Noninvasive Assessment of Airway Alterations in Smokers The Small Airways Revisited

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1 Noninvasive Assessment of Airway Alterations in Smokers The Small Airways Revisited Sylvia Verbanck, Daniël Schuermans, Marc Meysman, Manuel Paiva, and Walter Vincken Respiratory Division, Academic Hospital, Vrije Universiteit Brussel; and Biomedical Physics Laboratory, Université Libre de Bruxelles, Brussels, Belgium It has been shown that structural changes in small airways of smokers with average smoking histories greater than 35 pack-years could be reflected in the single-breath washout test. The more sophisticated multiple breath washout test (MBW) has the potential to anatomically locate the affected small airways in acinar and conductive lung zones through increased phase III slope indices S acin and S cond, respectively. Pulmonary function, S acin, and S cond were obtained in 63 normal never-smokers and in 169 smokers classified according to smoking history ( 10 pack-years; pack-years; packyears; 30 pack-years). Compared with never-smokers, significant changes in S acin (p 0.02), S cond (p 0.001), and diffusing capacity (DL CO ;p 0.001) were detected from greater than 10 pack-years onwards. Spirometric abnormality was significant only from greater than 20 pack-years onwards. In smokers with greater than 30 packyears and DL CO less than 60% predicted, the presence of emphysema resulted in disproportionally larger S acin than S cond increases. We conclude that S cond and S acin can noninvasively detect airway changes from as early as 10 pack-years onwards, locating the earliest manifestations of smoking-induced small airways alterations around the acinar entrance. In these early stages, the associated DL CO decrease may be a reflection of ventilation heterogeneity rather than true parenchymal destruction. In more advanced stages of smokinginduced lung disease, differential patterns of S acin and S cond are characteristic of the presence of parenchymal destruction in addition to peripheral airways alterations. Keywords: lung periphery; multiple-breath washout; phase III slope; ventilation distribution The study of Cosio and colleagues (1) is a landmark in the demonstration of a relationship between structure of the peripheral lung and noninvasive measurements of pulmonary function. In particular, the 34 smokers in that study were classified in four groups (I to IV) according to a total pathology score of the small airways ( 2 mm internal diameter), including inflammatory cell infiltrate, squamous cell metaplasia of the airway epithelium, and airway wall fibrosis. Increasing total pathology severity scores were accompanied by increasing ventilation heterogeneity as measured from the N 2 phase III slope of the VC single-breath washout test (SBW VC ). Group I (normal pathology score and an average smoking history of 17 pack-years) had normal spirometry and normal N 2 phase III slopes, whereas group II (abnormal (Received in original form January 9, 2004; accepted in final form May 3, 2004) Supported by the Fund for Scientific Research Flanders, and the Federal Office for Scientific Affairs (program PRODEX), Belgium. Correspondence and requests for reprints should be addressed to Sylvia Verbanck, AZ-VUB, Consultatie Pneumologie, Laarbeeklaan 101, 1090 Brussels, Belgium. sylvia.verbanck@az.vub.ac.be This article has an online supplement, which is accessible from this issue s table of contents online at Am J Respir Crit Care Med Vol 170. pp , 2004 Originally Published in Press as DOI: /rccm OC on May 6, 2004 Internet address: pathology score and an average smoking history of approximately 40 pack-years) still had normal spirometry but abnormally high N 2 phase III slopes; groups III and IV (with increasing pathology scores but comparable smoking history) had both abnormal spirometry and abnormal N 2 phase III slopes. It was concluded that a test of unevenness of ventilation can detect structural change in the small airways of smokers long before spirometry can, and possibly at a time when structural changes are still potentially reversible. Several decades of ventilation distribution studies have identified potential contributors to the N 2 phase III slope of a SBW VC other than the small airways, such as gravity (2), or airway closure in the first portion of a SBW VC inspiration (3). Nevertheless, the Cosio study (1) continues to be misinterpreted to mean that any increase in the N 2 phase III slope of the SBW VC is a demonstration of small airways alterations. In the context of smoking-induced lung disease, the early SBW VC studies had led to deceiving results in predicting decline in FEV 1 (4, 5), and the SBW VC test has been largely abandoned as a tool to monitor the small airways, except for one 13-year follow-up study (6). With our present understanding of washout tests, the N 2 phase III slope analysis of the multiple-breath washout (MBW) (7, 8) has several major advantages with respect to the SBW VC :(1) it is hardly affected by gravity (9) and, therefore, better suited to represent intrinsic airway structure than the SBW VC ;(2)itisnot affected by airway closure below functional residual capacity (FRC), which is known to also affect the SBW VC phase III slope in a complex fashion (3); and (3) it can distinguish between proximal and peripheral origins of ventilation heterogeneity, which is impossible with a SBW unless tracer gases of different diffusivities are used (10). These advantages probably contribute to the ability of the MBW test to indeed identify early structural changes, as this study will show, even in smokers with a smoking history of as little as 10 pack-years. Finally, in the particular case of monitoring the smoker s lung, which potentially involves a huge number of small airways (i.e., all airways with diameters 2 mm), the classification of airway deterioration in conductive and/or acinar lung compartments is a valuable distinction to make. The purpose of the present work is to revive the interest of using noninvasive tests of ventilation distribution in the original context of the study of small airways in the smoker s lung by applying a state-of-the-art MBW ventilation distribution technique that can be used to identify conductive and acinar ventilation defects, as has been recently done in a number of specific clinical settings (11 14). METHODS Lung function and MBW testing were performed in a standard fashion (see online supplement). Lung function indices included FEV 1, PEF, FVC, mean forced mid-expiratory flow (FEF ), forced expiratory flow after exhalation of 75% FVC (FEF 75 ), single-breath carbon monoxide transfer factor (Dl CO ), plethysmographic measurement of lung volume

2 Verbanck, Schuermans, Meysman, et al.: Small Airways in Smokers 415 at functional residual capacity (FRC pl ), and specific airway conductance (sgaw). From MBW testing, we obtained indices of conductive (S cond ) and acinar (S acin ) ventilation heterogeneity, and ventilated FRC (FRC MBW ). The theory of the MBW phase III slope analysis leading to indices S cond and S acin has been previously described (8), and the computation of S cond and S acin is reiterated in the online supplement. It implies that ventilation heterogeneity can be attributed to different lung depths, and that S cond and S acin are intrinsically independent (11). Because S cond and S acin are derived from phase III slopes, their value increases when ventilation heterogeneity increases. In particular, S acin will increase in value if ventilation heterogeneity is increased in the acinar lung zone, due to an alteration of the intraacinar asymmetry, irrespective of flow asynchrony. On the other hand, S cond will increase if the conductive airways and their subtended units undergo an increase in flow asynchrony (such that the best ventilated units empty preferentially early in expiration) and/or an increased difference of their specific ventilation. Subjects The study protocol was approved by the hospital s ethics committee. One hundred seventy two smokers (77 males/95 females) with a wide range of smoking histories participated in this prospective study covering a 2-year period. A detailed smoking history was obtained and expressed as number of pack-years (1 pack-year 20 cigarettes/day/ year). All smokers had been instructed to abstain from smoking for at least 4 hours before testing. Most smokers (125 out of 172) were recruited either from hospital personnel smoking rooms or from outpatients on their first visit to the smoking cessation clinic; they had never visited a pulmonary function laboratory before, and had no medical history of respiratory disease. These volunteers were classified according to smoking history ( 10 pack-years; 10 pack-years 20; 20 packyears 30; pack-years 30). In addition to the volunteers, another 47 patients with documented overt chronic obstructive pulmonary disease (COPD) and a smoking history of greater than 30 pack-years were recruited from the Respiratory Division s Out-Patient Clinic. These patients with COPD were contacted before their scheduled control visit to the Out-Patient Clinic. They were stable at the time of testing (no change in treatment, no exacerbations for greater than 3 months). Control values were obtained for lung function and MBW indices in normal never-smokers. To avoid the confounding effects from possible bronchial hyperresponsiveness (8), all control subjects had to test negative on a histamine bronchoprovocation test with a cumulative dose up to 2 mg histamine, to be included in the control group (n 63). Statistical Analysis Using Statistica 5.1 (StatSoft, Tulsa, OK), one-way analysis of variance was performed to detect differences in all MBW and pulmonary function variables between the different smoker subgroups. Bonferroni adjustment was used to test for post hoc differences with a significance level set at p Pearson correlation analyses were also performed. RESULTS The lung function and MBW data obtained from the neversmokers and from the smokers with a smoking history of less than 30 pack-years are summarized in Table 1; all smokers were current smokers. The average smoking history of the three smoker subgroups amounted to 5.7 pack-years, 16.6 pack-years, and 25.6 pack-years, respectively. In the smokers with a smoking history of 10 or more pack-years, Dl CO (p 0.001), S acin (p 0.02), and S cond (p 0.001) were significantly different from the never-smokers, whereas the corresponding spirometric indices, FEV 1 /FVC (p 0.1), FEF (p 0.1), and FEF 75 (p 0.07), were not significantly different. In the smokers with a smoking history of 20 or more pack-years, significant reductions were observed for FEV 1 /FVC (p 0.001), FEF (p 0.002), and FEF 75 (p 0.001). Other lung function measurements, such as FEV 1, sgaw, and FRC MBW, were similar between the four subgroups of Table 1, and trapped volume (i.e., FRC pl minus FRC MBW ) was not significantly different from zero in any of these subgroups. We also considered individual abnormality on the small airways indices (S cond,s acin,fef 75, and Dl CO ) by considering the number of subjects with values below a mean of 1.96 *SD (for abnormal FEF 75 and Dl CO ) or values over a mean of 1.96 *SD (for abnormal S cond and S acin ); respective mean and SD values were established on the basis of the 63 normal subjects. Of the 91 smokers with less than 30 pack-years, the number of abnormal subjects were: n 10 (FEF 75 ), n 19 (Dl CO ); n 24 (FEF 75 or Dl CO ), n 28 (S cond ), n 23 (S acin ), and n 39 (S cond or S acin ). Of the 24 subjects with an abnormal FEF 75 or abnormal Dl CO, 14 also had an abnormal S cond or S acin. Conversely, the remaining 25 of 39 subjects with abnormal S cond or S acin showed no abnormality in terms of FEF 75 or Dl CO whatsoever. Considering only the 91 smokers of Table 1, correlations between S acin and number of pack-years (r 0.38) and between S cond and number of pack-years (r 0.39) were highly significant (p for both). As expected, the smoker subgroups with the longer smoking history were somewhat older than the smokers with a smoking history of less than 10 pack-years and the never-smokers. We, therefore, needed to validate that the observed S acin and S cond changes were true reflections of early airway impairment rather than artifacts related to age. Among the 63 never-smokers whose ages ranged between 20 and 55 years, no significant correlations were found between S acin or S cond and age (p 0.1 for both). The smoker group with a smoking history of 30 or more packyears (n 81; 34 volunteers and 47 patients with COPD) was subdivided into three subgroups according to the presence of airway obstruction and/or the presence of emphysema. Smokers with an FEV 1 /FVC 70% were labeled as non-copd according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (15). Smokers with an FEV 1 /FVC of less than 70%, a Dl CO of less than 60% predicted, and emphysema confirmed by high resolution computed tomography scan were labeled as COPD/E to designate a typical COPD group with definite presence of emphysema. Finally, smokers with an FEV 1 / FVC of less than 70% and a Dl CO of 70% predicted or greater were labeled as COPD/ to indicate a typical COPD group with a low probability of major emphysematous lesions. We deliberately adopted a 10% margin on the Dl CO above the 60% predicted cutoff (below which a high-resolution computed tomography scan request is clinically indicated) to avoid a gray zone of patients with COPD with possible emphysema without high-resolution computed tomography scan confirmation. Out of the 34 undocumented (volunteer) smokers with a smoking history of 30 or more pack-years, none had a Dl CO below 60% predicted, and three subjects were discarded because their Dl CO was in the gray zone between 60 and 70% predicted. For all smokers with a smoking history of 30 or more pack-years, we adopted these stringent classification criteria to obtain subgroups with distinct pathologic features for a systematic comparison of lung function and MBW indices in more advanced smokinginduced lung disease. Note also that in the COPD groups, not all smokers were current smokers (with smoke cessation times, as indicated by the patients, ranging from 6 months to 20 years); in the COPD/ group 18 out of 26 were current smokers, and in the COPD/E group 11 out of 27 were current smokers. The lung function and MBW data obtained on the three smoker subgroups with a smoking history of 30 pack-years or more are summarized in Table 2. There was no significant difference in smoking history between these three smoker subgroups (one-way analysis of variance; p 0.07). Considering only the 78 smokers of Table 2, correlations between either S acin or S cond and number of pack-years were not significant (p 0.1 for both). Of all parameters listed in Table 2, only Dl CO and FRC MBW did not differ between the COPD/ group and the non-copd

3 416 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL TABLE 1. LUNG FUNCTION AND MULTIPLE-BREATH WASHOUT RESULTS OBTAINED IN SMOKERS WITH LESS THAN 30 PACK- YEARS SMOKING HISTORY AND NEVER-SMOKERS Never-Smokers Smokers (pack-years 10) Smokers (10 pack-years 20) Smokers (20 pack-years 30) (n 63) (n 27) (n 35) (n 29) Mean SD Mean SD Mean SD Mean SD Age, yr * 1 43* 1 Smoking history, pack-years * * * 0.6 Lung function FEV 1, % predicted FEV 1 /FVC, % * 1 FEF 25 75, % predicted * 5 FEF 75, % predicted * 5 DL CO, % predicted * 3 81* 3 sgaw, 1/cm H 2 O s MBW S acin,l * * S cond,l * * FRC MBW, ml FRCpl FRC MBW,ml Definition of abbreviations:dl CO carbon monoxide diffusing capacity; FEF forced expiratory flow between 25 and 75% FVC; FEF 75 forced expiratory flow after expiration of 75% FVC; FRC MBW FRC measured by MBW; FRCpl FRC measured by plethysmography; FRCpl FRC MBW FRCpl minus FRC MBW ; MBW multiple-breath washout test; S acin index of acinar conductive ventilation (see text); S cond index of acinar ventilation heterogeneity (see text); sgaw specific airway conductance. * Significantly different from never-smokers (one-way analysis of variance; post-hoc Bonferroni p 0.05). group. Despite the absence of FRC MBW change, FRC pl in the COPD/ group had increased to such an extent that the trapped volume assessed by the difference between both FRC measurements amounted to 675 ml, corresponding to approximately 20% of ventilated FRC. Between the COPD/ and COPD/E groups, significant differences were found in Dl CO (by design), in FEV 1, FEV 1 /FVC, and in S acin, but not in FEF 25 75,FEF 75,orinS cond. Trapped volume was almost double in COPD/E versus COPD/ groups for a similar ventilated FRC MBW. Figure 1 summarizes small airways behavior, as can be inferred from the combination of MBW analysis and spirometry, in the smoker s lung ranging from its earliest stages up to that in the patient with overt COPD. Given the pathophysiologic differences between the early and advanced disease process, and the overall age difference between the groups with less than 30 pack-years and 30 or more pack-years, a direct quantitative comparison between groups across either side of the 30 packyear line may not be meaningful here. Figure 1 is merely meant to illustrate the differential S acin and S cond response depending on the stage in the disease process. For instance, in smokers with a smoking history up to 30 pack-years, the progressive increase in airflow limitation in terms of FEV 1 /FVC (but not in FEV 1 ; see Table 1) is paralleled by a progressive impairment in both S acin and S cond. In the case of advanced smoking-induced alterations ( 30 pack-years), S cond, and S acin clearly show a differential pattern depending on whether or not additional obstruction in TABLE 2. LUNG FUNCTION AND MULTIPLE-BREATH WASHOUT RESULTS OBTAINED IN SMOKERS WITH 30 PACK-YEARS SMOKING HISTORY OR MORE Non-COPD (n 25) COPD/ (n 26) COPD/E (n 27) Mean SE Mean SE Mean SE Age, yr * 2 68*, 2 Smoking history, pack-years Lung function FEV 1, % predicted * 3 51*, 3 FEV 1 /FVC, % * 2 45*, 2 FEF 25 75, % predicted * 2 17* 1 FEF 75, % predicted * 2 17* 1 DL CO, % predicted *, 2 sgaw, 1/cm H 2 O s * * Multiple-breath washout S acin,l * *, S cond,l * * FRC MBW, ml FRCpl FRC MBW,ml * *, 139 Definition of abbreviations: COPD/ COPD subjects with FEV 1 /FVC less than 70% and DL CO 70% predicted or greater; COPD/E COPD subjects with FEV 1 /FVC less than 70%, DL CO less than 60% predicted and high-resolution computed topography confirmed emphysema; DL CO carbon monoxide diffusing capacity; FEF forced expiratory flow between 25 and 75% FVC; FEF 75 forced expiratory flow after expiration of 75% FVC; FRC MBW FRC measured by MBW; FRCpl FRC measured by plethysmography; MBW multiple-breath washout test; S acin index of acinar conductive ventilation (see text); S cond index of acinar ventilation heterogeneity (see text); sgaw specific airway conductance. * Significantly different from non-copd group (one-way analysis of variance; post-hoc Bonferroni p 0.05). Significant difference between COPD/ and COPD/E groups (one-way analysis of variance; post-hoc Bonferroni p 0.05).

4 Verbanck, Schuermans, Meysman, et al.: Small Airways in Smokers 417 Figure 1. Lung function and ventilation distribution indices as a function of smoking history for FEV 1 /FVC, FEF 75,DL CO, FRCpl FRC MBW,S cond, and S acin. Right of the vertical dashed lines are the smokers with a 30 or more pack-years smoking history classified into three categories: non-copd, subjects with FEV 1 /FVC 70% or greater (solid square); COPD/, COPD subjects with FEV 1 /FVC less than 70% and DL CO 70% predicted or greater (solid triangle); COPD/E, COPD subjects with FEV 1 /FVC less than 70%, DL CO less than 60% predicted and high-resolution computed topography confirmed emphysema (open triangle). COPD, chronic obstructive pulmonary disease; DL CO, carbon monoxide diffusing capacity; FEF 25 75, forced expiratory flow between 25 and 75% FVC; FEF 75, forced expiratory flow after expiration of 75% FVC; MBW, multiple-breath washout test; FRC MBW, functional residual capacity measured by MBW; FRCpl, functional residual capacity measured by plethysmography; FRCpl FRC MBW, FRCpl minus FRC MBW ;S acin, index of acinar conductive ventilation; S cond, index of acinar ventilation heterogeneity. pack-years. terms of FEV 1 /FVC (and also FEV 1 ; see Table 2) originates from parenchymal loss as indicated by Dl CO (and affecting only S acin ). Direct comparisons between indices of lung function (reflecting global malfunction at a given lung depth) and indices of ventilation distribution (reflecting heterogeneous malfunction at a given lung depth) should be regarded with caution due to their intinsic differences. Nevertheless, we provide some scatterplots of selected lung function indexes (FEV 1,FEF 75, sgaw, and Dl CO ) against S acin and S cond across all smokers in the online data supplement (Figures E1 to E3 in the online supplement). As expected, these plots show a picture of increasing S acin and S cond, with decreasing FEV 1,FEF 75, Sgaw, or DL CO. DISCUSSION The most important findings of the present work can best be separated into observations related to early detection (smokers with a smoking history of less than 30 pack-years) and to more advanced smoking-induced lung injury (in smokers with a smoking history of 30 or more pack-years). In the smokers with a smoking history less than 30 packyears: (1) increased S cond and S acin in smokers with as little as 10 pack-years smoking history, but normal spirometry and normal Sgaw, indicate early changes of small airways in both the conductive and acinar lung zone compartments, probably reflecting inflamed airways located around the acinar entrance; (2) decreased spirometric mid- and end-expiratory flows (FEF 25 75, FEF 75 ) in smokers with a smoking history of 20 or more packyears reflect a more advanced deterioration in the small airways of which the anatomical location remains uncertain, but it is unlikely that even FEF 75 would reflect small airways beyond the conductive lung zone compartment (16); (3) the decrease in Dl CO in smokers with as little as a 10 pack-years smoking history is probably, at least in part, the result of a measurement artifact due to the underlying ventilation heterogeneity (as indicated by increased S cond and S acin ) (17), rather than the reflection of actual parenchymal damage in these very early stages of smokinginduced airways injury. Finally, we speculate that the progessive small airways dysfunction with increased smoking history observed here could be the noninvasive and functional equivalent of the correlation observed between increased expression of inflammatory mediators in epithelium harvested from small airways and smoking history of asymptomatic smokers (18). The fact that the transitory zone around the acinar entrance is so vulnerable to accumulation of particulate matter and associated airway wall thickening as opposed to the larger conducting airways (19), explains why indices reflecting small airways around the acinar entrance are so apt to identify the smoking-induced lung structure changes. In the smokers with a smoking history of 30 pack-years or more, two striking analogies can be observed (Figure 1): (1) between FEF 75 and S cond, the similar FEF 75 in both COPD groups and the FEF 75 difference between the non-copd group and the

5 418 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL two COPD groups is mimicked by S cond ; and (2) between Dl CO and S acin, the marked Dl CO difference between COPD/ and COPD/E groups is mimicked by S acin. The fact that in the two COPD groups, both S cond and S acin are increased with respect to the non-copd group, and that S cond is increased to the same extent in both COPD groups, indicates a similar deterioration of the small airways around the acinar entrance in the patients with COPD with similar smoking histories, irrespective of the presence of emphysema. This can be brought in agreement with observations by Saetta and coworkers (20) of increased CD8 T-lymphocytes and remodeling of peripheral airways in patients with COPD with respect to the asymptomatic smoker. The additional S acin increase without further S cond increase in the COPD/E group then reflects an additional destruction of the alveolated air spaces. Taken together across all smokers, S acin and S cond patterns indicate that these noninvasive indices are not only very sensitive in picking up early changes, but maintain the ability to actually link ventilation distribution changes to anatomical structures in the more advanced stages of smoking-induced lung disease. The advantage of the combined use of S cond and S acin (derived from the same MBW test) over the combined use of FEF 75 and Dl CO (derived from two different test maneuvers) can be best appreciated from the comparison between non-copd and COPD/ groups (Figure 1). On the one hand, Dl CO is similar between both groups, and FEF 75 follows S cond behavior to represent deteriorated small conductive airways in the COPD/ group versus the non-copd group. However, the deterioration of small acinar airways peripheral to the conductive compartment (as evidenced by a twofold S acin increase between non-copd and COPD/ ) is information that cannot be gained from Dl CO or FEF 75, unless one speculates that the above-mentioned FEF 75 decrease between COPD/ and non-copd not only reflects conductive but also acinar small airways, which seems very unlikely (16). Also, in the early detection stages, the Dl CO abnormality observed after 10 pack-years (when FEF 75 is still unaffected) is very likely due to a ventilation heterogeneity related artifact. We contend that direct measures of ventilation heterogeneity, such as S acin and S cond, present a more attractive alternative to an index, such as Dl CO, that is indirectly affected by ventilation heterogeneity. Finally, it is interesting to note that trapped volume at FRC, assessed as the difference between FRC pl and FRC MBW, is only apparent in the patient with overt COPD (Table 2). Another hallmark of the patient with COPD is the decreased sgaw, also an index derived from plethysmography but which is less subject to mouth versus alveolar pressure artifacts when measured in obstructed patients than FRC pl (21). The normal sgaw values in all non-copd smokers and the sudden drop in sgaw in the patient with COPD, clearly shows that large airway obstruction is involved only in the patient with overt COPD, irrespective of the presence of emphysema. This highlights the importance of being able to noninvasively assess small airways dysfunction in the screening of smokers before they develop overt COPD. The large airway obstruction can be brought in agreement with immunopathology data by Lams and colleagues (22) showing increased numbers of CD8 cells infiltrating the large airway epithelium of patients with COPD with respect to asymptomatic smokers. We must point out, however, that any suggested associations between lung functional data (from spirometry or ventilation distribution) and biological markers of a lung disease process need to be interpreted with caution. Indeed, the way in which different markers of airway wall inflammation result in a functional change by affecting either static airway caliber or the dynamics of airway expansion is not trivial. In the advanced stages of smoking-induced lung disease, small airways changes can be identified by histologic inspection or immunohistochemical assessment of lung resections (1, 19, 23). By attributing pathology scores, as in the study of Cosio and colleagues (1), small airway histomorphometry can then be correlated with indices of ventilation distribution, such as the VC SBW N 2 phase III slope (note that in the Cosio study, the group with a normal pathology score had an average smoking history of 17 pack-years). Although this study demonstrated that a morphologic change in the small airways did affect the SBW, other contributors to the VC nitrogen SBW test have now been identified (2, 3), precluding its usage today as an unequivocal reflection of small airways. When a modified SBW with He and SF 6 tracer gases was used to accentuate the small airways content in the phase III slope to study smokers lungs, Van Muylem and coworkers (23) found significant correlations with scores of fibrosis and inflammation of the respiratory bronchioles. Besides the practical aspects of performing He and SF 6 SBW tests (mass spectrometer) as an alternative to measuring MBW derived S cond and S acin (N 2 analyzer), there is another critical issue. The individual He and SF 6 SBW phase III slopes, reflecting structural changes in the proximal and peripheral acinus, respectively (24), are bound to be contaminated by the conductive airways contribution to the SBW phase III slope (which is isolated by means of S cond in the MBW test). The SF 6 -He phase III slope difference and any change thereof can be unequivocally attributed to a change within the acinar lung zone. However, some degree of acinar structure change could be missed by the SF 6 -He SBW phase III slope difference: if structure changes in the proximal and peripheral acinar lung zone are such that they affect respective He and SF 6 slopes to a similar extent, no net effect on the SF 6 -He slope difference will be observed (such structure changes should be detected by S acin in the MBW test). In the early stages of smoking-induced lung disease, a gold standard of peripheral lung damage to validate noninvasive indices of small airways dysfunction is as yet impossible to obtain in human subjects. However, animal studies have provided some validation of MBW related indices. Tsang and coworkers (25) demonstrated a differential response of proximal and peripheral MBW indices to oleic acid induced pulmonary edema in mongrel dogs where indeed only the peripheral MBW index was affected. In rats with different types of induced emphysema (26) and smoking-induced lesions of the nonalveolated small airways (27), significant correlations could be obtained between ventilation distribution tests and histomorphometry. The cutoff between the proximal and peripheral MBW index in each species is dictated by the location of the O 2 N 2 diffusion front and can be computed on the basis of a realistic lung morphometry. In human adult lungs, the diffusion front is situated at the level of the acinar entrance (24), hence, proximal and peripheral indices of ventilation heterogeneity are referred to as conductive and acinar ventilation heterogeneity and quantified by S cond and S acin, respectively. We conclude that the use of MBW to noninvasively probe early smoking-induced lung alterations has addressed the expectations put forward by the Cosio group study (1) that tests of unevenness of ventilation, could offer the possibility to show abnormalities at a time when pathologic changes are still potentially reversible. To the best of our knowledge no other studies have since emerged that were able to noninvasively detect smoking-induced ventilation heterogeneities originating in the small airways, from as early as 10 pack-years smoking history onwards. This makes the MBW test an eligible screening tool in the management of smoking-induced lung disease, and responds to the need for improved noninvasive mechanical tests of lung function expressed in recent recommendations for future research in COPD (28).

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