Inert gas washout was first described more than 60 years ago and 2 principal tests have been developed from it: the single breath and multiple breath washout (MBW) techniques. The invention of fast responding gas analysers almost 60 years ago and small computers 30 years later have facilitated breath-by-breath analysis and the development of sophisticated analysis techniques. It is now possible to detect not only the degree of pulmonary ventilation inhomogeneity, but also to gain important insight into the location of the underlying disease process. While single breath washout requires a full vital capacity effort, tidal breathing during the multiple breath test requires minimal co-operation and co-ordination, and is feasible in subjects of all ages. Available MBW normative data from parameters, such as the lung clearance index, appears to vary minimally with age, making MBW particularly useful to follow children longitudinally. Multiple breath inert gas washout has demonstrated improved sensitivity, in comparison to spirometry, in the early detection of a number of important disease processes, including cystic fibrosis. Despite this, these important techniques remain under-utilised in the clinical setting and there is a lack of commercially available devices currently available. The recent resurgence of research in this area has produced a large number of important studies and a pronounced international interest has developed in these techniques. This review article will provide an overview of the theoretical background of inert gas washout and analysis indices, review important physiological and clinical insights gained from research to date (as well as from our own experience) to illustrate its utility, and outline the challenges that lie ahead in incorporating these techniques into the mainstream clinical setting.

Carroll N, Elliot J, Morton A, James A: The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 1993;147:405–410.
Hamid Q, Song Y, Kotsimbos TC, Minshall E, Bai TR, Hegele RG, Hogg JC: Inflammation of small airways in asthma. J Allergy Clin Immunol 1997;100:44–51.
Hamutcu R, Rowland JM, Horn MV, Kaminsky C, MacLaughlin EF, Starnes VA, Woo MS: Clinical findings and lung pathology in children with cystic fibrosis. Am J Respir Crit Care Med 2002;165:1172–1175.
Hogg JC, Macklem PT, Thurlbeck WM: Site and nature of airway obstruction in chronic obstructive lung disease. N Engl J Med 1968;278:1355–1360.
Darling RC, Cournand A, Mansfield JS, Richards DW: Studies on the intrapulmonary mixture of gases. I. Nitrogen elimination from blood and body tissues during high oxygen breathing. J Clin Invest 1940;19:591–597.
Cosio MG: Looking at the acinus with function tests: can you believe it? Am J Respir Crit Care Med 2006;174:847–848.
Weibel ER: Principles and methods for the morphometric study of the lung and other organs. Lab Invest 1963;12:131–155.
Paiva M: Gas transport in the human lung. J Appl Physiol 1973;35:401–410.
Macklem PT, Mead J: Resistance of central and peripheral airways measured by a retrograde catheter. J Appl Physiol 1967;22:395–401.
McNamara JJ, Castile RG, Glass GM, Fredberg JJ: Heterogeneous lung emptying during forced expiration. J Appl Physiol 1987;63:1648–1657.
Macklem PT: The physiology of small airways. Am J Respir Crit Care Med 1998;157:S181–S183.
Siri WE: A mass spectroscope for analysis in the low mass range. Rev Sci Instrum 1947;18:540.
Lilly JC: Mixing of gases within respiratory system with a new type nitrogen meter. Am J Physiol 1950;161:342–351.
Fowler KT, Hugh-Jones P: Mass spectrometry applied to clinical practice and research. Br Med J 1957;1:1205–1211.
Fowler KT: The respiratory mass spectrometer. Phys Med Biol 1969;14:185–199.
Fowler WS: Lung function studies; uneven pulmonary ventilation in normal subjects and in patients with pulmonary disease. J Appl Physiol 1949;2:283–299.
Robertson JS, Siri WE, Jones HB: Lung ventilation patterns determined by analysis of nitrogen elimination rates; use of mass spectrometer as a continuous gas analyzer. J Clin Invest 1950;29:577–590.
Kjellman B: Lung function in children with pneumonia: with special reference to distribution of inspired air and regional lung function. Scand J Respir Dis 1968;49:185–201.
Wall MA: Moment analysis of multibreath nitrogen washout in young children. J Appl Physiol 1985;59:274–279.
Kraemer R, Meister B: Fast real-time moment-ratio analysis of multibreath nitrogen washout in children. J Appl Physiol 1985;59:1137–1144.
Hjalmarson O, Sandberg K: Abnormal lung function in healthy preterm infants. Am J Respir Crit Care Med 2002;165:83–87.
Aurora P, Bush A, Gustafsson P, Oliver C, Wallis C, Price J, Stroobant J, Carr S, Stocks J: Multiple-breath washout as a marker of lung disease in preschool children with cystic fibrosis. Am J Respir Crit Care Med 2005;171:249–256.
Schibler A, Schneider M, Frey U, Kraemer R: Moment ratio analysis of multiple breath nitrogen washout in infants with lung disease. Eur Respir J 2000;15:1094–1101.
Robinson PD, Cooper P, Van Asperen P, Fitzgerald D, Selvadurai H: Using index of ventilation to assess response to treatment for acute pulmonary exacerbation in children with cystic fibrosis. Pediatr Pulmonol 2009, in press.
Latzin P, Sauteur L, Thamrin C, Schibler A, Baldwin D, Hutten GJ, Kyburz M, Kraemer R, Riedel T, Frey U: Optimized temperature and deadspace correction improve analysis of multiple breath washout measurements by ultrasonic flowmeter in infants. Pediatr Pulmonol 2007;42:888–897.
Fuchs SI, Sturz J, Junge S, Ballmann M, Gappa M: A novel sidestream ultrasonic flow sensor for multiple breath washout in children. Pediatr Pulmonol 2008;43:731–738.
Horsley AR, Gustafsson PM, Macleod KA, Saunders C, Greening AP, Porteous DJ, Davies JC, Cunningham S, Alton EW, Innes JA: Lung clearance index is a sensitive, repeatable and practical measure of airways disease in adults with cystic fibrosis. Thorax 2008;63:135–140.
van Meerten RJ: Expiratory gas concentration curves for examination of uneven distribution of ventilation and perfusion in the lung: first communication: theory. Respiration 1970;27:552–564.
Dutrieue B, Lauzon AM, Verbanck S, Elliott AR, West JB, Paiva M, Prisk GK: Helium and sulfur hexafluoride bolus washin in short-term microgravity. J Appl Physiol 1999;86:1594–1602.
Guy HJ, Prisk GK, Elliott AR, Deutschman RA 3rd, West JB: Inhomogeneity of pulmonary ventilation during sustained microgravity as determined by single-breath washouts. J Appl Physiol 1994;76:1719–1729.
Van Muylem A, De Vuyst P, Yernault JC, Paiva M: Inert gas single-breath washout and structural alteration of respiratory bronchioles. Am Rev Respir Dis 1992;146:1167–1172.
Venegas JG, Winkler T, Musch G, Vidal Melo MF, Layfield D, Tgavalekos N, Fischman AJ, Callahan RJ, Bellani G, Harris RS: Self-organized patchiness in asthma as a prelude to catastrophic shifts. Nature 2005;434:777–782.
Burger EJ Jr, Macklem P: Airway closure: demonstration by breathing 100 percent O2 at low lung volumes and by N2 washout. J Appl Physiol 1968;25:139–148.
Engel LA, Grassino A, Anthonisen NR: Demonstration of airway closure in man. J Appl Physiol 1975;38:1117–1125.
Hughes JM, Rosenzweig DY, Kivitz PB: Site of airway closure in excised dog lungs: histologic demonstration. J Appl Physiol 1970;29:340–344.
Cosio M, Ghezzo H, Hogg JC, Corbin R, Loveland M, Dosman J, Macklem PT: The relations between structural changes in small airways and pulmonary-function tests. N Engl J Med 1978;298:1277–1281.
Michaelson ED, Wanner A, Watson H, Silva G, Zapata A, Serafini-Michaelson M, Sackner MA: Distribution of ventilation in normal children. Chest 1979;76:658–662.
Lum S, Gustafsson P, Ljungberg H, Hulskamp G, Bush A, Carr SB, Castle R, Hoo AF, Price J, Ranganathan S, Stroobant J, Wade A, Wallis C, Wyatt H, Stocks J: Early detection of cystic fibrosis lung disease: multiple-breath washout versus raised volume tests. Thorax 2007;62:341–347.
Gustafsson PM, Aurora P, Lindblad A: Evaluation of ventilation maldistribution as an early indicator of lung disease in children with cystic fibrosis. Eur Respir J 2003;22:972–979.
Verbanck S, Schuermans D, Van Muylem A, Paiva M, Noppen M, Vincken W: Ventilation distribution during histamine provocation. J Appl Physiol 1997;83:1907–1916.
Beydon N, Davis SD, Lombardi E, Allen JL, Arets HG, Aurora P, Bisgaard H, Davis GM, Ducharme FM, Eigen H, Gappa M, Gaultier C, Gustafsson PM, Hall GL, Hantos Z, Healy MJ, Jones MH, Klug B, Lodrup Carlsen KC, McKenzie SA, Marchal F, Mayer OH, Merkus PJ, Morris MG, Oostveen E, Pillow JJ, Seddon PC, Silverman M, Sly PD, Stocks J, Tepper RS, Vilozni D, Wilson NM, American Thoracic Society/European Respiratory Society Working Group on Infant and Young Children Pulmonary Function Testing: An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007;175:1304–1345.
Fowler WS: Lung function studies; the respiratory dead space. Am J Physiol 1948;154:405–416.
Langley F, Even P, Duroux P, Nicolas RL, Cumming G: Ventilatory consequences of unilateral pulmonary artery occlusion. Coll Inst Natl Santé Rech Méd 1975;51:209–212.
Larsson A, Jonmarker C, Werner O: Ventilation inhomogeneity during controlled ventilation: which index should be used? J Appl Physiol 1988;65:2030–2039.
Bouhuys A: Pulmonary nitrogen clearance in relation to age in healthy males. J Appl Physiol 1963;18:297–300.
Saidel GM, Salmon RB, Chester EH: Moment analysis of multibreath lung washout. J Appl Physiol 1975;38:328–334.
Cumming G, Jones JG: The construction and repeatability of lung nitrogen clearance curves. Respir Physiol 1966;1:238–248.
Shao H, Sandberg K, Hjalmarson O: Impaired gas mixing and low lung volume in preterm infants with mild chronic lung disease. Pediatr Res 1998;43:536–541.
Nye RE Jr: Theoretical limits to measurement of uneven ventilation. J Appl Physiol 1961;16:1115–1124.
Verbanck S, Paiva M: Model simulations of gas mixing and ventilation distribution in the human lung. J Appl Physiol 1990;69:2269–2279.
Dutrieue B, Vanholsbeeck F, Verbanck S, Paiva M: A human acinar structure for simulation of realistic alveolar plateau slopes. J Appl Physiol 2000;89:1859–1867.
Crawford AB, Makowska M, Paiva M, Engel LA: Convection- and diffusion-dependent ventilation maldistribution in normal subjects. J Appl Physiol 1985;59:838–846.
Engel LA: Gas mixing within the acinus of the lung. J Appl Physiol 1983;54:609–618.
Paiva M, Verbanck S, van Muylem A: Diffusion-dependent contribution to the slope of the alveolar plateau. Respir Physiol 1988;72:257–270.
Aurora P, Kozlowska W, Stocks J: Gas mixing efficiency from birth to adulthood measured by multiple-breath washout. Respir Physiol Neurobiol 2005;148:125–139.
Macleod KA, Horsley AR, Bell NJ, Greening AP, Innes JA, Cunningham S: Ventilation heterogeneity in well controlled asthmatic children, with normal spirometry, indicates residual airways disease. Thorax 2009;64:33–37.
Horsley AR, Macleod KA, Robson AG, Lenney J, Bell NJ, Cunningham S, Greening AP, Gustafsson PM, Innes JA: Effects of cystic fibrosis lung disease on gas mixing indices derived from alveolar slope analysis. Respir Physiol Neurobiol 2008;162:197–203.
Aljassim F, Robinson PD, Sigurs N, Gustafsson PM: A whisper from the silent lung zone. Pediatr Pulmonol 2009, in press.
Hankinson JL, Stocks J, Peslin R: Reproducibility of lung volume measurements. Eur Respir J 1998;11:787–790.
Schaanning CG, Gulsvik A: Accuracy and precision of helium dilution technique and body plethysmography in measuring lung volumes. Scand J Clin Lab Invest 1973;32:271–277.
Gustafsson PM, Johansson HJ, Dahlback GO: Pneumotachographic nitrogen washout method for measurement of the volume of trapped gas in the lungs. Pediatr Pulmonol 1994;17:258–268.
Gustafsson PM, Kallman S, Ljungberg H, Lindblad A: Method for assessment of volume of trapped gas in infants during multiple-breath inert gas washout. Pediatr Pulmonol 2003;35:42–49.
Gustafsson PM: Peripheral airway involvement in CF and asthma compared by inert gas washout. Pediatr Pulmonol 2007;42:168–176.
Downie SR, Salome CM, Verbanck S, Thompson B, Berend N, King GG: Ventilation heterogeneity is a major determinant of airway hyperresponsiveness in asthma, independent of airway inflammation. Thorax 2007;62:684–689.
Prisk GK, Lauzon AM, Verbanck S, Elliot AR, Guy HJ, Paiva M, West JB: Anomalous behavior of helium and sulfur hexafluoride during single-breath tests in sustained microgravity. J Appl Physiol 1996;80:1126–1132.
Bryan AC, Milic-Emili J, Pengelly D: Effect of gravity on the distribution of pulmonary ventilation. J Appl Physiol 1966;21:778–784.
Milic-Emili J, Henderson JA, Dolovich MB, Trop D, Kaneko K: Regional distribution of inspired gas in the lung. J Appl Physiol 1966;21:749–759.
Olson LE, Rodarte JR: Regional differences in expansion in excised dog lung lobes. J Appl Physiol 1984;57:1710–1714.
Wilson TA, Olson LE, Rodarte JR: Effect of variable parenchymal expansion on gas mixing. J Appl Physiol 1987;62:634–639.
Paiva M, van Muylem A, Ravez P, Yernault JC: Inspired volume dependence of the slope of the alveolar plateau. Respir Physiol 1984;56:309–325.
Van Muylem A, Baran D: Overall and peripheral inhomogeneity of ventilation in patients with stable cystic fibrosis. Pediatr Pulmonol 2000;30:3–9.
Saniie J, Saidel GM, Chester EH: Real-time moment analysis of pulmonary nitrogen washout. J Appl Physiol 1979;46:1184–1190.
Gustafsson PM, Ljungberg HK, Kjellman B: Peripheral airway involvement in asthma assessed by single-breath SF6 and He washout. Eur Respir J 2003;21:1033–1039.
Altes TA, Powers PL, Knight-Scott J, Rakes G, Platts-Mills TA, de Lange EE, Alford BA, Mugler JP 3rd, Brookeman JR: Hyperpolarized 3He MR lung ventilation imaging in asthmatics: preliminary findings. J Magn Reson Imaging 2001;13:378–384.
Samee S, Altes T, Powers P, de Lange EE, Knight-Scott J, Rakes G, Mugler JP 3rd, Ciambotti JM, Alford BA, Brookeman JR, Platts-Mills TA: Imaging the lungs in asthmatic patients by using hyperpolarized helium-3 magnetic resonance: assessment of response to methacholine and exercise challenge. J Allergy Clin Immunol 2003;111:1205–1211.
Venegas J: Linking ventilation heterogeneity and airway hyperresponsiveness in asthma. Thorax 2007;62:653–654.
Sonnappa S, Bastardo CM, McKenzie SA, Bush A, Aurora P: Conductive airways ventilation inhomogeneity is a frequent finding in preschool wheezers. Am J Respir Crit Care Med 2008;177:A701.
Bourdin A, Paganin F, Prefaut C, Kieseler D, Godard P, Chanez P: Nitrogen washout slope in poorly controlled asthma. Allergy 2006;61:85–89.
Verbanck S, Schuermans D, Noppen M, Van Muylem A, Paiva M, Vincken W: Evidence of acinar airway involvement in asthma. Am J Respir Crit Care Med 1999;159:1545–1550.
Payne DN, Rogers AV, Adelroth E, Bandi V, Guntupalli KK, Bush A, Jeffery PK: Early thickening of the reticular basement membrane in children with difficult asthma. Am J Respir Crit Care Med 2003;167:78–82.
Verbanck S, Schuermans D, Paiva M, Vincken W: The functional benefit of anti-inflammatory aerosols in the lung periphery. J Allergy Clin Immunol 2006;118:340–346.
Aurora P, Gustafsson P, Bush A, Lindblad A, Oliver C, Wallis CE, Stocks J: Multiple breath inert gas washout as a measure of ventilation distribution in children with cystic fibrosis. Thorax 2004;59:1068–1073.
Dakin CJ, Numa AH, Wang H, Morton JR, Vertzyas CC, Henry RL: Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 2002;165:904–910.
Brenner DJ, Hall EJ: Computed tomography an increasing source of radiation exposure. N Engl J Med 2007;357:2277–2284.
Gustafsson PM, De Jong PA, Tiddens HA, Lindblad A: Multiple-breath inert gas washout and spirometry versus structural lung disease in cystic fibrosis. Thorax 2008;63:129–134.
Kraemer R, Blum A, Schibler A, Ammann RA, Gallati S: Ventilation inhomogeneities in relation to standard lung function in patients with cystic fibrosis. Am J Respir Crit Care Med 2005;171:371–378.
Aljassim F, Lindblad A, Gustafsson PM: Long-term lung clearance index (LCI) monitoring in young children with CF. Eur Respir J 2007;30:33s.
Verbanck S, Schuermans D, Meysman M, Paiva M, Vincken W: Noninvasive assessment of airway alterations in smokers: the small airways revisited. Am J Respir Crit Care Med 2004;170:414–419.
Verbanck S, Schuermans D, Paiva M, Meysman M, Vincken W: Small airway function improvement after smoking cessation in smokers without airway obstruction. Am J Respir Crit Care Med 2006;174:853–857.
Verbanck S, Schuermans D, Van Muylem A, Melot C, Noppen M, Vincken W, Paiva M: Conductive and acinar lung-zone contributions to ventilation inhomogeneity in COPD. Am J Respir Crit Care Med 1998;157:1573–1577.
Verbanck S, Schuermans D, Vincken W: Small airways ventilation heterogeneity and hyperinflation in COPD: response to tiotropium bromide. Int J Chron Obstruct Pulmon Dis 2007;2:625–634.
Thompson BR, Lewis A, Raven J, Borg B, Abramson M: Acinar heterogeneity is detected in patinets with chronic bronchitis. Respirology 2008;13(suppl 2):A4.
Van Muylem A, Knoop C, Estenne M: Early detection of chronic pulmonary allograft dysfunction by exhaled biomarkers. Am J Respir Crit Care Med 2007;175:731–736.
Estenne M, Van Muylem A, Knoop C, Antoine M: Detection of obliterative bronchiolitis after lung transplantation by indexes of ventilation distribution. Am J Respir Crit Care Med 2000;162:1047–1051.
Van Muylem A, Paiva M, Estenne M: Involvement of peripheral airways during methacholine-induced bronchoconstriction after lung transplantation. Am J Respir Crit Care Med 2001;164:1200–1203.
Van Muylem A, Verbanck S, Estenne M: Monitoring the lung periphery of transplanted lungs. Respir Physiol Neurobiol 2005;148:141–151.
Schoeffel R, Reid C, Greenwood M, Downie S, King GG: Peripheral airway function declines following allogeneic bone marrow transplantation. Respirology 2008;13(suppl 2):A4.
Khalid Y, Mellgren K, Fasth A, Lindblad A, Gustafsson P: Obliterative bronchiolitis after stem cell transplantation: inert gas washout makes early diagnosis of this serious complication possible (in Swedish). Lakartidningen 2007;104:3373–3376.
Pillow JJ, Frerichs I, Stocks J: Lung function tests in neonates and infants with chronic lung disease: global and regional ventilation inhomogeneity. Pediatr Pulmonol 2006;41:105–121.
Hulskamp G, Pillow JJ, Dinger J, Stocks J: Lung function tests in neonates and infants with chronic lung disease of infancy: functional residual capacity. Pediatr Pulmonol 2006;41:1–22.
Wauer RR, Maurer T, Nowotny T, Schmalisch G: Assessment of functional residual capacity using nitrogen washout and plethysmographic techniques in infants with and without bronchopulmonary dysplasia. Intensive Care Med 1998;24:469–475.
Hulskamp G, Lum S, Stocks J, Wade A, Hoo AF, Costeloe K, Hawdon J, Deeptha K, Pillow JJ: Association of prematurity, lung disease and body size with lung volume and ventilation inhomogeneity in unsedated neonates: a multicentre study. Thorax 2009;64:240–245.
Kraemer R, Zehnder M, Meister B: Intrapulmonary gas distribution in healthy children. Respir Physiol 1986;65:127–137.
Orzalesi MM, Hart MC, Cook CD: Distribution of ventilation in normal subjects from 7 to 45 years of age. J Appl Physiol 1965;20:77–78.
Svenonius E, Kautto R: The volume of trapped gas in healthy children aged 7–16. Lung 1979;156:71–77.
Hutchison AA, Erben A, McLennan LA, Landau LI, Phelan PD: Intrasubject variability of pulmonary function testing in healthy children. Thorax 1981;36:370–377.
McCarthy DS, Craig DB, Cherniak RM: Intra-individual variability in maximum expiratory flow-volume and closing volume in asymptomatic subjects. Am Rev Respir Dis 1975;112:407–411.
Roberts CM, MacRae KD, Winning AJ, Adams L, Seed WA: Reference values and prediction equations for normal lung function in a non-smoking white urban population. Thorax 1991;46:643–650.
Sixt R, Bake B, Oxhoj H: The single-breath N2-test and spirometry in healthy non-smoking males. Eur J Respir Dis 1984;65:296–304.
Solymar L, Aronsson PH, Bake B, Bjure J: Nitrogen single breath test, flow-volume curves and spirometry in healthy children, 7–18 years of age. Eur J Respir Dis 1980;61:275–286.
Knudson RJ, Lebowitz MD, Burton AP, Knudson DE: The closing volume test: evaluation of nitrogen and bolus methods in a random population. Am Rev Respir Dis 1977;115:423–434.
Hedenstrom H, Malmberg P, Fridriksson HV: Reference values for lung function tests in men: regression equations with smoking variables. Ups J Med Sci 1986;91:299–310.
Hedenstrom H, Malmberg P, Agarwal K: Reference values for lung function tests in females. Regression equations with smoking variables. Bull Eur Physiopathol Respir 1985;21:551–557.
Teculescu DB, Varona-Lopez W, Bruant A, Pham QT, Locuty J, Deschamps JP: Determinants of alveolar nitrogen slope and closing volumes in healthy adolescents. Am Rev Respir Dis 1990;142:607–615.
King GG, Downie SR, Verbanck S, Thorpe CW, Berend N, Salome CM, Thompson B: Effects of methacholine on small airway function measured by forced oscillation technique and multiple breath nitrogen washout in normal subjects. Respir Physiol Neurobiol 2005;148:165–177.
Aurora P: Multiple-Breath Inert Gas Washout to Detect Inhomogeneity of Ventilation Distribution in Preschool Children with Cystic Fibrosis; PhD thesis, University College London, London, 2005.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.