gas mixing efficiency from birth to adulthood measured by multiple-breath washout

Respiratory Physiology & Neurobiology 148 (2005) 125–139

Gas mixing efficiency from birth to adulthoodmeasured by multiple-breath washout

Paul Auroraa,b,∗, Wanda Kozlowskaa, Janet Stocksa

a Portex Anaesthesia, Intensive Therapy and Respiratory Medicine Unit, Institute of Child Health, London, UKb Cardiorespiratory and Critical Care Division, Great Ormond Street Hospital for Children, London, UK

Accepted 26 May 2005

Abstract

Efficient mixing of inspired gas with the resident gas of the lung is an essential requirement of effective respiration. Thisreview focuses on one method for quantifying ventilation inhomogeneity: the multiple-breath inert gas washout (MBW).

MBW has been employed as a research tool in adults and school age children for more than 50 years. Modifications allowingdata collection in infants and preschoolers have been described recently. Indices of overall ventilation inhomogeneity, such asthe lung clearance index and moment ratios, are raised in many infants with lung disease of prematurity, and in young childrenwith cystic fibrosis. These indices may be more sensitive than other lung function measures for the early detection of airwaydisease. We describe, for the first time, a development of the MBW analysis that allows calculation of acinar and conductivezone inhomogeneity indices in spontaneously breathing children.

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Although methodological and analytical issues remain, the future clinical and research applications of MBW justify acesearch in this field.

2005 Elsevier B.V. All rights reserved.

eywords:MBW; Child; Child, preschool; Infant

. Introduction

Mixing of inspired gas with the resident gas ofhe lung is an essential requirement of effective res-iration. If gas mixing is inefficient or inhomoge-

∗ Corresponding author. Tel.: +44 20 7905 2382;ax: +44 20 7829 8634.E-mail address:[email protected] (P. Aurora).

neous, then an increase in minute ventilation is reqto deliver inspired gas to the alveoli and to efadequate gas exchange. The efficiency of gasing is dependent upon the architecture of the lparticularly of the peripheral airways; and uponative time constants of parallel lung units, whichturn are determined by heterogeneity of airway retance and lung compliance (Engel, 1983). Instrumentthat measure the efficiency of gas mixing in ch

569-9048/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.resp.2005.05.027

126 P. Aurora et al. / Respiratory Physiology & Neurobiology 148 (2005) 125–139

hood have enormous potential application, most obvi-ously for the early detection of lung disease, butalso to record the growth and development of thelungs.

Such instruments may now exist. Recent reportshave described the use of electrical impedance tomog-raphy (Frerichs, 2000), aerosol deposition studies(Darquenne and Prisk, 2004) and single-breath inertgas washout (Ljungberg and Gustafsson, 2003) forthe quantification of ventilation inhomogeneity. How-ever, none of these techniques have been extensivelyemployed in unsedated children to date. This reviewwill therefore focus on a fourth method: the multiple-breath inert gas washout (MBW).

Although initially described many years ago, MBWhas only been used intermittently to assess gas mix-ing efficiency or ventilation inhomogeneity in infantsand young children (Wall, 1985), possibly reflectingthe complexity of data analysis and the lack of com-mercially available equipment and software. Duringrecent years, technological advances combined withincreasing awareness that conventional measures ofairway function may not detect early changes in periph-eral airway function until lung disease is well estab-lished have led to a resurgence of interest in this field(Gustafsson et al., 2003). The MBW is applicableto subjects of all ages, including unsedated infants(Hjalmarson and Sandberg, 2002), as measurementsare performed during spontaneous tidal breathing. Avariety of indices have been reported from MBW.S mo-g ismsb ebyp el-o datac ren( 02a ithr ist dataf forf t isn unc-t dgei nge ter-p outc

2. Developmental respiratory physiology andanatomy

2.1. Prenatal lung development

The prenatal phase of life is a crucial period for thedevelopment of the bronchial tree and airspaces and it isnow recognized that a significant portion of respiratorysymptoms in childhood and adulthood can be attributedto lung development during pre and early postnatal life(Stick, 2000; Stocks and Hislop, 2002; von Mutius,2001). The lung appears as a ventral diverticulum ofthe foregut during the fourth week of gestation. By16 weeks of gestation, branching of this epitheliumlined lung bud into surrounding mesenchyme has pro-duced all pre-acinar airways seen in the adult lung,with parallel development of the pulmonary circula-tion. Intra-acinar airways are formed by further divisionup to 30 weeks of gestation. Although gas exchange canoccur from around 22 to 23 weeks gestation, true alve-oli do not appear until about 28–30 weeks. During thelast trimester of gestation, alveolarisation is extremelyrapid, such that approximately 100–150 million alve-oli (representing 30–50% of the adult complement) arepresent by term (i.e. 40 weeks gestation) (Hislop et al.,1986).

2.2. Postnatal lung development

Thus, at birth the lung has a small volume relatedt ith am areao gasb

llyc eolis -m 02H ur-i uents Inm inuet t hasb lthya thata ,2 ionsi kers.

ome of these describe overall ventilation inhoeneity, but others may describe specific mechany which inhomogeneity is generated, and therrovide detailed indirect information on lung devpment and pathology. Specific adaptations toollection methods are required for young childAurora et al., 2005; Hjalmarson and Sandberg, 20),nd a number of challenges remain, particularly wegard to data analysis. The aim of this reviewo describe these methods, summarise publishedrom studies in children and discuss prioritiesuture research. Prior to exploring this theme, iecessary to briefly consider both structural and f

ional aspects of lung development. Such knowles fundamental not only for lung modelling duriarly life but for the measurement, analysis and inretation of the efficiency of gas mixing throughhildhood.

o body surface area and all airways are present wature wall structure. There is sufficient surfacef alveoli for efficient gas exchange and the bloodarrier is the same thickness as in the adult.

Alveolarisation continues after birth and is usuaomplete by the age of 2–3 years, after which alvimply enlarge with childhood growth (ATS-ERS stateent, 2004; Hislop, 2002; Stocks and Hislop, 20).ence postnatal lung growth is particularly rapid d

ng the first 3 years of life, but there is also a subseqpurt during and immediately following puberty.ales, but not females, thoracic dimensions cont

o increase for several years after final adult heigheen attained (i.e. into the mid twenties). In headults, alveolar volume is relatively constant, solveolar number is proportional to lung size (Ochs et al.004). With senescence however, alveolar dimens

ncrease, an effect that is exaggerated in active smo

P. Aurora et al. / Respiratory Physiology & Neurobiology 148 (2005) 125–139 127

The lung at birth or in the first few years of lifeis not simply a miniature of the adult, as this alveolargrowth and development is of a greater magnitude thanairway – growth so called dysanaptic growth (Stocksand Hislop, 2002). Extensive reviews of factors influ-encing lung growth have been published (AmericanThoracic Society, 1991; ATS-ERS statement, 2004;Merkus, 2003; Stocks and Hislop, 2002). Further-more, although most parameters of respiratory functionremain remarkably constant when related to surfacearea or body size, the underlying factors determin-ing these parameters may vary considerably accordingto age and maturity of the individual, as discussedbelow.

2.3. Functional differences during early life

The relatively large surface area to body massratio and rapid somatic growth in infants are suchthat oxygen consumption and metabolic rate are rel-atively high when compared with those in adults. Theincreased minute ventilation in relation to body sizethat is required to meet this increased demand foroxygen in early life is achieved primarily through anincreased respiratory rate during the first months oflife, with weight corrected tidal volume remaining rel-atively constant (Fig. 1A and B). The rapid lung andsomatic growth that occurs during the first year of lifeis also accompanied by major developmental changes

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ig. 1. Relationships between functional residual capacity, tidal voluealthy children measured at the Institute of Child Health, London, beecruitment. Note the dramatic increase in functional residual capacit

me, respiratory rate and age in healthy children. Data collected from 91tween November 2000 and December 2004. See text for details on subjecty and tidal volume, and the fall in respiratory rate with age.


in respiratory physiology, particularly with respect tothe influence of the upper airways, the highly compli-ant chest wall and dynamic elevation of FRC (Stocks,2005; Stocks and Hislop, 2002).

Infants are preferential nose breathers with nasalresistance representing approximately 50% of total air-way resistance. Consequently, changes in lower air-way resistance with disease or therapeutic interventionsmay be masked. This makes lung function tests suchas the MBW, which are relatively independent of theupper airways, particularly attractive in this age group.At end expiration, intra-pleural pressure is consider-ably less negative in both the infant and the elderly thanin the young adult. In the elderly, this is due to emphyse-matous changes with loss of lung recoil, whereas in theinfant, although lung recoil is similar to that in an adult,the chest wall is extremely compliant, resulting in mini-mal outward recoil with which to keep the lungs and air-ways distended. This results in instability of FRC anda tendency for peripheral airway closure during tidalbreathing. The latter not only impairs gas exchangeand ventilation-perfusion balance, particularly in thedependent parts of the lung, but together with the smallabsolute size of the airways, renders the infant andyoung child particularly susceptible to airway obstruc-tion and wheezing disorders. The potential difficultiesimposed by the compliant chest wall are at least par-tially compensated by the tendency of infants to breathein before they reach the lung volume determined by thepassive mechanics of their respiratory system, therebyd di-t usel vityt wb nessm ys,i s, buti asticr sio-l pt-i lifeoa

od-o gc bee lop-m

3. MBW in children: apparatus and datacollection

3.1. Basic MBW apparatus and methodology

The open circuit multiple-breath washout techniquewas first introduced in the 1940s for measuring func-tional residual capacity (FRC) and for assessing over-all ventilation inhomogeneity. The methodology forperforming MBW in older children and adults hasbeen described in detail (Gustafsson et al., 2003), andmodifications that allow successful data collection ininfants (under 2 years) (Hjalmarson and Sandberg,2002; Schibler et al., 2000) and preschool children (2–5years inclusive) (Aurora et al., 2005) have also beendescribed recently.

In essence, MBW requires the subject to breathetidally, whilst washing a tracer gas from their lungs.The original form of this test was nitrogen (N2) MBW,where the resident N2 in the lung is diluted by 100%oxygen (O2). An alternative method is to use an inerttracer gas, such as helium (He), sulphur hexafluoride(SF6) or argon (Gustafsson et al., 2003). This inert gasis first “washed in” to the lungs, and then washed outusing air.

With their rapid respiratory rate and higher ratioof tidal volume/FRC (Fig. 1D), wash-in and washoutsare generally much faster in younger subjects. Bothphases of the technique are generally completed within1–2 min in healthy infants or preschool children andwe let thin2

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ynamically elevating the end expiratory level. In adion to changes in expiratory timing, infants oftenaryngeal and post inspiratory diaphragmatic actio slow (or brake) expiratory flow. In infants, a loaseline airway function or heightened responsiveay be due not only to anatomically small airwa

ncreased smooth muscle tone or excess secretionncreased airway wall thickness or decreased elecoil of both the chest wall and airways. These phyogical differences are highly significant when attemng to model the respiratory system during earlyr investigate structure–function relationships (Stocksnd Hislop, 2002).

The remainder of this review will describe methlogy for collecting and analyzing MBW in younhildren, and discuss how this technique couldmployed to answer important clinical and deveental questions.

ithin 5 min in those with airway disease (Aurorat al., 2005). Consequently, it is usually possib

o complete three technically successful runs wi0 min.

The apparatus required for performing SF6 MBWn a young child is displayed inFig. 2. This apparatus employed in the authors’ laboratory, and generhe data presented in this manuscript. Specifice use a size 0 (infants) or size 1 Fleisch pneumhometer (PNT), connected to a differential presransducer (Validyne, Model MP 45-14-871, Validyorp., CA, USA) to measure flow. This flow sign

s demodulated and amplified (Validyne MC1-alidyne Corp., CA, USA). Older children and adure required to wear a nose-clip and breathe thr

he PNT via a mouthpiece. Preschool childrennfants breathe via a facemask apparatus, whicpplied to the face using therapeutic putty to obtai


Fig. 2. An infant and a 3-year-old child performing the washin phase of multiple-breath washout. (A) The investigator is holding the facemaskapparatus to the child’s face with his left hand. This ensures an airtight seal, which must be maintained throughout the washin and washout.He is able to monitor flow and gas concentration on a computer screen (off picture). At the end of washin the bias flow is disconnected andthe washout phase commences. (B) The investigator is supporting the back of the child’s head with her right hand, and holding the facemaskapparatus to the child’s face with her left hand. The child is distracted by an entertainment video playing on a television screen off the left ofthe picture. A second investigator monitors flow and gas concentration. NB.Fig. 2A courtesy of Dr. Per Gustafsson.Fig. 2B reproduced fromAurora et al. (2005), with permission.

airtight seal. The mouthpiece/facemask is connectedto the PNT via a short connector, into which thesampling tube from a respiratory mass spectrometer(AMIS 2000, Innovision A/S, Odense, Denmark) isplaced. This mass spectrometer has a sampling rateof 20 mL min−1 and the gas concentration signals areupdated at a rate of 33.3 Hz. The system dead-space isseparated into two components (Aurora et al., 2005)namely:

(i) The pre-capillary dead-space is defined as thedead-space between the child’s lips and the cap-illary inlet, and is estimated as 12.5 mL for thepreschool facemask system, 7.5 or 10 mL, depen-dant on mask size, for the infant facemask system,and 5 mL for the mouthpiece system.

(ii) The post-capillary dead-space is defined as thedead-space between the capillary inlet and the endof the expiratory port of the PNT, and is measuredas 15 mL for both the preschool and mouthpieceapparatus, and 5 mL for the infant apparatus.

Each SF6 MBW consists of two phases. During thewash-in phase the subject inspires a dry gas mixturecontaining a low concentration of SF6, 21% O2 andbalance N2. In some cases this mixture may contain

low concentrations of other inert gases, such as He (seebelow). The gas is provided via a bias flow apparatus,with flow set at a level greater than the maximum inspi-ratory flow produced by the subject, so that rebreathingdoes not occur. Wash-in is continued until the inspira-tory and expiratory SF6 concentrations are stable andequal. At this moment the bias flow is stopped dur-ing expiration, either by disconnecting the bias flowmanually, or by switching a valve, and washout isstarted. The washout phase continues until the end-tidal SF6 concentration is below 1/40th of the startingconcentration.

The output from the washout phase of a SF6 MBWis presented inFig. 3. This recording was taken froma preschool child in the authors’ laboratory. The fig-ure displays flow and gas concentration plotted againsttime, and shows an exponential decay in end-tidalgas concentration. This is termed thewashout curve.Fig. 4displays tracer gas concentration plotted againstexpired volume, from a single-breath of an MBW (cor-responding to breath 3 inFig. 3). The third phase ofthis plot (Kjellmer et al., 1959; Paiva and Engel, 1981),termed the alveolar plateau, or the phase III slope, alsoprovides an important measure of ventilation inhomo-geneity (see below).


Fig. 3. Washout curve. The black trace represents flow (left axis). The green trace represents SF6 concentration (right axis). A is the end ofthe wash-in, where SF6 concentration during inspiration and expiration is identical, and equal to the SF6 concentration in the test gas cylinder.The bias flow is disconnected at point B, and from this point the SF6 concentration during inspiration is zero, as the child is inhaling air. TheSF6 concentration on expiration falls with each breath (C), as the resident gas of the lung is progressively diluted by the inspired air. Figurereproduced fromAurora et al. (2005), with permission.

3.2. Alternative apparatus for MBWmeasurement: advantages and disadvantages

The choice of tracer gas may be important, partic-ularly in young children (Pillow et al., in press). Most

Fig. 4. Phase III slope fitted over 65–95% of expired volume. PhaseIII slope in a healthy 3-year-old child. The black trace is tracer gasconcentration plotted against expired volume. The blue line repre-sents least-square regression over the interval 65–95% of expiredvolume. The red line is continuation of the regression line. The slopeof the regression, i.e. the slope of phase III, is 0.28 L−1.

of the early studies (Bolton, 1979) and some publishedmore recently (Hjalmarson and Sandberg, 2005; Krae-mer et al., 2004) have utilized N2 MBW. Advantages ofN2 MBW are that N2 analysers are relatively inexpen-sive, as is the washout gas (100% O2). Disadvantagesrelate to the effect of 100% O2 upon breathing pat-tern in young children, the potential toxicity of 100%O2 (from free radical formation and from induction ofatelectasis), and the difficulty of analysing N2 washoutin subjects who are dependent on supplemental O2 foradequate oxygenation. The most commonly employedalternatives are He and SF6 (Ostlund et al., 1992).

In addition to choice of tracer gas, alternative flowmeters and gas analysers are available. Particularly inyoung children, minimization of apparatus deadspaceis essential. Some investigators have used nasal masks(Watts et al., 1977) or placed the flowmeter in thebias flow (Hjalmarson and Sandberg, 2002) to mini-mize equipment deadspace. Tracer gas detection haspredominantly included infrared N2 and SF6 analysersin addition to mass spectrometry. Mass spectrometryhas the advantage of online analysis of multiple tracergases, allowing simultaneous measurement of gasesof differing diffusivities. Like the infrared analyser


(Jonmarker et al., 1985), physical separation of pointof measurement of the tracer gas and flow necessitatescorrection factors for delay and flow side sampling,whilst low sampling frequency may limit accuracy ofmeasurements in infants with high respiratory rates.

Analysis of MBW may be complex, and mostresearch laboratories use customized software pro-grammes specifically tailored to their hardwareconfigurations and research needs. More recently, acommercially available ultrasonic flowmeter (USFM)offers the possibility of MBW measurement by mea-suring flow and tracer gas molar mass (Pillow et al.,2004). The advantages of this system are mainstreamtracer gas measurement, and commercial software,which potentially facilitates collaboration betweenlaboratories. A major limitation of the system is that itcurrently does not allow calculation of phase III slopeparameters (see below).

3.3. Specific challenges for MBW measurement inyoung children

The majority of lung function tests employed inadults and school age children (those aged 6–16 years)require a large measure of co-operation, and a degree ofco-ordination. Neither co-operation nor co-ordinationis readily available when studying younger children.In infants and in very young children up to the ageof 2 years this problem can be overcome by perform-ing measurements when the child is asleep. It is thenp tidalb tus,m nts ofl ea-sI on,p bed wom entsc ea-s hing,i bym iqued s-s . Form tion,s red.I ld to

perform the appropriate manoeuvres by incorporatingthem in play (Aurora et al., 2004a; Stocks, 2005).

These modifications to data collection may impactupon the results obtained. For MBW in both infants andpreschool children a facemask apparatus is employed,rather than a mouthpiece and nose-clip. This modifica-tion increases the apparatus dead-space, and providesa further compartment in which incomplete gas mix-ing may occur. At present there are no published dataexamining the impact of the subject–apparatus inter-face upon MBW indices. However, as in older sub-jects, infants compensate for increases in dead-spaceand resistance by alterations in breathing pattern, mostcommonly by increases in tidal volume. In addition,whilst measurements in preschool children are madewith subject in the upright, seated position, measure-ments in infants are made during sleep, with the infantin the supine position. Studies in adults have demon-strated that supine posture results in elevation of thediaphragm, and alters the effect of gravity upon ven-tilation distribution (Gronkvist et al., 2002). However,the anatomy of the infant thorax differs from that ofthe adult (Stocks and Hislop, 2002) and it cannot beassumed that supine posture will have the same effect.

4. Analytical issues

4.1. Washout curve analysis

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ossible to take measurements that only requirereathing (such as MBW). With specialised apparaeasurements of airway resistance, measureme

ung volumes by body plethysmography, and even mures of forced expiration are possible (Stocks, 2005).n pre-school children daytime sleep is uncommarticularly in a laboratory environment, and mayifficult to obtain even with sedation. There are tain methods by which lung function measurem

an therefore be obtained in this age group. For murements that can be obtained during tidal breatt may be sufficient to distract the child, for example

usic or an entertainment video. This is the technemonstrated inFig. 2. In this situation the child is paive whilst the investigator obtains measurementseasurements that require a degree of co-opera

uch as spirometry, some form of incentive is requin essence the investigator encourages the chi

A number of indices to describe the washout cuave been proposed (Larsson et al., 1988), the mosommonly reported of which are the lung clearandex (LCI), the mixing ratio (MR) and moment ratio

The lung clearance index (LCI) is the numberung volume turnovers required to reduce end tidaoncentration to 1/40th of starting value

O = CEV

FRC

here TO, turnovers; CEV, cumulative expired vme; FRC, functional residual capacity.

The mixing ratio (MR) is the ratio between tbserved and the predicted number of breaths req

o reduce the end tidal tracer concentration to 1/40tarting value, where the predicted number of bres that which would be needed to complete the wasn ideal mixing conditions.


Moment ratios represent the ratio between the firstand zeroth moment of the washout (M1/M0) or the ratiobetween the second and zeroth moment of the washout(M2/M0).

WhereM0 is the area under the gas concentrationversus TO plot;M1 is the area under the gas concen-tration× TO versus TO plot; M2 is the area under thegas concentration× * TO2 versus TO plot.

A large number of other indices have been reported,but there have been very few studies comparing themdirectly to determine which gives the most informa-tion. From the limited published data available, LCI,MR and moment ratios appear to have similar ability todiscriminate between health and disease states. In addi-tion, unpublished data from our own laboratory haveshown that the LCI is relatively unaffected by breath-ing pattern (tidal volume, FRC and respiratory rate) inpreschool and school age children.

4.2. The progression of the phase III slope duringMBW

Diverse alterations in anatomy or lung function canlead to similar alterations of the washout curve. Inparticular, washout curves do not provide informa-tion about the mechanisms involved in the produc-tion of ventilation inhomogeneity, or the magnitude oflung units involved. However, it has been suggested(Crawford et al., 1985; Engel, 1983; Paiva and Engel,1981; Paiva et al., 1982; Verbanck et al., 1998) that thei inedf oc II isn res-s ght tioni ech-a ho-m enti et-i idedp ivaa al.,1 rlyt toc thya lessw the

Fig. 5. The contribution of CDI and DCDI to overall ventilationinhomogeneity. According to the Paiva–Engel model, CDI has a lin-ear relationship with expired volume (whether expressed as breathnumber or TO). DCDI rises for the first five breaths, or 1.5 turnovers,and then is unchanged. These characteristics allow separation of theSnIII vs. breath number (or SnIII vs. TO plot) into the two com-ponents. TheScond index is calculated as the slope of the SnIII vs.turnover CDI relationship.Sacin is the DCDI component of the firstbreath SnIII, calculated by subtracting the CDI component (see text).

fifth breath (or 1.5 TO). The relationship between SnIIIand TO from 1.5 TO upwards therefore describes CDI,and subtracting this from the overall inhomogeneitymeasured gives an index of DCDI (Fig. 5). Verbancket al. (1998)have recently refined these two indices,describing the slope of the linear portion of the SnIIIversus turnover relationship (due to CDI) asScond(rep-resenting gas transport in the conducting airways, anddefining Sacin (an index of DCDI, representing gastransport at the mouth of the acinus) as

Sacin = SIIIB1 − ScondTOB1

where SIIIB1 is the total phase III slope of first breath;TOB1 the TO corresponding to the first exhalation.

4.3. Specific challenges when calculating Sacin

and Scond in children

TheSacinandScondanalysis is developed from mod-elling and experimental studies performed in adults.Some assumptions of these analyses may be impracti-cal or invalid when analysing MBW from young chil-dren. Most importantly, in the previous studies of adultsubjects from which these indices were reported, sub-jects were required to maintain a fixed tidal volume

dentification of specific mechanisms could be obtarom the MBW if the phase III slope (SIII) is alsomputed for each breath. In this analysis, the SIormalized for tracer gas concentration, and progion of this normalized phase III slope (SnIII) throuhe course of a MBW allows the observed ventilanhomogeneity to be attributed to two separate mnisms, originally termed convection dependent inogeneity (CDI) and diffusion–convection depend

nhomogeneity (DCDI). Full description of the theorcal basis of these two mechanisms has been provreviously (Crawford et al., 1985; Engel, 1983; Pand Engel, 1981; Paiva et al., 1982; Verbanck et998). Briefly, CDI is predicted to increase linea

hrough the course of a MBW. DCDI is predictedontribute greatly to overall inhomogeneity in healdults at the beginning of a MBW, but increaseith subsequent breaths, reaching asymptote by


during MBW (Crawford et al., 1985; Verbanck et al.,1998). This volume was most commonly defined as1.0 L. During pilot studies in the authors’ laboratory itwas noted that young children rarely maintain the sametidal volume throughout MBW. Two issues related tothis were identified. First, if tidal volume falls below acertain minimum, it will not be possible to identify aphase III slope. Second, even if a phase III slope canbe identified, the value may be influenced by changesin tidal volume in a non-linear way.

For this reason, quality control criteria in ourown laboratory stipulate a minimum expired vol-ume for SIII to be reported, where minimum breathsize = (3.5× body weight (kg) + precapillary dead-space)×2 (mL).

The rationale for this total breath volume is thatairway dead-space can be estimated at 2 mL/kg body-weight, and a minimum phase III volume of 3 mL/kgbodyweight is required to obtain an adequate regres-sion. The phase III starting point is set at double the air-way and instrument dead-space. It is accepted that thisfigure is arbitrary, but it is argued that some minimumregression volume must be set. Individual SIII datapoints that were inadequate, whether because of inad-equate breath volume or signal noise, were removedfrom the SnIII versus TO plot beforeScond andSacincalculation.Fig. 6 shows the SnIII versus TO plot ofthe washout inFig. 3 prior to deletion of inaccurateSIII data points. By applying this criterion it has been

F

possible for us to calculateSacin andScond from MBWperformed in healthy preschool and school age children(results presented below). It has not yet been possibleto perform these analyses routinely in infants, in whomthe phase III may be more difficult to identify, particu-larly in the presence of rapid shallow breathing.

5. MBW in healthy subjects from birth toadulthood

Data presented in this section were collected in ourown laboratory between November 2000 and Decem-ber 2004. Details of recruitment of healthy subjects,data collection and analysis have been described indetail previously (Aurora et al., 2004b, 2005).

5.1. Washout curve analysis

Ninety-one healthy children (46 [51%] boys) com-pleted MBW. Their median age was 4.9 years (range0.4–16.8 years) and height and weight was appropriatefor age (Freeman et al., 1995). Fourteen (15%) wereinfants and 43 (47%) were of preschool age. The groupmean LCI was 6.8 (S.D. 0.6), and there was a weaknegative relationship between LCI and age (r2 = 0.11;p< 0.001;Fig. 7). The mean LCI for the infants was7.2 (S.D. 0.6), for the preschool children 6.8 (S.D.0.4) and for the school age children 6.4 (S.D. 0.5). Bymultivariate regression analysis, including age, height,w botha ela-t tot wasm onlye yc

5

ngc herea ticalia in3 lderc as an age
ig. 6. SnIII plotted against TO for washout curve presented inFig. 3.
eight, sex and age group as predictor variables,ge (positive relationship) and height (negative r

ionship) made small but significant contributionshe model fit. However, the greatest contributionade by the constant, and the multivariate model

xplained 35% of the variability of LCI in healthhildren.

.2. SnIII analysis

Results from SnIII analysis in infants and youhildren under 2 years of age will not be presenteds we are still resolving methodological and analy

ssues in these small subjects. First breath SnIII,ScondndSacin could be reported from three MBW runs4/43 (79%) preschool children and 29/34 (85%) ohildren in whom the test was attempted. There wegative relationship between first breath SnIII and


Fig. 7. Lung clearance index plotted against age in healthy chil-dren and children with cystic fibrosis from birth to 16 years. Brokenlines represent cut-offs between infants and preschool children, andpreschool and school age children. There are differences in data col-lection methodology between these age groups. Relationships arepresented in the text. Healthy children represented by open circles,children with CF by crosses. The dotted line represents the upper limitof normality for LCI, calculated as mean + 1.96S.D. The differencein LCI between healthy and CF groups dwarfs the between-subjectvariability of LCI within the healthy population. On close inspectionit is also noted that there is a positive relationship between LCI andage in children with CF. This is not immediately obvious because thenumber of subjects in the preschool range is so much greater than thatin the school-age range. In healthy preschool and school age chil-dren there is no relationship between LCI and age (r2 = 0.03,p= 0.2,andr2 = 0.00,p= 0.8, respectively) though the preschool group hada slightly higher mean LCI than the school-age group. A markednegative age relationship was seen in the infant group (r2 = 0.45,p= 0.003).

(r2 = 0.22,p< 0.001). By multivariate regression anal-ysis, including the same predictor variables as for LCI,43% of the variability of SnIII was explained by subjectcharacteristics. However, a strong hyperbolic relation-ship between first breath SnIII and expired volumewas noted. When the product of first breath SnIII andexpired volume, here termed first breath SnIIIcorr, wasplotted against age, no relationship was seen (r2 = 0.01,p= 0.1). Furthermore, by multiple regression analysis,none of the five explanatory variables was a significantpredictor of SnIIIcorr, either independently or in com-bination (r2 for multivariate model 0.02,p 0.3). Analternative volume correction was also performed bycalculating the product of first breath SnIII and FRC.This index was not completely independent of sub-ject characteristics, being positively correlated with age(r2 = 0.07,p= 0.01). These analyses were repeated inthe preschool and school age groups separately. Similarresults were obtained.

By similar analyses, it was possible to calculateexpired volume corrected indices fromSacin (Sacin,corr)andScond (Scond,corr). BothSacin,corrandScond,corrwerefound to be virtually age independent in healthy chil-dren aged from 2 to 16 years (Figs. 8 and 9).

5.3. Interpretation of results

5.3.1. LCI analysisThe majority of lung function measures are highly

dependent on age and body size in healthy subjects(c ool

Fig. 8. (A)Scond and (B)Sacin plotted against age in healthy children agp= 0.009.Sacin vs. age:r2 = 0.27,p< 0.01).

American Thoracic Society, 1991; Stocks, 2005). Byontrast, the LCI is relatively constant from presch

ed 2–16 years. Negative relationships are seen (Scond vs. age:r2 = 0.11,


Fig. 9. (A)Scond,corrand (B)Sacin,corrplotted against age in healthy children aged 2–16 years.Scondcorris calculated as the product ofScond andexpired volume.Sacin,corris calculated as the product ofSacin and expired volume. There is no relationship betweenScond,corr(r2 = 0.05,p= 0.09)orSacin,corr(r2 = 0.01,p= 0.59) and age (see text).

to school age years and is only slightly higher in infants.As discussed below, the variation in LCI within thehealthy population across the age groups is minor whencompared to differences in LCI between healthy chil-dren and those with cystic fibrosis (CF) (Fig. 7).

5.3.2. SnIII analysisWhilst the negative relationships between SnIII and

age, and SnIII and lung volume (Ljungberg, 2004) havebeen described previously, they have not been ques-tioned. The rationale for volume correction of phaseIII indices is based upon the hyperbolic relationshipbetween SnIII and expired volume, and the observa-tion that indices derived from the washout curve, suchas the LCI, are virtually age independent in health, atleast in the 2–16 year age range. Volume correction ofSnIII offers the possibility to study relationships withage and with other lung function parameters in chil-dren with lung disease, without the need for regressionequations and calculation ofz-scores. This has not beenpossible previously.

6. Clinical value of MBW analyses in children

6.1. MBW in infants

There are almost 20 published studies of MBWin infancy (Pillow et al., in press). All have reportedindices (LCI or MR) from the washout curve. Theseh in

infants when compared with adults; ventilation inho-mogeneity on day 1 postpartum, which subsequentlyimproves (possibly related to retention of lung fluid);and increased inhomogeneity in infants born pretermand in those with respiratory distress syndrome, whencompared to healthy newborns. Intervention studieshave reported improvements in ventilation inhomo-geneity after administration of surfactant to ventilatedpreterm infants (Pillow et al., in press).

There is need for caution when interpreting suchstudies in ventilated infants. A normal result fromwashout curve analysis does not necessarily imply nor-mal lung function, as several authors have reported sin-gle compartment ventilation patterns in subjects whohave clear signs of respiratory distress. A normal LCIin combination with a low FRC, for example, may indi-cate collapse of part of the lung, or widespread closureof peripheral airways, associated with gas trapping.Provided the remaining lung ventilates homogenously,the washout curve can appear normal. Alternatively,a study protocol that performs MBW after manoeu-vres that recruit lung volume (such as inflation to totallung capacity), may transiently normalize an inhomo-geneously ventilated lung and impair the ability to dis-criminate between healthy and diseased groups (Pillowet al., in press).

In addition, interpretation of MBW in apparentlyhealthy newborns, and those comparing infants withlung disease to healthy control groups are often com-plicated by the choice of indices. Whilst some washoutc tors
ave described relatively inefficient gas mixing urve indices (such as MR) include correction fac


for airway dead-space, others (such as LCI) do not(Larsson et al., 1988). In older children and adults, littledifference in repeatability and variability between com-pensated and uncompensated indices is seen. However,breathing pattern in healthy newborn infants is far morevariable than in older infants, and it has been suggestedthat compensated indices allow better discriminationbetween health and disease in this population (Pillowet al., in press).

6.2. MBW in preschool children

There have been relatively few published studies ofMBW involving pre-school children. All have reportedindices from washout curve analysis rather than fromSnIII analysis.Couriel et al. (1985)reported MBWin 58 healthy children and 24 children with CF aged3.9–6.8 years. The test was performed using a mouth-piece and nose-clip apparatus, and the authors reportedgreat difficulty in collecting adequate measurements inthis age group. Acceptable results were obtained in only40 children (49%) of whom only 10 were aged less than5 years (29% of the 34 children in this age group whoattempted the test). Moment ratios obtained in childrenwith CF were significantly higher than those obtainedin controls, and moment ratios obtained in childrenwith CF who had evidence of severe lung disease weresignificantly higher than those obtained in childrenwith fewer symptoms. In the same year,Wall (1985)reported measurements in 36 healthy children and 10c iecea tudyt ablem m thiss nifi-c andt nM CF.I latedt oulda en.G -e en.E , andu piecea omemM th

CF had normal spirometry results, but many of thesehad abnormal LCI or MR. Finally, in 2004, our ownlaboratory reported results from MBW, spirometry andbody plethysmography in 30 preschool children withCF and 30 matched controls (Aurora et al., 2005). Suc-cess rate for MBW on first attempt was 79%. Abnormalresults in children with CF were detected in 22/30(73%) children by MBW, 14/30 (47%) by plethysmog-raphy but only 4/30 (13%) by spirometry.

6.3. MBW studies in older children

In addition to the above, several studies have inves-tigated ventilation distribution in school age and adultCF subjects using this or related methods (Aurora etal., 2004b; Gozal et al., 1993; Habib and Lutchen,1991; Kraemer et al., 2004). These studies have shownthat indices of ventilation inhomogeneity are raisedin CF subjects compared with healthy subjects, thatMBW may be abnormal in these subjects even whenspirometry results are normal, and that ventilation inho-mogeneity is correlated to airway resistance and FEV1in CF subjects.

7. The future: challenges and opportunities

It is clear that MBW holds significant promise asan objective tool for the individual patient, as well asfor clinical trials seeking to document the impact oft er, an

7

ool,a ncesi antr t, andt stureh ,2 hil-d n thep ece-n ld bep lderc ourl

hildren with CF aged 3–6 years. Again, a mouthpnd nose-clip apparatus was employed, but in this s

he children were distracted by means of a portusic system and headphones. Success rates fro

tudy were not presented. Children with CF had sigantly higher moment ratios than healthy children,here was a negative relationship (r2 = 0.49) betwee

1/M0 and Shwachman score in the children withn the discussion of these results, the author specuhat use of a facemask system, sealed with putty, wllow MBW measurement in even younger childrustafsson et al. (2003)reported MBW and spiromtry in 43 children with CF and 28 healthy childright of these children were aged 6 years or lesssed a facemask apparatus rather than the mouthnd nose-clip used by the older children. Main outceasures were MR and LCI from MBW, and FEV1 andEF25 from spirometry. The majority of children wi

reatments on functional disease severity. Howevumber of important research questions remain.

.1. Methodological issues

Comparison of results between infant, preschnd school age groups is complicated by differe

n data collection methodology. The most importelate to subject body posture during measuremenhe subject–apparatus interface. The effect of poas already been studied in adults (Gronkvist et al.002). Ideally, this study should be repeated in cren, but this presents a considerable challenge ireschool age group. Comparison of a mouthpioseclip apparatus with a facemask apparatus couerformed during a controlled breathing study in ohildren and adults, and this study is ongoing inaboratory.


Whilst adults and older school-age children can per-form MBW during controlled breathing, this is notpossible for younger children. Published studies exam-ining the effect of changes inVT and FRC upon washoutcurve indices (such as LCI and MR) have investigatedalterations that are beyond the boundaries of physiolog-ical variation. Preliminary analysis of data collected inour own laboratory suggests that changes in breathingpattern have little effect upon LCI. Ideally this analysisshould be followed up by a series of controlled studies.

7.2. Normal lung development

Fig. 7 demonstrates small differences in LCIbetween age groups in health, with young infantsappearing to produce the highest values. Is this phe-nomenon related to the relatively large apparatus andairway deadspace relative to lung volume in infants,or is it demonstrating a decrease in ventilation inho-mogeneity over the first few months of life? In otherwords, is this pattern a methodological artefact, or isit providing new information on lung development?Using the slightly different method of comparing SF6and He slopes from SBW,Van Muylem et al. (1996)obtained data consistent with an increase in branch-ing asymmetry of the acinus throughout childhood andadolescence. However, SBW is impractical in infantsand younger children, and Van Muylem’s study com-menced from mid school age. Could MBW provideinformation on lung development earlier in life?

7

them lthyc fer-ei chil-d s ares m-e biba ,i withC CF(e rmalM ase,r cted

to future prognosis. There is no gold standard for theidentification of CF lung disease, so the only way thisquestion can be answered is by longitudinal studies,tracking MBW changes in individuals, and interventionstudies, assessing the effect of treatment upon MBWindices. Prior to such studies, a better understandingof between occasion variability of MBW indices isnecessary.

There have been few studies of MBW in childrenwith asthma, but these have demonstrated that MBWindices correlate with spirometry indices (Wall et al.,1987), and that ventilation distribution improves afterbronchodilation (Lutchen et al., 1990). In addition, oneadult lung transplant center has reported that SBW isable to detect post-transplant obliterative bronchiolitisearlier than serial spirometry (Estenne et al., 2000).There is potential for using MBW to track lung functionin young children post lung transplantation, though thenecessarily small numbers of subjects in such a studywould complicate analysis.

8. Conclusion

As described at the beginning of this review, aninstrument to measure the efficiency of gas mixingin childhood has enormous potential application, mostobviously for the early detection of lung disease, butalso to record the growth and development of the lungs.MBW is potentially such an instrument. Althoughi ain,t Wj

A

sis-t rt inter-p ers,M oo,D eP fora d int iket eses

.3. Could these tests be of clinical value?

An important note from these analyses is thatagnitude of differences in LCI seen between hea

hildren and those with CF far outweighs the difnces seen within healthy populations (Fig. 7). Studies

n adults, school age children and now preschoolren have demonstrated that abnormal LCI resulteen in subjects with CF who still have normal spirotry (Aurora et al., 2005; Gustafsson et al., 2003; Hand Lutchen, 1991; Kraemer et al., 2004). Furthermore

t has been demonstrated that school-age childrenF have higher LCI than preschool children with

Aurora et al., 2004b; Gustafsson et al., 2003). How-ver, it has not yet been demonstrated that abnoBW represents early changes of CF lung dise

ather than an epiphenomenon, which is unconne

mportant methodological and analytical issues remhe future clinical and research applications of MBustify accelerated research in this field.

cknowledgements

The authors would like to acknowledge the asance of Dr. Per Gustafsson, Goteborg, Sweden, foechnical assistance and advice regarding dataretation and Ms. Cara Oliver, Ms. Clare Saunds. Emma Scrase, Dr. Sooky Lum, Ms. Ah Fong Hr. Henrik Ljungberg, Dr. Georg Hulskamp, Dr. Janillow, Dr. Anders Lindblad and Dr. P.J. Subbaraossistance in collecting the original data presente

his paper. Finally, and most importantly, we would lo thank the children and families who took part in thtudies.


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