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Establishing normative nasal nitric oxide values in infants
Respiratory Medicine 109 (2015) 1126e1130
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Establishing normative nasal nitric oxide values in infants Phillip S. Adams a, Xin Tian b, Maliha Zahid c, Omar Khalifa c, Linda Leatherbury d, Cecilia W. Lo c, * a
Division of Pediatric Anesthesiology, Department of Anesthesiology, University of Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, PA, 15224, USA Office of Biostatistics Research, National Heart, Lung and Blood Institute, 6701 Rockledge Dr MSC 7913, Bethesda, MD, 20892, USA c Department of Developmental Biology, University of Pittsburgh School of Medicine, 4401 Penn Ave, 8120 Rangos Research Center, Pittsburgh, PA, 15201, USA d Division of Cardiology, Department of Pediatrics, Children's National Medical Center, 111 Michigan Ave NW #200, Washington, DC, 20310, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 April 2015 Received in revised form 2 July 2015 Accepted 13 July 2015 Available online 15 July 2015
Introduction: Primary ciliary dyskinesia (PCD), a disease of impaired respiratory cilia motility, is often difficult to diagnose. Recent studies show low nasal nitric oxide (nNO) is closely linked to PCD, allowing the use of nNO measurement for PCD assessments. Nasal NO cutoff values for PCD are stratified by age, given nNO levels normally increase with age. However, normative values for nNO have not been established for infants less than 1 year old. In this study, we aim to establish normative values for nNO in infants and determine their utility in guiding infant PCD assessment. Methods and results: We obtained 42 nNO values from infants less than 1 year old without a history of PCD or recurrent sinopulmonary disease. Using regression analysis, we estimated the mean age-adjusted nNO values and established a 95% prediction interval (PI) for normal nNO. Using these findings, we were able to show 14 of 15 infant PCD patients had abnormally low nNO with values below the 95% PI. Conclusions: In this study we determined a regression model that best fits normative nNO values for infants less than 1 year old. This model identified the majority of PCD infants as having abnormally low nNO. These findings suggest nNO measurement can help guide PCD assessment in infants, and perhaps other pulmonary diseases with a link to low nNO. With early assessments, earlier clinical intervention may be possible to slow disease progression and help reduce pulmonary morbidity. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Primary ciliary dyskinesia Nasal nitric oxide Infant Control values Stratification method
1. Introduction Primary ciliary dyskinesia (PCD) is a disease of impaired respiratory cilia motility causing mucociliary clearance defects and high morbidity from recurrent sinopulmonary disease [1,2]. Unfortunately, currently one-third of PCD patients are not identified until adulthood, and with unrecognized disease comes a high risk of
Abbreviations: PCD, primary ciliary dyskinesia; EM, electron microscopy; nNO, nasal nitric oxide; CHP, Children's Hospital of Pittsburgh of UPMC; MRI, magnetic resonance imaging; ppb, part per billion; CNMC, Children's National Medical Center; PI, prediction interval; LPL, lower prediction limit; CHD, congenital heart disease; CED, cranioectodermal dysplasia; CF, cystic fibrosis. * Corresponding author. Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, 8120 Rangos Research Center, Pittsburgh, 15201, PA, USA. E-mail addresses: [email protected] (P.S. Adams), [email protected] (X. Tian), [email protected] (M. Zahid), [email protected] (O. Khalifa), lleather@ childrensnational.org (L. Leatherbury), [email protected] (C.W. Lo). http://dx.doi.org/10.1016/j.rmed.2015.07.010 0954-6111/© 2015 Elsevier Ltd. All rights reserved.
recurrent pulmonary infections that ultimately can progress to irreparable chronic respiratory impairment [3]. The diagnosis of PCD can be complex, and often involves a combination of different types of assessments that may include nasal or bronchial biopsies for cilia motion evaluation utilizing video microscopy as well as cilia ultrastructure analysis by electron microscopy (EM). However, nasal biopsies can be uncomfortable for patients and they can have variable quality that is not always suitable for cilia motion or EM analysis. In addition, not all PCD patients exhibit abnormal EM cilia ultrastructure. Genetic testing is also now feasible for PCD diagnosis, as nearly 30 genes have been identified to cause PCD [4]. Despite this nearly 30% of patients undergoing genetic testing fail to have a PCD causing mutation identified [4]. More recently, the measurement of nasal nitric oxide (nNO) has been shown to be useful for PCD screening, as PCD patients are observed to have nNO levels that are only 10e20% of normal, healthy controls [5,6]. Nasal NO values are detectable in neonates within hours of being born and measurements are relatively easy to
P.S. Adams et al. / Respiratory Medicine 109 (2015) 1126e1130
obtain, reproducible, and noninvasive [7e9]. Several nNO sampling methods exist whereby a small sampling cannula is placed just into the naris and the amount of nNO being produced by the nasal sinuses is analyzed [10]. Many of these methods require patient cooperation to exhale against a resistance or phonate to close the velum during sampling. However, quiet tidal breathing has been validated in younger children unable to cooperate [7,8,11]. Nasal NO values obtained by such tidal breath sampling has shown good reliability, sensitivity, and high within-subject repeatability [11e14]. As methods for nNO measurement have become standardized, and nNO cutoff values established and validated for PCD, nNO is becoming a useful screening tool for PCD assessments [6,13,15,16]. The PCD level nNO cutoff values are stratified by age, given nNO levels normally rise with age. However, there is very little data on infant nNO values except for a few case reports. This is despite the fact that approximately 75% of patients with PCD have a history of neonatal respiratory distress syndrome, suggesting the need for early diagnosis and early intervention to slow disease progression [17e19]. In this study, we investigate and show the feasibility of establishing normative nNO values in children less than 1 year of age to guide infant nNO assessments. 2. Methods 2.1. Patient recruitment With Institutional Review Board approval, we enrolled infants and young children (ages 0e13 months) from the Children's Hospital of Pittsburgh (CHP) of UPMC. Infants were presenting on an outpatient basis for either evaluation for, or follow-up from general surgical procedures, outpatient surgical procedures, as patients presenting for MRI imaging, or as healthy volunteers (Supplemental Table 1). Infants were not eligible if they had a diagnosis of PCD or history, signs, or symptoms of recurrent or chronic sinopulmonary disease. Infants were excluded if their underlying diagnosis was consistent with a defect associated with abnormal nitric oxide levels or ciliary dysfunction. Late-preterm infants (defined as 34e37 weeks post-conceptual age) were enrolled, but infants born prior to 34 weeks post-conceptual age were excluded [20]. 2.2. Data collection With parental written informed consent, nNO values were acquired using a CLD 88sp NO analyzer (Eco Physics AG, Ann Arbor, Michigan) according to established protocols [21]. All values were obtained via low continuous sampling suction at a rate of 0.3 L/min from each naris using a nasal olive sampling cannula during tidal breathing. Five peak values from each naris during 50 s of tidal breathing were averaged to yield a final value in nl/min [22,23]. Values in nl/min are equal to values in parts per billion (ppb) multiplied by the sampling flow rate in L/min (with standard sampling flow rates recommended between 0.25 and 3 L/min) [21]. To ensure consistency, a single investigator (OK) collected all the nNO values at CHP. Additional values were obtained from subjects using the same inclusion criteria and identical methods in a previous study at the Children's National Medical Center (CNMC) in Washington, D.C. (collected by coauthor LL). 2.3. Statistical analysis The nNO values plotted against ages demonstrated increased variability with the larger nNO values in the older infants. Therefore, we applied a log transformation to the nNO values to
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normalize the distribution and then performed a regression analysis. A quadratic age effect was included in the model to explore the possible nonlinear age trend. The regression model coefficients were calculated by the least squares method and the associated prediction interval (PI) was generated to obtain the plausible nNO values from this study population. Statistical analysis was performed using the R statistical software, version 3.1.2. 3. Results A total of 42 values were used in this study (Table 1). Ten of the 42 values were from CNMC, with the remaining 32 values obtained from CHP. Ages ranged from 8 to 459 days old (mean 151, median 91) and nNO values from 9.9 nl/min to 96.3 nl/min (mean 38.7 nl/ min, median 30.6 nl/min) (Table 1). CHP patient characteristics included 19/32 males (59%) and 27/32 Caucasians (84%) (Supplemental Table 1). No patients had a diagnosis of PCD, cystic fibrosis, or situs abnormalities/heterotaxy. The regression analysis showed that the quadratic age term was statistically significant (p ¼ 0.025 using an F-test), suggesting a quadratic relationship between nNO and age. Thus, the nNO values increase more rapidly between 0 and 6 months of age, compared to 6e12 months of age. The log-transformation nNO values were
Table 1 Infant nNO values (CHP, CNMC). # of values
Origin of value
Age (d)
nNO (nl/min)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
CNMC CNMC CNMC CNMC CNMC CNMC CHP CHP CHP CHP CHP CNMC CHP CHP CHP CHP CHP CHP CHP CNMC CHP CHP CHP CNMC CHP CHP CHP CHP CHP CHP CHP CHP CHP CNMC CHP CHP CHP CHP CHP CHP CHP CHP
8 10 15 18 23 24 24 30 36 41 47 53 58 63 64 65 81 82 88 90 91 91 98 150 158 169 187 190 227 238 247 262 267 270 283 301 322 335 343 364 373 459
10.2 9.9 19.9 18.6 13.8 11.7 19.0 13.4 23.5 16.6 23.5 22.2 19.8 23.5 40.9 19.6 18.7 30.5 27.9 23.1 34.1 48.5 23.9 32.8 38.6 67.5 78.0 52.2 39 30.6 60.4 37.7 90.0 33.4 75.9 30.4 42.3 69.5 77.7 81.5 79.1 96.3
Infant nNO values and ages obtained from Children's Hospital of Pittsburgh of UPMC (CHP) and Children's National Medical Center (CNMC).
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evenly distributed around the fitted regression curve (Fig. 1) with R2 ¼ 0.73, suggesting 73% of observed variation in nNO can be explained by the chosen model of nNO with age. The estimated mean value of log nNO at a given age (in months) can be calculated from the regression equation:
logðnNOÞ ¼ 2:6511 þ 0:2324 age 0:00829 ðageÞ2 We can then take the exponential to obtain the mean nNO,
i h nNO ¼ exp 2:6511 þ 0:2324 age 0:00829 ðageÞ2 : The 95% PI was calculated to obtain an interval of plausible nNO values for independent future observations of “normal” infants within the observed age range. At each given age, the PI is expected to contain the future nNO values from this population 95% of the time. This PI can be calculated for any age from 1 to 365 days old using the equation:
PI ¼ exp Mean logðnNOÞ±t0:025; 39 SðageÞ ; where t0.025, 39is the critical value of 0.025 upper tail probability for a t-distribution with a degree of freedom of 39, and S(age) is the prediction error for log(nNO) at a given age. Specifically, we can compute the lower prediction limit (LPL) of the 95% PI for nNO at m months of age with the equation:
LPL ¼ exp ðA BÞ; where A ¼ 2:6511 þ 0:2324m 0:00829m2 ;
The predicted nNO and 95% PI are displayed for each of the first 12 months of life (Table 2). Note age in days needs to be converted to months (divided by 30.4) before placing in the formula above. To evaluate the validity of this model for determining normal nNO values for guiding infant PCD assessments, we used published nNO values available for 12 infants with PCD [24e28]. In addition, we obtained nNO values from 3 infants recruited at CHP diagnosed with PCD. PCD diagnosis in these infants was based on various combinations of findings including clinical observation of chronic sinopulmonary symptoms, visceral organ situs abnormalities, cilia EM ultrastructural defects, and mutations in known PCD genes (Supplemental Table 2). Plotting the nNO values from these 15 PCD infants on the same curve showed 14 were below the lower limit of the 95% PI of the age-adjusted nNO values. This indicates the linear regression model of normative infant nNO values has a detection sensitivity of 93.3% for PCD (Fig. 1, Supplemental Table 2). To determine the efficacy of the linear regression model, we also plotted the nNO values previously reported for four healthy infants in two publications that also provided sampling flow rates [12,24] (Fig. 1). These four normal infant values were within our prediction interval, independently confirming the efficacy of the model. 4. Discussion We obtained infant nNO values and established a regression equation to estimate age-adjusted nNO values throughout infancy up to 1 year of age. This regression model provided a good fit to the data (R2 ¼ 0.73, model P < 0.0001) and correctly identified 14 of 15 PCD infants as having abnormally low nNO, a 93.3% detection sensitivity. In a meta-analysis of nNO screening for PCD in 988 subjects across a wide age range performed by Collins et al., we
1=2 B ¼ 0:680 1 þ 0:1114 0:07505m þ 0:02398m2 0:002807m3 þ 0:000111m4 :
Fig. 1. Normative nNO values for infants less than one year of age. nNO values (black circles) for the first year of life are plotted in a log-scale. The estimated mean curve and 95% predicted intervals (PI) are illustrated by the blue and brown lines, respectively. Values below the lower limit of the 95% PI are considered abnormally low. Note of the 15 infants with PCD (red *), 14 (>93%) exhibit nNO below the lower 95% PI; four nNO values from healthy infants in the published literature [12,22] are displayed with green dots.
noted a mean sensitivity of 94% ± 7% was achieved (nNO of 65 ± 27 nl/min) [13]. These findings may have potential relevance for other diseases, as we recently showed a high prevalence of cilia dysfunction comprising abnormal ciliary motion and low nNO in patients with congenital heart disease (CHD). This was associated with a high prevalence of sinopulmonary symptoms and respiratory diseases [29,30]. These findings reflect the potential role for cilia in the pathogenesis of CHD [31]. Studies in patients and mice have also shown mutations causing PCD can result in a high prevalence of heterotaxy and complex CHD [32,33]. Low nNO also has been observed in a patient with cranioectodermal dysplasia (CED), a classic ciliopathy previously thought to arise from a nonmotile primary cilia defect [34]. This patient was found to have compound heterozygous WDR35 mutations, and was observed to have obstructive airway disease not commensurate with the relatively mild rib cage dysplasia. Interestingly, this patient was shown to have abnormal respiratory ciliary motion and nNO at the PCD cutoff value, indicating overlap in genes required for motile and primary cilia function [34]. Further indicating the potential prognosticating value for nNO assessment is another intriguing study showing CHD children with very low nNO requiring single ventricle surgical palliation are 14 times more likely to fail and require heart transplantation [35]. Patients with cystic fibrosis (CF) also have been reported to have low nNO, suggesting nNO may be valuable for stratifying CF
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Table 2 Age-adjusted nNO values for the first 12 months of life (and 95% PI)a. Age (m)
Age (d)
Age-adjusted nNO (nl/min)
Upper limit 95% PI
Lower limit 95% PI
1 2 3 4 5 6 7 8 9 10 11 12
30 60 90 120 150 180 210 240 270 300 330 360
17.68 21.70 26.22 31.17 36.45 41.95 47.51 52.94 58.05 62.63 66.49 69.45
35.58 43.38 52.37 62.41 73.25 84.53 95.83 106.74 116.92 126.18 134.48 142.03
8.78 10.86 13.13 15.56 18.14 20.82 23.55 26.26 28.82 31.08 32.87 33.96
a Based on regression model with age ¼ m months, and the equation: Age adjusted nNO ¼ expð2:6511 þ 0:2324m 0:00829m2 Þ; upper limit of 95% PI ¼ expðA þ BÞ; lower limit of 95% PI ¼ expðA BÞ; whereA ¼ 2:6511 þ 0:2324m 0:00829m2 ; B ¼ 0:680ð1 þ 0:1114 0:07505m þ 0:02398m2 0:002807m3 þ 0:000111m4 Þ:
patients [36e38]. Together, these findings suggest nNO assessment may have potential value in guiding the clinical care of patients with a wide spectrum of diseases not traditionally linked to PCD. Our study has several limitations, one being the paucity of measurements from older infants ages 6e12 months (median age of 3 months). There are publications with mention of infant nNO values, however these could not be included as direct comparison with the current study was not possible given missing information (flow rate not given for nNO assessment, age at nNO measurement not indicated). PCD infant nNO values were even more difficult to obtain. Hence, the normative infant nNO model generated can guide whether nNO values are in the normal range, but they are not able to diagnose PCD. More measurements from infants with PCD will be needed for a similar modeling of infant PCD nNO values to guide PCD screening. Another limitation is the recruitment of infants from the surgical and radiology departments for obtaining the normative nNO values. While PCD was excluded from these subjects, these were not all normal healthy infants. This study population was necessitated given the difficulty in transporting the nNO machine off-site and the lack of a wellness clinic in CHP. Hence future replication of these findings in normal healthy infants would be desirable. 5. Conclusion This is the first study to show the feasibility of standardizing normative nNO values for infants less than 1 year of age. The nNO values with age generated by our regression model can be used in a similar fashion to other growth curves (i.e. height, weight, head circumference) to assess nNO in subjects throughout infancy. Findings from the nNO assessments can help determine whether additional assessments are warranted, such as genetic testing and EM analysis, to establish a PCD diagnosis. Earlier diagnosis will provide the opportunity to improve outcome by allowing earlier therapeutic intervention to slow progression to chronic pulmonary disease. In addition to its clinical applicability, these findings will provide the means to stratify and classify infants in studies within the evolving fields of PCD, CHD, and ciliopathy research. Conflict of interest None declared. Acknowledgments This work was supported by the PA Department of Health. In addition, we would like to thank Stacy Gibson, BSN, RN, CPN
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S.K. Dutcher, T.W. Ferkol, Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia, Am. J. Hum. Genet. 91 (4) (2012) 685e693. N. Nakhleh, R. Francis, R.A. Giese, X. Tian, Y. Li, M.A. Zariwala, H. Yagi, O. Khalifa, S. Kureshi, B. Chatterjee, S.L. Sabol, M. Swisher, P.S. Connelly, M.P. Daniels, A. Srinivasan, K. Kuehl, N. Kravitz, K. Burns, I. Sami, H. Omran, M. Barmada, K. Olivier, K.K. Chawla, M. Leigh, R. Jonas, M. Knowles, L. Leatherbury, C.W. Lo, High prevalence of respiratory ciliary dysfunction in congenital heart disease patients with heterotaxy, Circulation 125 (2012) 2232e2242. A.S. Garrod, M. Zahid, X. Tian, R.J. Francis, O. Khalifa, W. Devine, G.C. Gabriel, L. Leatherbury, C.W. Lo, Airway ciliary dysfunction and sinopulmonary symptoms in patients with congenital heart disease, Ann. Am. Thorac. Soc. 11 (9) (2014) 1426e1432. Y. Li, N.T. Klena, G.C. Gabriel, X. Liu, A.J. Kim, K. Lemke, Y. Chen, B. Chatterjee, W. Devine, R.R. Damerla, C.F. Chang, H. Yagi, J.T. San Augustin, M. Thahir, S. Anderton, C. Lawhead, A. Vescovi, H. Pratt, J. Morgan, L. Haynes, C.L. Smith, J.T. Eppig, L. Reinholdt, R. Francis, L. Leatherbury, M. Ganapathiraju, K. Tobita, G.J. Paozur, C.W. Lo, Global genetic analysis in mice unveils central role for cilia in congenital heart disease, Nature 521 (7553) (2015 May 28) 520e524. M.P. Kennedy, H. Omran, M.W. Leigh, S. Dell, L. Morgan, P.L. Molina, B.V. Robinson, S.L. Minnix, H. Olbrich, T. Severin, P. Ahrens, L. Lange, H.N. Morillas, P.G. Noone, M.A. Zariwala, M.R. Knowles, Congenital heart disease and other heterotaxic defects in a large cohort of patients with primary ciliary dyskinesia, Circulation 115 (2007) 2814e2821. S.Y. Tan, J. Rosenthal, X.Q. Zhao, R.J. Francis, B. Chatterjee, S.L. Sabol, K.L. Linask, L. Bracero, P.S. Connelly, M.P. Daniels, Q. Yu, H. Omran, L. Leatherbury, C.W. Lo, Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia, J. Clin. Invest 117 (12) (2007) 3742e3752. Y. Li, A.S. Garrod, S. Madan-Khetarpal, G. Sreedher, M. McGuire, H. Yagi, N.T. Klena, G.C. Gabriel, O. Khalifa, M. Zahid, A. Panigrahy, D.J. Weiner, C.W. Lo, Respiratory motile cilia dysfunction in a patient with cranioectodermal dysplasia and biallelic WDR35 mutations, Am. J. Med. Genet. (2015) [in press]. C. Yau, M. Zahid, O. Khalifa, W. Devine, L. Leatherbury, P. Wearden, C. Lo, Low nasal nitric oxide as a prognosticator of failing single ventricle physiology in congenital heart disease patients? [abstract], Circulation 130 (2014) A16375. K.M. de Winter-de Groot, S. van Haren Noman, L. Speleman, A.G. Schilder, C.K. van der Ent, Nasal nitric oxide levels and nasal polyposis in children and adolescents with cystic fibrosis, JAMA Otolaryngol. Head. Neck Surg. 139 (9) (2013) 931e936. V.M. Struben, W.V. Sewbalak, M.H. Wieringa, C.J. Mantingh, L.M. van den Toorn, M. Bakker, L. Feenstra, J.C. de Jongste, Nasal nitric oxide in cystic fibrosis with and without humming, Eur. J. Clin. Invest 37 (2) (2007) 140e144. R.K. Michl, J. Hentschel, C. Fischer, J.F. Beck, J.G. Mainz, Reduced nasal nitric oxide production in cystic fibrosis patients with elevated systemic inflammation markers, PLoS One 8 (11) (2013;13) e79141.
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Establishing normative nasal nitric oxide values in infants Phillip S. Adams a, Xin Tian b, Maliha Zahid c, Omar Khalifa c, Linda Leatherbury d, Cecilia W. Lo c, * a
Division of Pediatric Anesthesiology, Department of Anesthesiology, University of Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, PA, 15224, USA Office of Biostatistics Research, National Heart, Lung and Blood Institute, 6701 Rockledge Dr MSC 7913, Bethesda, MD, 20892, USA c Department of Developmental Biology, University of Pittsburgh School of Medicine, 4401 Penn Ave, 8120 Rangos Research Center, Pittsburgh, PA, 15201, USA d Division of Cardiology, Department of Pediatrics, Children's National Medical Center, 111 Michigan Ave NW #200, Washington, DC, 20310, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 April 2015 Received in revised form 2 July 2015 Accepted 13 July 2015 Available online 15 July 2015
Introduction: Primary ciliary dyskinesia (PCD), a disease of impaired respiratory cilia motility, is often difficult to diagnose. Recent studies show low nasal nitric oxide (nNO) is closely linked to PCD, allowing the use of nNO measurement for PCD assessments. Nasal NO cutoff values for PCD are stratified by age, given nNO levels normally increase with age. However, normative values for nNO have not been established for infants less than 1 year old. In this study, we aim to establish normative values for nNO in infants and determine their utility in guiding infant PCD assessment. Methods and results: We obtained 42 nNO values from infants less than 1 year old without a history of PCD or recurrent sinopulmonary disease. Using regression analysis, we estimated the mean age-adjusted nNO values and established a 95% prediction interval (PI) for normal nNO. Using these findings, we were able to show 14 of 15 infant PCD patients had abnormally low nNO with values below the 95% PI. Conclusions: In this study we determined a regression model that best fits normative nNO values for infants less than 1 year old. This model identified the majority of PCD infants as having abnormally low nNO. These findings suggest nNO measurement can help guide PCD assessment in infants, and perhaps other pulmonary diseases with a link to low nNO. With early assessments, earlier clinical intervention may be possible to slow disease progression and help reduce pulmonary morbidity. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Primary ciliary dyskinesia Nasal nitric oxide Infant Control values Stratification method
1. Introduction Primary ciliary dyskinesia (PCD) is a disease of impaired respiratory cilia motility causing mucociliary clearance defects and high morbidity from recurrent sinopulmonary disease [1,2]. Unfortunately, currently one-third of PCD patients are not identified until adulthood, and with unrecognized disease comes a high risk of
Abbreviations: PCD, primary ciliary dyskinesia; EM, electron microscopy; nNO, nasal nitric oxide; CHP, Children's Hospital of Pittsburgh of UPMC; MRI, magnetic resonance imaging; ppb, part per billion; CNMC, Children's National Medical Center; PI, prediction interval; LPL, lower prediction limit; CHD, congenital heart disease; CED, cranioectodermal dysplasia; CF, cystic fibrosis. * Corresponding author. Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, 8120 Rangos Research Center, Pittsburgh, 15201, PA, USA. E-mail addresses: [email protected] (P.S. Adams), [email protected] (X. Tian), [email protected] (M. Zahid), [email protected] (O. Khalifa), lleather@ childrensnational.org (L. Leatherbury), [email protected] (C.W. Lo). http://dx.doi.org/10.1016/j.rmed.2015.07.010 0954-6111/© 2015 Elsevier Ltd. All rights reserved.
recurrent pulmonary infections that ultimately can progress to irreparable chronic respiratory impairment [3]. The diagnosis of PCD can be complex, and often involves a combination of different types of assessments that may include nasal or bronchial biopsies for cilia motion evaluation utilizing video microscopy as well as cilia ultrastructure analysis by electron microscopy (EM). However, nasal biopsies can be uncomfortable for patients and they can have variable quality that is not always suitable for cilia motion or EM analysis. In addition, not all PCD patients exhibit abnormal EM cilia ultrastructure. Genetic testing is also now feasible for PCD diagnosis, as nearly 30 genes have been identified to cause PCD [4]. Despite this nearly 30% of patients undergoing genetic testing fail to have a PCD causing mutation identified [4]. More recently, the measurement of nasal nitric oxide (nNO) has been shown to be useful for PCD screening, as PCD patients are observed to have nNO levels that are only 10e20% of normal, healthy controls [5,6]. Nasal NO values are detectable in neonates within hours of being born and measurements are relatively easy to
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obtain, reproducible, and noninvasive [7e9]. Several nNO sampling methods exist whereby a small sampling cannula is placed just into the naris and the amount of nNO being produced by the nasal sinuses is analyzed [10]. Many of these methods require patient cooperation to exhale against a resistance or phonate to close the velum during sampling. However, quiet tidal breathing has been validated in younger children unable to cooperate [7,8,11]. Nasal NO values obtained by such tidal breath sampling has shown good reliability, sensitivity, and high within-subject repeatability [11e14]. As methods for nNO measurement have become standardized, and nNO cutoff values established and validated for PCD, nNO is becoming a useful screening tool for PCD assessments [6,13,15,16]. The PCD level nNO cutoff values are stratified by age, given nNO levels normally rise with age. However, there is very little data on infant nNO values except for a few case reports. This is despite the fact that approximately 75% of patients with PCD have a history of neonatal respiratory distress syndrome, suggesting the need for early diagnosis and early intervention to slow disease progression [17e19]. In this study, we investigate and show the feasibility of establishing normative nNO values in children less than 1 year of age to guide infant nNO assessments. 2. Methods 2.1. Patient recruitment With Institutional Review Board approval, we enrolled infants and young children (ages 0e13 months) from the Children's Hospital of Pittsburgh (CHP) of UPMC. Infants were presenting on an outpatient basis for either evaluation for, or follow-up from general surgical procedures, outpatient surgical procedures, as patients presenting for MRI imaging, or as healthy volunteers (Supplemental Table 1). Infants were not eligible if they had a diagnosis of PCD or history, signs, or symptoms of recurrent or chronic sinopulmonary disease. Infants were excluded if their underlying diagnosis was consistent with a defect associated with abnormal nitric oxide levels or ciliary dysfunction. Late-preterm infants (defined as 34e37 weeks post-conceptual age) were enrolled, but infants born prior to 34 weeks post-conceptual age were excluded [20]. 2.2. Data collection With parental written informed consent, nNO values were acquired using a CLD 88sp NO analyzer (Eco Physics AG, Ann Arbor, Michigan) according to established protocols [21]. All values were obtained via low continuous sampling suction at a rate of 0.3 L/min from each naris using a nasal olive sampling cannula during tidal breathing. Five peak values from each naris during 50 s of tidal breathing were averaged to yield a final value in nl/min [22,23]. Values in nl/min are equal to values in parts per billion (ppb) multiplied by the sampling flow rate in L/min (with standard sampling flow rates recommended between 0.25 and 3 L/min) [21]. To ensure consistency, a single investigator (OK) collected all the nNO values at CHP. Additional values were obtained from subjects using the same inclusion criteria and identical methods in a previous study at the Children's National Medical Center (CNMC) in Washington, D.C. (collected by coauthor LL). 2.3. Statistical analysis The nNO values plotted against ages demonstrated increased variability with the larger nNO values in the older infants. Therefore, we applied a log transformation to the nNO values to
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normalize the distribution and then performed a regression analysis. A quadratic age effect was included in the model to explore the possible nonlinear age trend. The regression model coefficients were calculated by the least squares method and the associated prediction interval (PI) was generated to obtain the plausible nNO values from this study population. Statistical analysis was performed using the R statistical software, version 3.1.2. 3. Results A total of 42 values were used in this study (Table 1). Ten of the 42 values were from CNMC, with the remaining 32 values obtained from CHP. Ages ranged from 8 to 459 days old (mean 151, median 91) and nNO values from 9.9 nl/min to 96.3 nl/min (mean 38.7 nl/ min, median 30.6 nl/min) (Table 1). CHP patient characteristics included 19/32 males (59%) and 27/32 Caucasians (84%) (Supplemental Table 1). No patients had a diagnosis of PCD, cystic fibrosis, or situs abnormalities/heterotaxy. The regression analysis showed that the quadratic age term was statistically significant (p ¼ 0.025 using an F-test), suggesting a quadratic relationship between nNO and age. Thus, the nNO values increase more rapidly between 0 and 6 months of age, compared to 6e12 months of age. The log-transformation nNO values were
Table 1 Infant nNO values (CHP, CNMC). # of values
Origin of value
Age (d)
nNO (nl/min)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
CNMC CNMC CNMC CNMC CNMC CNMC CHP CHP CHP CHP CHP CNMC CHP CHP CHP CHP CHP CHP CHP CNMC CHP CHP CHP CNMC CHP CHP CHP CHP CHP CHP CHP CHP CHP CNMC CHP CHP CHP CHP CHP CHP CHP CHP
8 10 15 18 23 24 24 30 36 41 47 53 58 63 64 65 81 82 88 90 91 91 98 150 158 169 187 190 227 238 247 262 267 270 283 301 322 335 343 364 373 459
10.2 9.9 19.9 18.6 13.8 11.7 19.0 13.4 23.5 16.6 23.5 22.2 19.8 23.5 40.9 19.6 18.7 30.5 27.9 23.1 34.1 48.5 23.9 32.8 38.6 67.5 78.0 52.2 39 30.6 60.4 37.7 90.0 33.4 75.9 30.4 42.3 69.5 77.7 81.5 79.1 96.3
Infant nNO values and ages obtained from Children's Hospital of Pittsburgh of UPMC (CHP) and Children's National Medical Center (CNMC).
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evenly distributed around the fitted regression curve (Fig. 1) with R2 ¼ 0.73, suggesting 73% of observed variation in nNO can be explained by the chosen model of nNO with age. The estimated mean value of log nNO at a given age (in months) can be calculated from the regression equation:
logðnNOÞ ¼ 2:6511 þ 0:2324 age 0:00829 ðageÞ2 We can then take the exponential to obtain the mean nNO,
i h nNO ¼ exp 2:6511 þ 0:2324 age 0:00829 ðageÞ2 : The 95% PI was calculated to obtain an interval of plausible nNO values for independent future observations of “normal” infants within the observed age range. At each given age, the PI is expected to contain the future nNO values from this population 95% of the time. This PI can be calculated for any age from 1 to 365 days old using the equation:
PI ¼ exp Mean logðnNOÞ±t0:025; 39 SðageÞ ; where t0.025, 39is the critical value of 0.025 upper tail probability for a t-distribution with a degree of freedom of 39, and S(age) is the prediction error for log(nNO) at a given age. Specifically, we can compute the lower prediction limit (LPL) of the 95% PI for nNO at m months of age with the equation:
LPL ¼ exp ðA BÞ; where A ¼ 2:6511 þ 0:2324m 0:00829m2 ;
The predicted nNO and 95% PI are displayed for each of the first 12 months of life (Table 2). Note age in days needs to be converted to months (divided by 30.4) before placing in the formula above. To evaluate the validity of this model for determining normal nNO values for guiding infant PCD assessments, we used published nNO values available for 12 infants with PCD [24e28]. In addition, we obtained nNO values from 3 infants recruited at CHP diagnosed with PCD. PCD diagnosis in these infants was based on various combinations of findings including clinical observation of chronic sinopulmonary symptoms, visceral organ situs abnormalities, cilia EM ultrastructural defects, and mutations in known PCD genes (Supplemental Table 2). Plotting the nNO values from these 15 PCD infants on the same curve showed 14 were below the lower limit of the 95% PI of the age-adjusted nNO values. This indicates the linear regression model of normative infant nNO values has a detection sensitivity of 93.3% for PCD (Fig. 1, Supplemental Table 2). To determine the efficacy of the linear regression model, we also plotted the nNO values previously reported for four healthy infants in two publications that also provided sampling flow rates [12,24] (Fig. 1). These four normal infant values were within our prediction interval, independently confirming the efficacy of the model. 4. Discussion We obtained infant nNO values and established a regression equation to estimate age-adjusted nNO values throughout infancy up to 1 year of age. This regression model provided a good fit to the data (R2 ¼ 0.73, model P < 0.0001) and correctly identified 14 of 15 PCD infants as having abnormally low nNO, a 93.3% detection sensitivity. In a meta-analysis of nNO screening for PCD in 988 subjects across a wide age range performed by Collins et al., we
1=2 B ¼ 0:680 1 þ 0:1114 0:07505m þ 0:02398m2 0:002807m3 þ 0:000111m4 :
Fig. 1. Normative nNO values for infants less than one year of age. nNO values (black circles) for the first year of life are plotted in a log-scale. The estimated mean curve and 95% predicted intervals (PI) are illustrated by the blue and brown lines, respectively. Values below the lower limit of the 95% PI are considered abnormally low. Note of the 15 infants with PCD (red *), 14 (>93%) exhibit nNO below the lower 95% PI; four nNO values from healthy infants in the published literature [12,22] are displayed with green dots.
noted a mean sensitivity of 94% ± 7% was achieved (nNO of 65 ± 27 nl/min) [13]. These findings may have potential relevance for other diseases, as we recently showed a high prevalence of cilia dysfunction comprising abnormal ciliary motion and low nNO in patients with congenital heart disease (CHD). This was associated with a high prevalence of sinopulmonary symptoms and respiratory diseases [29,30]. These findings reflect the potential role for cilia in the pathogenesis of CHD [31]. Studies in patients and mice have also shown mutations causing PCD can result in a high prevalence of heterotaxy and complex CHD [32,33]. Low nNO also has been observed in a patient with cranioectodermal dysplasia (CED), a classic ciliopathy previously thought to arise from a nonmotile primary cilia defect [34]. This patient was found to have compound heterozygous WDR35 mutations, and was observed to have obstructive airway disease not commensurate with the relatively mild rib cage dysplasia. Interestingly, this patient was shown to have abnormal respiratory ciliary motion and nNO at the PCD cutoff value, indicating overlap in genes required for motile and primary cilia function [34]. Further indicating the potential prognosticating value for nNO assessment is another intriguing study showing CHD children with very low nNO requiring single ventricle surgical palliation are 14 times more likely to fail and require heart transplantation [35]. Patients with cystic fibrosis (CF) also have been reported to have low nNO, suggesting nNO may be valuable for stratifying CF
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Table 2 Age-adjusted nNO values for the first 12 months of life (and 95% PI)a. Age (m)
Age (d)
Age-adjusted nNO (nl/min)
Upper limit 95% PI
Lower limit 95% PI
1 2 3 4 5 6 7 8 9 10 11 12
30 60 90 120 150 180 210 240 270 300 330 360
17.68 21.70 26.22 31.17 36.45 41.95 47.51 52.94 58.05 62.63 66.49 69.45
35.58 43.38 52.37 62.41 73.25 84.53 95.83 106.74 116.92 126.18 134.48 142.03
8.78 10.86 13.13 15.56 18.14 20.82 23.55 26.26 28.82 31.08 32.87 33.96
a Based on regression model with age ¼ m months, and the equation: Age adjusted nNO ¼ expð2:6511 þ 0:2324m 0:00829m2 Þ; upper limit of 95% PI ¼ expðA þ BÞ; lower limit of 95% PI ¼ expðA BÞ; whereA ¼ 2:6511 þ 0:2324m 0:00829m2 ; B ¼ 0:680ð1 þ 0:1114 0:07505m þ 0:02398m2 0:002807m3 þ 0:000111m4 Þ:
patients [36e38]. Together, these findings suggest nNO assessment may have potential value in guiding the clinical care of patients with a wide spectrum of diseases not traditionally linked to PCD. Our study has several limitations, one being the paucity of measurements from older infants ages 6e12 months (median age of 3 months). There are publications with mention of infant nNO values, however these could not be included as direct comparison with the current study was not possible given missing information (flow rate not given for nNO assessment, age at nNO measurement not indicated). PCD infant nNO values were even more difficult to obtain. Hence, the normative infant nNO model generated can guide whether nNO values are in the normal range, but they are not able to diagnose PCD. More measurements from infants with PCD will be needed for a similar modeling of infant PCD nNO values to guide PCD screening. Another limitation is the recruitment of infants from the surgical and radiology departments for obtaining the normative nNO values. While PCD was excluded from these subjects, these were not all normal healthy infants. This study population was necessitated given the difficulty in transporting the nNO machine off-site and the lack of a wellness clinic in CHP. Hence future replication of these findings in normal healthy infants would be desirable. 5. Conclusion This is the first study to show the feasibility of standardizing normative nNO values for infants less than 1 year of age. The nNO values with age generated by our regression model can be used in a similar fashion to other growth curves (i.e. height, weight, head circumference) to assess nNO in subjects throughout infancy. Findings from the nNO assessments can help determine whether additional assessments are warranted, such as genetic testing and EM analysis, to establish a PCD diagnosis. Earlier diagnosis will provide the opportunity to improve outcome by allowing earlier therapeutic intervention to slow progression to chronic pulmonary disease. In addition to its clinical applicability, these findings will provide the means to stratify and classify infants in studies within the evolving fields of PCD, CHD, and ciliopathy research. Conflict of interest None declared. Acknowledgments This work was supported by the PA Department of Health. In addition, we would like to thank Stacy Gibson, BSN, RN, CPN
of the pediatric surgery department for her assistance with patient recruitment as well as all of the physicians, nurses and staff of the pediatric surgery, radiology, and anesthesiology departments for their assistance in accommodating recruitment and sampling. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.rmed.2015.07.010. References [1] J.S. Lucas, A. Burgess, H.M. Mitchison, E. Moya, M. Williamson, C. Hogg, National PCD Service, UK, Diagnosis and management of primary ciliary dyskinesia, Arch. Dis. Child. 99 (9) (2014) 850e856. [2] M.R. Knowles, L.A. Daniels, S.D. Davis, M.A. Zariwala, M.W. Leigh, Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease, Am. J. Respir. Crit. Care Med. 188 (8) (2013) 913e922. [3] M. Boon, I. Meyts, M. Proesmans, F.L. Vermeulen, M. Jorissen, K. De Boeck, Diagnostic accuracy of nitric oxide measurements to detect primary ciliary dyskinesia, Eur. J. Clin. Invest 44 (5) (2014) 477e485. [4] J. Lobo, M.A. Zariwala, P.G. Noone, Primary ciliary dyskinesia, Semin. Respir. Crit. Care Med. 36 (2) (2015) 169e179. [5] M.W. Leigh, C. O'Callaghan, M.R. Knowles, The challenges of diagnosing primary ciliary dyskinesia, Proc. Am. Thorac. Soc. 8 (5) (2011) 434e437. [6] M.W. Leigh, M.J. Hazucha, K.K. Chawla, B.R. Baker, A.J. Shapiro, D.E. Brown, L.M. Lavange, B.J. Horton, B. Qaqish, J.L. Carson, S.D. Davis, S.D. Dell, T.W. Ferkol, J.J. Atkinson, K.N. Olivier, S.D. Sagel, M. Rosenfeld, C. Milla, H.S. Lee, J. Krischer, M.A. Zariwala, M.R. Knowles, Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia, Ann. Am. Thorac. Soc. 10 (6) (2013) 574e581. [7] J.K. Marthin, K.G. Nielsen, Choice of nasal nitric oxide technique as first-line test for primary ciliary dyskinesia, Eur. Respir. J. 37 (2011) 559e565. [8] D. Mateos-Corral, R. Coombs, H. Grasemann, F. Ratjen, S.D. Dell, Diagnostic value of nasal nitric oxide measured with non-velum closure techniques for children with primary ciliary dyskinesia, J. Pediatr. 159 (2011) 420e424. [9] U. Schedin, M. Norman, L.E. Gustafsson, P. Herin, C. Frostell, Endogenous nitric oxide in the upper airways of healthy newborn infants, Pediatr. Res. 40 (1) (1996) 148e151. r, P.J. Cole, S.A. Kharitonov, [10] I. Horv ath, S. Loukides, T. Wodehouse, E. Csisze P.J. Barnes, Comparison of exhaled and nasal nitric oxide and exhaled carbon monoxide levels in bronchiectatic patients with and without primary ciliary dyskinesia, Thorax 58 (1) (2003) 68e72. [11] A. Manna, S. Montella, M. Maniscalco, M. Maglione, F. Santamaria, Clinical application of nasal nitric oxide measurement in pediatric airway diseases, Pediatr. Pulmonol. 50 (1) (2015) 85e99. [12] C.C. Chang, S.H. Lien, T.Y. Liu, Y.M. Hua, C.M. Lee, Y.S. Yuh, Nasal nitric oxide in infants before and after extubation, Pediatr. Pulmonol. 43 (11) (2008) 1072e1077. [13] S.A. Collins, K. Gove, W. Walker, J.S. Lucas, Nasal nitric oxide screening for primary ciliary dyskinesia: systematic review and meta-analysis, Eur. Respir. J. 44 (6) (2014) 1589e1599. [14] R. Corbelli, J. Hammer, Measurement of nasal nitric oxide, Paediatr. Respir. Rev. 8 (3) (2007) 269e272. [15] C. Hogg, Primary ciliary dyskinesia: When to suspect the diagnosis and how to confirm it, Paediatr. Respir. Rev. 10 (2) (2009) 44e50. [16] T. Wodehouse, S.A. Kharitonov, I.S. Mackay, P.J. Barnes, R. Wilson, P.J. Cole, Nasal nitric oxide measurements for the screening of primary ciliary
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