmean (95% CI ULN) measured FeNO was significantlyoverestimated (P < 0.05).

Figure 3 shows, for the same age range, the Blandand Altman31 comparisons between measured FeNO andFeNO predicted from the published reference equa-tions.13–15 There was a systematic bias between themeasured and predicted values; that is to say, the differ-ence with measured FeNO increased as the predictedFeNO increased. In addition, our mean � SD measuredFeNO was significantly overestimated by 9.6 � 3.8 ppb(P < 0.05), by 6.3 � 3.2 ppb (P < 0.05), and by4.8 � 3.2 ppb (P < 0.05), with, respectively, the Cana-dian (Fig. 3A),13 the Taiwan (Fig. 3B)15, and theFinlandian (Fig. 3C)14 reference equations.

Comparison After Values Adjustment According toBrooks et al.33

Supplementary E. Figure 2 shows individually mea-sured FeNO plotted against the corresponding predictedand adjusted value for the same age range, using Kovesiet al.13 (Supplementary E. Fig. 2A), Yao et al.15 (Sup-plementary E. Fig. 2B), and Malmberg et al.14 (Supple-mentary E. Fig. 2C) reference equations, respectively.As can be seen, the data showed wide disparity com-pared with the identity line. Supplementary E. Figure2D shows the measured FeNO of different age groupscompared with those measured/adjusted in the multi-centre study (Europe and USA).16 Our mean (95% CIULN) measured FeNO was significantly overestimated(P < 0.05) only for the {10–13} and {14–16} ageranges.

Supplementary E. Figure 3 shows, for the same agerange, the Bland and Altman31 comparisons betweenmeasured FeNO and FeNO predicted and adjusted fromthe published reference equations.13–15 There was a sys-tematic bias between the measured and predicted/adjusted values: our mean � SD measured FeNO wassignificantly overestimated by 4.02 � 3.40 ppb(P < 0.05), by 2.04 � 2.98 ppb (P < 0.05) and by1.02 � 3.06 ppb (P < 0.05), with, respectively, theCanadian (Supplementary E. Fig. 3A),13 the Taiwan(Supplementary E. Fig. 3B)15, and the Finlandian (Sup-plementary E. Fig. 3C)14 reference equations.

North-African Children FeNO Norms

Due to the inadequacy of the published norms,13–16

we established local norms adapted to our population.TA

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986 Rouatbi et al.

Pediatric Pulmonology

Since age did not correlate with FeNO, we simply give anormal values range for FeNO for North African chil-dren aged 6–16 years. It is much simpler for cliniciansto remember and device manufacturers to program.

The FeNO mean � SD, 95% CI ULN and minimum–maximum of the 211 children are presented in Table 5.In practice, three ways can be used to interpret a mea-sured FeNO value:

(i) Use of the total sample 95% CI ULN as a thresh-old. In this case, each child aged 5–16 years

FeNO value higher than 5.4 ppb, is considered asabnormal.

(ii) For more accuracy, a specific threshold (95% CIULN) for each age range is applied. For example,for a given child aged 12.0–12.9 years, each FeNO

value higher than 7.7 ppb, is considered asabnormal.

(iii) Use of the total sample maximum FeNO value asa threshold. In this case, each child aged 5–16 years FeNO value higher than 17.0 ppb, is con-sidered as abnormal.

Fig. 1. The FeNO in subgroups of children, according to age (A), height (B) and weight (C)

ranges. n, number of children. Data are shown as box-and-whiskers-plots illustrating the

mean ( ), standard deviation ( ) and minimum–maximum ( ). �P < 0.05: Comparison (t-test)

from one range to the next. NS, not significant.

FeNO Norms of Healthy North African Children 987

Pediatric Pulmonology

Reliability of North-African FeNO Norms

The mean � SD (95% CI) FeNO prospectively mea-sured in the 24 children (10.6 � 3.6 years, 141 � 18 cmand 37 � 14 kg) was 6.0 � 3.0 ppb (4.8–7.3).

When we apply the normal values mentioned inTable 5, we found no child with a FeNO higher than thethreshold (ULN) of 17.0 ppb or higher than the 95% CIULN specific for each age range.

DISCUSSION

The FeNO of a large group of healthy North African/Arab children between 6 and 16 years old was prospec-tively measured. The available published FeNO norms didnot reliably predict FeNO in our population and FeNO val-ues are lower in healthy North African/Arab childrenthan in other healthy populations. So, we can reject thenull hypothesis that we would see no difference in the

TABLE 3— Univariate Analysis Between the Fraction of Exhaled Nitric Oxide (FeNO) and Children’s Data

Girls (n ¼ 104) Boys (n ¼ 107) Total sample (n ¼ 211)

Univariate spearman correlation coefficients between FeNO data and continuous measures

Age (yr) 0.04 0.19� 0.12

Height (m) 0.07 0.17 0.12

Weight (kg) �0.02 0.14 0.06

Body mass index (kg m�2) �0.10 0.06 �0.01

Body surface area (m2) 0.02 0.16 0.1

Birth weight (mg) �0.04 0.12 0.03

Birth height (kg) �0.06 �0.03 �0.05

Duration of gestation (weeks) �0.06 0.13 0.07

Pubertal stage by Tanner scale 0.03 0.16 0.09

Qualitative scale of Tanner 0.01 0.16 0.08

Socioeconomic score �0.12 �0.11 �0.11

Forced vital capacity (FVC) (l) �0.01 0.20 0.20

FVC (%) �0.04 0.07 0.01

1st second forced expiratory volume (FEV1) (l) 0.03 0.21� 0.12

FEV1 (%) �0.01 0.04 �0.02

FEV1/FVC (absolute value) 0.06 �0.11 �0.01

Forced expiratory flow from 25 to 75% of FVC (FEF25–75%; l sec�1) 0.10 0.19 0.15�

FEF25–75% (%) 0.14 0.02 0.08

Univariate analysis between FeNO data and categorical variables

Socioeconomic level

Unfavorable 4.9 � 3.1 5.4 � 2.9 5.2 � 3.0

Favorable 4.2 � 1.7 4.4 � 3.0 4.3 � 2.1

Sports activity

Active 5.4 � 3.2 5.5 � 2.7 5.4 � 3.0

Non-active 4.1 � 2.1�� 5.1 � 3.0 4.7 � 2.7

Obesity status

Normal weight 4.8 � 3.0 5.4 � 3.1 5.1 � 3.1

Overweight or obesity 4.7 � 2.5 4.8 � 2.1 4.8 � 2.3

Bold values: statistically significant correlation.�P < 0.05 (univariate spearmen correlation coefficients).��P < 0.05 (t-tests) boys versus girls.

TABLE 4— Independent Variables Included in the Forward Linear Stepwise Multiple Regression Model for the Fraction ofExhaled Nitric Oxide

Independent variables

Non-standardized

regression

coefficient

Cumulative

determination

coefficient P level

Standard

error

1.64 residual

standard

deviation

Girls (n ¼ 104)

No variable is included

Boys (n ¼ 107)

Constant 3.17682 0.0000 0.7715 4.8

1st second forced expiratory volume (l) 0.75009 0.0451 0.0281

Total sample (n ¼ 211)

Constant 3.91283 0.0000 0.6778 4.6

Forced expiratory flow from 25 to

75% of forced vital capacity (l sec�1)

0.46666 0.0236 0.0516

988 Rouatbi et al.

Pediatric Pulmonology

means of the measured and predicted FeNO mean values.Our results strongly suggest that existing FeNO norms13–

16 need to be modified for North African/Arab children.Thus, we established a table of normal values accordingto age ranges. For North African children of any age, any

FeNO value greater than 17.0 ppb may be consideredabnormal. Finally, in an additional group of children pro-spectively assessed, we found no child with a FeNO

higher than the threshold of 17.0 ppb or higher than the95% CI ULN specific for each age range.

Fig. 2. Comparison with published regression equations or normal values (without adjustment

according to Brooks et al.32). Comparison of measured and predicted FeNO determined from

Kovesi et al.13 (race: Caucasian; A), from Yao et al.15 (race: Asian; B) and from Malmberg et al.14

(race: Caucasian; C) for the same age range or the same height range. Comparison of measured

FeNO from the present study (data are mean (&) and 95% CI ( ) with those measured by Buch-

vald et al.16 (race: Caucasian; D; data are mean (~) and 95% CI upper limit of normal (~)), in

different age subgroups. n, number of children having the age range of the predicted FeNO

study. Solid line (—): regression line. Dashed line (. . .): identity line. r2: coefficient of determina-

tion. r: correlation coefficient. �P (probability) < 0.05: comparison (unpaired t-test) of the FeNO

measured in the present study (&) and in the study of Buchvald et al.16 (~) for the same age

subgroup.

FeNO Norms of Healthy North African Children 989

Pediatric Pulmonology

Children GroupAs for all the studies aiming to publish FeNO

norms,13–16 ours was not a random population sample.Some caution should be warranted when interpretingthe results of cross-sectional studies in volunteers, be-cause of a possible selection bias and cohort effects.34

Thus, longitudinal studies analyzed by appropriate sta-tistical models are necessary to correctly describe thefunctional changes associated with age.23 Although nostatistical methods were used to choose the children,the number studied and the fact that many schoolsin different areas of Sousse were included give a

Fig. 3. The Bland and Altman representation (without adjustment according to Brooks et

al.33), for the same age and height range, of measured and predicted FeNO determined from

the reference equations of Kovesi et al.13 (race: Caucasian; A), Yao et al.15 (race: Asian; B)

and of Malmberg et al.14 (race: Caucasian; C). The correlation between mean difference

(y-axis) and mean value (x-axis) is significant in all instances, indicating a proportional error

of FeNO predicted with the corresponding reference equation. r, Correlation coefficient;

p, probability; n, number of children of the present study in the age range. —, mean; . . .,

mean � 1.96 � SD; and , regression line.

990 Rouatbi et al.

Pediatric Pulmonology

reasonable degree of confidence in the data. Our re-cruitment mode and subject age range were similar toprevious studies having comparable aims than ours(Supplementary E. Table 1).

According to international recommendations,23 alarge number of subjects (i.e., n > 100) is needed toensure no significant difference between the publishednorms and the values from the local community. Oursample size (n ¼ 211) appears to be satisfactory sincethe calculated one is 193 children.

Because of the frequency of asthma (7–10%) andatopy in children population,35 we have chosen healthychildren for this study. The childhood asthma is a majorcause of pediatric emergency department visit, repre-senting 5–6%.36 Thus, the early detection of this diseaseby measuring an easy and reliable parameter (FeNO) isdesirable.35,37 Non-inclusion criteria, applied in the pres-ent study, were inspired of similar studies according toATS/ERS guidelines.12 In contrast of Buchvald et al.16

study where outliers were defined as FeNO values abovearithmetic mean þ 2 SD, we have fixed the cut-off foroutliers to the arithmetic mean þ 3 SD (equal to 17 ppbfor girls aged 11.0–13.9 years where the mean of FeNO

is 5.7 ppb; Table 2). In addition, when applied in oursample (i.e., age range of 15–16 years, FeNO mean� SD ¼ 5.6 � 4.0), Buchvald et al.16 definition doesnot modify the number of our outliers.

The present study sample size (n ¼ 211) appeared tobe satisfactory when it is compared with those of otherstudies (n ¼ 65713; n ¼ 11414; n ¼ 66115; n ¼ 40516;Supplementary E. Table 1). We prospectively measuredthe FeNO in a second group of additional healthy chil-dren meeting the inclusion criteria of the present study.The validation group number (n ¼ 24) seems small, butwe think that it is sufficient to provide adequate valida-tion since it is closer to these included in similar studiesaiming to validate North African reference equations

for spirometric, peak nasal inspiratory flow, and for6-min walk distance data, respectively (n ¼ 2817,n ¼ 2838, and n ¼ 41).18 Our study is the first one thatuses an evaluation group, to verify the reliability ofchildren FeNO norms.

The exact definition of a ‘‘healthy’’ group is debat-able in children but we avoided confounding clinicalsituations, according to the ATS recommendation.23

Our children were free from chronic disease, but 48children (24 boys) showed overweight or obesity. How-ever, as did other authors, children having thinness orobesity were not excluded.39–41 Cebella et al.39 showedthat, in children, overweight or obesity was not associ-ated with increased FeNO levels, but they were an inde-pendent risk factor for asthma and allergic sensitization.Other authors40,41 demonstrated that BMI in asthmaticsmay increase airway oxidative stress and could explainthe BMI-related reductions in FeNO. Therefore, ourgroup composition reflects this ‘‘healthy’’ population.

In similar studies13–16 (Supplementary E. Table 1),two questionnaires, the most recommended for epide-miological research, were used (ATS-DLD-78-C andISSAC children’s questionnaires).24,25 In the presentstudy, both were combined and used to evaluate chil-dren characteristics, with some modifications to fit thesocio-cultural needs.

FeNO and Spirometry Measurements

Several techniques have been used to assess FeNO.We used an electrochemical analyzer (Medisoft) dueto availability of the distributor. To our knowledge, allstudies concerning children FeNO norms13–16 have usedthe chemiluminesence analyzers.42

Because spirometric maneuvers transiently reducethe FeNO levels, and as recommended by the ATS/ERS,12 NO analysis was performed before spirometry.

TABLE 5— Fraction of Exhaled Nitric Oxide (FeNO) Norms: FeNO Data (ppb) in Different Age Groups (n ¼ 211)

Age range (yr)

Number of

children

FeNO

Mean � standard

deviation

95% confidence interval

upper limit of normal Minimum–maximum

5.0–5.9 21 4.2 � 2.5 5.4 1.0–10.0

6.0–6.9 13 4.8 � 2.0 6.0 1.0–8.0

7.0–7.9 20 4.7 � 2.8 5.9 1.0–12.0

8.0–8.9 19 4.7 � 2.2 5.8 2.0–11.0

9.0–9.9 16 3.9 � 1.8 4.8 1.0–7.0

10.0–10.9 23 6.3 � 2.9 7.6 2.0–14.0

11.0–11.9 20 5.5 � 3.1 7.0 2.0–13.0

12.0–12.9 15 5.7 � 3.6 7.7 2.0–17.0

13.0–13.9 9 4.2 � 1.5 5.4 2.0–6.0

14.0–14.9 24 4.7 � 2.5 5.7 2.0–12.0

15.0–16.0 31 5.6 � 4.0 7.1 2.0–16.0

5.0–16.0 total sample 211 5.0 � 2.9 5.4 1.0–17.0

FeNO Norms of Healthy North African Children 991

Pediatric Pulmonology

Because environmental NO can reach high levels rela-tive to those in exhaled breath, standardized techniquesmust prevent the contamination of biological sampleswith ambient NO.12 As recommended by the ATS/ERS,12 not withstanding which technique is used; ambi-ent NO at the time of each test should be recorded. Inthe present study, mean � SD (minimum–maximum)ambient NO concentration is 1.3 � 1.3 ppb (0–4 ppb).Medisoft device has an absorption column with high ca-pacities for detecting and eliminating ambient NO. Thusits function is not limited by the values of ambient NO.

As it is uncertain whether measurements need tobe standardized for time of day (circadian rhythmeffects),12 we were prudent and FeNO measurementswere performed in the same period of the day (8 a.m.to 12 a.m.).

Interpretation of FeNO values relies upon comparisonwith predicted value available from published norms.13–16

To our knowledge, the present study is the first thatreported FeNO norms for healthy North African childrenaged from 6 to 16 years. Therefore, there is a continuingneed for such clinical research.

Non-Disease-Related Subject Factors InfluencingFeNO Values

In the total sample, gender, anthropometric data, pu-bertal status, sports activity, and SEL did not signifi-cantly affect the FeNO. Only spirometric data influencesignificantly FeNO levels. These factors will be analyzedone by one in the following sections.

Gender effect: As in published studies (Supplementa-ry E. Table 1), gender did not affect FeNO of Tunisianchildren. This factor could be reported after pubertalage.12 Additional information about the gender effect isdetailed in the ‘‘Supplementary data’’.

Age effect: As in two studies13,15 (Supplementary E.Table 1), age did not correlate with Tunisian childrenFeNO values (Table 4). Buchvald et al.,16 in a popula-tion of 405 children, have found that the upper limit ofthe 95% CI was age dependent, ranging from 15.7 ppbat the age of 4 years to 25.2 ppb for adolescents. Whilesome studies reported that FeNO increases withage12,14,16,43 (Supplementary E. Table 1), the mecha-nism for the age dependence of FeNO is largely un-known.15 Additional information about the age effect isdetailed in the ‘‘Supplementary data.’’

Body weight and BSA effects: In contrast of somechildren studies14–16 (Supplementary E. Table 1) orhealthy adults studies,44,45 we have not reported a sig-nificant correlation between body weight or BSA andFeNO levels in either gender. As mentioned by Yaoet al.,15 a preliminary consensus reached between theirstudy and that by Linn et al.46 is that the influence ofweight on FeNO levels is relatively small.

Pubertal status effect: Pubertal status, a factor thathad not been evaluated before, did not significantly af-fect the FeNO. It seems that hormonal modification didnot affect the FeNO values.

Physical activity effect: Sports activity, a conditionthat had not been evaluated before, did not significantlyaffect the FeNO. During exercise, according to one re-port, FeNO increases, and this effect may last up to1 hr.47 Others have reported that FeNO remains stableafter exercise. It would seem prudent to avoid strenuousexercise for 1 hr before the measurement.23,47

SEL effect: SEL, a factor that had not been evaluatedbefore, did not significantly affect the FeNO. The effectsof SEL on the spirometric variables are well docu-mented in industrialized countries48: a low SEL acceler-ates their decline and is associated with small airwayobstruction.48

Airway caliber effect: For the included boys and thetotal sample; respectively, only FEV1 (l) and onlyFEF25–75% (l sec�1) explain a slight but significantFeNO variability’s (Table 4). This result is not in agree-ment with previous published norms (Supplementary E.Table 1). However, it has been demonstrated that FeNO

levels may vary with the airway caliber,12 perhaps be-cause of a mechanical effect on NO output. Given thatFEV1 correlated with FeNO only in boys (Table 4), onewonders whether boy’s larger airways produced moreNO?

Why the Findings About the FeNO Determinants AreNot Consistent With Previous Literature?

There is good evidence that FeNO mainly reflects ato-py in population studies.12,49 For that reason, we havedetermined (using questionnaires and each child healthrecord) the atopic status of study subjects. Only healthynon-atopic children were included. However, for moreaccuracy, it was preferable to determine the serum levelof total immunoglobulin E (IgE) and specific IgE asdone by Yao et al.15 or to estimate sensitization to aller-gens with prick testing as done by Malmberg et al.14

There is much remaining variation that is unex-plained, and several factors may be involved in additionto methodological factors, for example, subclinicalairway inflammation of various causes, nutritionalhistory50, and race or genetic factors.51

As FeNO values obtained with different devices arenot directly comparable and may differ to a clinicallyrelevant, as the device is used,32 we have adjusted ourdata according to Brooks et al.33 As can be seen (Sup-plementary E. Figs. 2 and 3) and even after adjustment,our mean � SD measured FeNO was significantly over-estimated by the Canadian, the Taiwan, and the Finlan-dian reference equations.13–15 Also, when comparedwith the adjusted values of the multicentre study,16 our

992 Rouatbi et al.

Pediatric Pulmonology

mean (95% CI ULN) measured FeNO remains signifi-cantly overestimated but only for the {10–13} and {14–16}age ranges. Therefore, care must be taken when com-paring the present study FeNO results with those usingdifferent machines in different studies. Thus, the use ofother studies FeNO norms may lead to misinterpretationof the FeNO values.

Among the published FeNO norms for children13–16

(Supplementary E. Table 1), none have proposeda method of interpreting the measured FeNO or haveprovided a prospective verification of their studiedpopulations.

FeNO Norms and Interpretation

Due to the inadequacy of the published norms,13–16

we established local normal values adapted to our pop-ulation. For practical and routine interpretation of FeNO,three ways were proposed.

A reference equation should include only easily mea-sured anthropometric data that appears to influenceFeNO. Spirometric parameters have been studied in ourstudy and by some authors15,43 but were not retainedfor our and their final reference equations for a practicaland simple interpretation. In the present study, no easilymeasured anthropometric data appear to explain FeNO

variability. For that reason, we have not established areference equation for FeNO and like in the study ofBuchvald et al.16 (Supplementary E. Table 1), we haveproposed local FeNO normal values (Table 5). In addi-tion, compared to the published reference equations,13–

15 we found significant differences between measuredand predicted FeNO (Figs. 2 and 3). The measured FeNO

of different age groups was compared with those mea-sured in the multicentric study.16 In all instances, ourmean (95% CI ULN) measured FeNO was significantlyoverestimated (Fig. 2). This can be explained by theracial factor13 and the method of measure (Medisoft vs.NIOX).33

Reliability of the Local FeNO Norms

The prospectively evaluated population demonstratedthe problems of using normal ranges established in oth-er populations. The reliability of the normal range weestablished was confirmed in the prospectively studiedpopulation, confirming the continuing need of establish-ing regional reference norms as stated by the ATS/ERS.12 This argues for the use of a specific referencenorms in the present population. The implications ofthis for children with bronchial asthma may be consid-erable, resulting in a false-positive misdiagnosis ofbronchial inflammation.

FeNO measurements offer a step forward in the as-sessment of airways disease. As an ‘‘inflammometer,’’

FeNO provides the clinician with hitherto unavailableinformation regarding the nature of underlying airwayinflammation, thus complementing conventional physio-logical testing, including the measurement of airwayhyper-responsiveness. FeNO measurements are easy toperform, reproducible, and technically less demandingthan induced sputum analysis. They are unreliable incurrent smokers and, when used diagnostically, inpatients who have been taking inhaled or oral steroidsrecently.5

In conclusion, we have established reliable norms tointerpret the results of FeNO in healthy North Africanchildren. The FeNO can easily be predicted accordingage-table ranges. Local FeNO norms enrich the globalrepository of FeNO norms the pediatrician can use tochoose the most appropriate norms based on children’slocation or ethnicity.

ACKNOWLEDGMENTS

Authors are thankful to directors of the Saladin pri-mary school of Sousse and the Jawhara college ofSousse for their helping in the recruitment of children.Authors also wish to thank professors Lamia Bougham-moura, Ahmed Abdelghani, and Iheb Bougmiza fortheir invaluable contribution in the preparation of themanuscript.

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