Inert gas washout tests, performed using the single-or multiple-breath washout technique, were first described over 60 years ago. As measures of ventilation distribution inhomogeneity, they offer complementary information to standard lung function tests, such as spirometry, as well as improved feasibility across wider age ranges and improved sensitivity in the detection of early lung damage. These benefits have led to a resurgence of interest in these techniques from manufacturers, clinicians and researchers, yet detailed guidelines for washout equipment specifications, test performance and analysis are lacking. This manuscript provides recommendations about these aspects, applicable to both the paediatric and adult testing environment, whilst outlining the important principles that are essential for the reader to understand. These recommendations are evidence based, where possible, but in many places represent expert opinion from a working group with a large collective experience in the techniques discussed.Finally, the important issues that remain unanswered are highlighted. By addressing these important issues and directing future research, the hope is to facilitate the incorporation of these promising tests into routine clinical practice.

Background: Airway hyperresponsiveness is the ability of airways to narrow excessively in response to inhaled stimuli and is a key feature of asthma. Airway inflammation and ventilation heterogeneity have been separately shown to be associated with airway hyperresponsiveness. A study was undertaken to establish whether ventilation heterogeneity is associated with airway hyperresponsiveness independently of airway inflammation in subjects with asthma and to determine the effect of inhaled corticosteroids on this relationship. Methods: Airway inflammation was measured in 40 subjects with asthma by exhaled nitric oxide, ventilation heterogeneity by multiple breath nitrogen washout and airway hyperresponsiveness by methacholine challenge. In 18 of these subjects with uncontrolled symptoms, measurements were repeated after 3 months of treatment with inhaled beclomethasone dipropionate. Results: At baseline, airway hyperresponsiveness was independently predicted by airway inflammation (partial r 2 = 0.20, p,0.001) and ventilation heterogeneity (partial r 2 = 0.39, p,0.001). Inhaled corticosteroid treatment decreased airway inflammation (p = 0.002), ventilation heterogeneity (p = 0.009) and airway hyperresponsiveness (p,0.001). After treatment, ventilation heterogeneity was the sole predictor of airway hyperresponsiveness (r 2 = 0.64, p,0.001). Conclusions: Baseline ventilation heterogeneity is a strong predictor of airway hyperresponsiveness, independent of airway inflammation in subjects with asthma. Its persistent relationship with airway hyperresponsiveness following anti-inflammatory treatment suggests that it is an important independent determinant of airway hyperresponsiveness. Normalisation of ventilation heterogeneity is therefore a potential goal of treatment that may lead to improved long-term outcomes.

We investigated ventilation inhomogeneity during provocation with inhaled histamine in 20 asymptomatic nonsmoking subjects. We used N2 multiple-breath washout (MBW) to derive parameters Scond and Sacin as a measurement of ventilation inhomogeneity in conductive and acinar zones of the lungs, respectively. A 20% decrease of forced expiratory volume in 1 s (FEV1) was used to distinguish responders from nonresponders. In the responder group, average FEV1 decreased by 26%, whereas Scond increased by 390% with no significant change in Sacin. In the nonresponder group, FEV1 decreased by 11%, whereas Scond increased by 198% with no significant Sacin change. Despite the absence of change in Sacin during provocation, baseline Sacin was significantly larger in the responder vs. the nonresponder group. The main findings of our study are that during provocation large ventilation inhomogeneities occur, that the small airways affected by the provocation process are situated proximal to the acinar zone where the diffusion front stands, and that, in addition to overall decrease in airway caliber, there is inhomogeneous narrowing of parallel airways.

Rationale Small airways function studies in lung disease have used three promising multiple breath washout (MBW) derived indices: indices of ventilation heterogeneity in the acinar (S acin ) and conductive (S cond ) lung zones, and the lung clearance index (LCI). Since peripheral lung structure is known to change with age, ventilation heterogeneity is expected to be affected too. However, the age dependence of the MBW indices of ventilation heterogeneity in the normal lung is unknown. Objectives The authors systematically investigated S acin , S cond or LCI as a function of age, testing also the robustness of these relationships across two laboratories. Methods MBW tests were performed by never-smokers (50% men) in the age range 25e65 years, with data gathered across two laboratories (n¼120 and n¼60). For comparison with the literature, the phase III slopes from classical single breath washout tests were also acquired in one group (n¼120). Measurements and main results All three MBW indices consistently increased with age, representing a steady worsening of ventilation heterogeneity in the age range 25e65. Age explained 7e16% of the variability in S acin and S cond and 36% of the variability in LCI. There was a small but significant gender difference only for S acin . Classical single breath washout phase III slopes also showed age dependencies, with gender effects depending on the normalisation method used. Conclusions With respect to the clinical response, age is a small but consistent effect that needs to be factored in when using the MBW indices for the detection of small airways abnormality in disease.

The ability of the diaphragm to generate pressures at different lung volumes (VLs) in humans may be determined by the following factors: 1) its in vivo three-dimensional shape, radius of curvature, and tension according to Laplace law; 2) the relative degree to which it is apposed to the rib cage (i.e., zone of apposition) and lungs (i.e., diaphragm dome); and 3) its length-force properties. To gain more insight into these factors we have reconstructed from nuclear magnetic images the three-dimensional shape of the diaphragm of four normal subjects under supine relaxed conditions at four different VLs: residual volume, functional residual capacity, functional residual capacity plus one-half of the inspiratory capacity, and total lung capacity. Under our experimental conditions the shape of the diaphragm changes substantially in the anteroposterior plane but not in the coronal one. Multivariate regression analysis indicates that the zone of apposition is dependent on both diaphragm shortening and lower rib cage widening with lung inflation, although much more on the first of these two factors. Because of the changes in anteroposterior shape and expansion of the insertional origin at the costal margin with lung inflation, the data therefore suggest that the diaphragm may be more accurately modeled by a "widening piston" (Petroll's model) than a simple "piston in a cylinder" model. A significant portion of the muscular surface is lung apposed, suggesting that diaphragmatic force has radial vectors in the dome and vectors along the body axis in the zone of apposition. The muscular surface area of the diaphragm decreased linearly by approximately 41% with VL from residual volume to total lung capacity. Diaphragmatic fibers may shorten under physiological conditions more than any other skeletal muscle. The large changes in fiber length combined with limited shape changes with lung inflation suggest that the length-twitch force properties of the diaphragm may be the most important factor for the pressure-generating function of this respiratory muscle in response to bilateral phrenic shocks at different VLs.

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; 10-20 pack-years; 20-30 pack-years; > 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 pack-years 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 smoking-induced lung disease, differential patterns of S(acin) and S(cond) are characteristic of the presence of parenchymal destruction in addition to peripheral airways alterations.

We investigated acinar airway involvement in 20 patients with stable asthma, using the phase III slope analysis of the multiple breath N2 washout previously applied in a group of patients with COPD (Am. J. Respir. Crit. Care Med. 1998;157:1573-1577). This technique quantifies severity of conductive and acinar components of ventilation maldistribution separately, through indices S(cond) and S(acin), which increase when respective ventilation inhomogeneities increase. We also investigated the effect of salbutamol inhalation on S(cond) and S(acin) in patients with asthma and compared it with that obtained in patients with COPD. Baseline measurements in the patients with asthma show that (1) acinar ventilation inhomogeneity was indeed abnormal in patients with asthma (S(acin) = 0.195 +/- 0.026 L-1) despite the normal diffusing capacity in this group; S(acin) values were intermediate between those obtained in unaffected individuals and patients with COPD, and that (2) conductive ventilation inhomogeneity was abnormal in the patients with asthma (S(cond) = 0.076 +/- 0.006 L-1) but similar to that obtained in the patients with COPD. Measurements after salbutamol inhalations showed significant changes in S(cond) and S(acin) only in the patients with asthma (p < 0.001). This study primarily demonstrated significant, but partially reversible, acinar airway impairment in patients with asthma, as compared with the more severe baseline acinar airway impairment in patients with COPD, which was not reversible after salbutamol inhalation.

We investigated ventilation inhomogeneity in patients with chronic obstructive pulmonary disease (COPD) through use of the multiple breath N2 washout test (MBW). From an alveolar slope analysis throughout the MBW, we derived two indices, S(cond) and S(acin), as a measure of ventilation inhomogeneity in conductive and acinar zones of the lungs, respectively (J. Appl. Physiol. 1997;83:1807-1816). We evaluated the relationship of S(cond) and S(acin) to standard lung-function indices by means of a principal-components factor analysis, which linked correlated indices to independent factors accounting for 81% of the total variance within the COPD group. S(acin) was linked to the so-called acinar lung-zone factor, which also comprises diffusion capacity measurements. S(cond) was linked to the so-called conductive lung-zone factor, which also comprises specific airway conductance (S(Gaw)) and forced expiratory flows. FEV1 divided by FVC (FEV1/FVC) was the only variable linked to both the conductive and the acinar lung-zone factors. The fact that S(cond) and S(acin) were linked to independent factors provides statistical confirmation of the hypothesis that S(cond) and S(acin) reflect independent lung alterations, whereas FEV1/FVC behavior indicates a combined conductive and acinar contribution to airways obstruction.