The measurement of lung volumes using body plethysmography and helium dilution methods in COPD patients: a correlation and diagnosis analysis

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease characterized by persistent airflow limitation. Moreover, lung hyperinflation evaluated by lung volumes is also the key pathophysiologic process during COPD progression. Nevertheless, there is still no preferred method to evaluate lung volumes. For this study, we recruited 170 patients with stable COPD to assess lung volumes stratified by airflow limitation severity. Lung volumes including residual volume (RV) and total lung capacity (TLC) were determined by both body plethysmography and helium dilution methods. The discrepancies between these two methods were recorded as ΔRV%pred, ΔTLC%pred, and ΔRV/TLC. We found that ΔRV%pred, ΔTLC%pred, and ΔRV/TLC increased significantly with the severity of COPD. The differences of lung capacity between these two methods were negatively correlated with FEV1%pred, and diffusing capacity for carbon monoxide (DLCO%pred). Moreover, the receiver operating characteristic (ROC) for ΔTLC%pred to distinguish severe COPD from non-severe COPD had an area under curve (AUC) of 0.886. The differences of lung volume parameters measured by body plethysmography and helium dilution methods were associated with airflow limitation and can effectively differentiate COPD severity, which may be a supportive method to assess the lung function of stable COPD patients.

during panting maneuvers and excessive compliance of the extrathoracic airway 11,12 . Multi-breath Helium dilution method (MBHD) is an alternative method for measuring alveolar volume, but may cause underestimation for the uneven distribution of ventilation and the gas contained within the poorly ventilated regions not incorporating in the helium estimate of lung volume 8,9 . As the biases of these two methods are both associated with a degree of airflow limitation, the differences between these two methods may provide an alternative marker to reflect the degree of airway obstruction and be an optimal substitute to evaluate the severity of COPD.
Based on current evidence of lung volumetric parameters in COPD and potential estimation biases in WBP and MBHD methods, we conducted a prospective correlation and diagnosis analysis to further assess the value of lung volume as well as the differences between these two methods in distinguishing COPD severities, to clarify the influences of airflow limitation on lung volume measurement, and to evaluate the correlation with diffusing capacity.

Results
A total of 170 patients were included into this prospective study. All patients were confirmed with stable COPD according to GOLD standards 1 . None of the patients had an acute exacerbation during previous 4 weeks before pulmonary function tests, and all medications which may alter lung function were stopped for at least 72 hours. Pulmonary function variables are performed in Table 1 and Fig. 1. The subject population included 44 patients (25.9%) with GOLD Classification 1, 54 patients (31.8%) with GOLD Classification 2, 34 patients (20.0%) with GOLD Classification 3, and 38 patients (22.3%) with GOLD Classification 4.
As Fig. 1 and Table 1 shown, TLC%pred, RV%pred and RV/TLC measured by WBP were significantly increased in GOLD Classification 2, 3, and 4, compared with Classification 1. In contrast, these parameters measured by MBHD were no significant difference between different GOLD classifications ( Fig. 1). TLC%pred, RV%pred and RV/TLCmeasured by WBP were significant larger than that determined by MBHD in all COPD stages (37.8 ± 22.9%, 75.7 ± 55.0%, and 11.2 ± 12.5%, all p < 0.0001, Table 1). The differences of TLC%pred, RV%pred and RV/TLC were significant greater from the patients with GOLD 3 and 4 diseases, compared to the differences from the patients with GOLD 1 and 2 diseases (Table 1). To further confirm the influences of airflow obstruction on lung volume measurement, we studied the correlation of Δ RV%pred, Δ TLC%pred, and Δ RV/ TLC with FEV 1 %pred. We found that the difference of TLC%pred measured by these two methods was strong correlated with FEV 1 %pred (r = − 0.685, p < 0.001, Fig. 2A). There was also moderate but significant correlation between differences of RV%pred and FEV 1 %pred (r = − 0.579, p < 0.001, Fig. 2B). However, the correlation between differences of RV/TLC and FEV 1 %pred was weak (r = − 0.290, p < 0.001, Fig. 2C).
As Δ RV%pred, Δ TLC%pred, and Δ RV/TLC were correlated with FEV 1 %pred, we depicted ROC curves and calculated the area under curve (AUC) to evaluate the accuracy of Δ RV%pred, Δ TLC%pred, and Δ RV/TLC in discriminating different COPD severities. Because of limited patient samples, we combined mild and moderate COPD patients, defined by GOLD classification of airflow limitation, as 'non-severe' group; while the severe and very severe COPD patients were combined as 'severe' group. We found that Δ RV%pred, Δ TLC%pred, and Δ RV/ TLC could efficiently distinguish severe COPD from non-severe COPD with a high AUC (Fig. 3). The most effective variable was Δ TLC%pred with the AUC up to 0.886 (95% CI 0.834~0.939). The Δ TLC%pred value of 34.2 would have a sensitivity of 93.1% and specificity of 79.6%, with the positive and negative likelihood ratio of 4.56 and 0.09, respectively (Table 2). Similarly, as shown in Table 2, Δ RV%pred, and Δ RV/TLC also had a relatively high sensitivity and specificity to differentiate COPD severity.
In our study, we also found that the diffusing capacity measured with diffusing capacity for carbon monoxide (D L CO%pred) significantly decreased with increased severity of COPD (Table 1). D L CO%pred was   Table 1. Spirometric and lung volume variables in 170 subjects stratified by airflow limitation severity. Data were described as mean ± standard deviation (SD). *p < 0.05 Compared with GOLD classification stage 1; # p < 0.05 Compared with GOLD classification stage 2; $ p < 0.05 Compared with GOLD classification stage 3. significant lower from the patients with GOLD 2, 3, and 4 disease, compared to the patients with GOLD 1 disease (all p < 0.05). Interestingly, the difference of TLC%pred as measured by WBP and MBHD was negatively correlated with DLCO%pred (r = − 0.505, p < 0.05, Fig. 2D), therefore suggesting that this discrepancy may also be associated with the degree of lung parenchymal destruction.

Discussion
In our study, we found that lung volume variables including TLC%pred, RV%pred and RV/TLC as measured by WBP were significantly increased as COPD exacerbated. These variables measured by WBP were significantly higher than those measured by MBHD method. In addition, differences measured by these two methods were negatively correlated with FEV 1 %pred and effectively differentiated COPD severity. Moreover, we also identified that the discrepancy of TLC measured by these different methods was negatively correlated with diffusing capacity. Thus, lung volume measurement of the differences between these two methods may be an alternative marker to reflect the degree of airflow obstruction and gas trapping. It has been widely acknowledged that spirometric measurements of FEV 1 and FEV 1 /FVC are key parameters in diagnosing and grading severity of COPD. However, COPD is a complex disease that is characterized by the presence of airflow limitation, air trapping and emphysema 1 . Decreased FEV 1 primarily results from small airway obstruction and emphysema 13 . In combination, both airway obstruction and parenchymal destruction lead to expiratory gas trapping, resulting in hyperinflation 1 . Our study found that TLC%pred, RV%pred and RV/ TLC measured by WBP significantly increased as airflow limitation worsened, indicating that airflow limitation and gas trapping share the common pathophysiologic change during COPD progression. This suggests that testing of lung volumes could be an effective addition to spirometry in comprehensive assessment of COPD. These , and RV/TLC (C) as measured by whole-body plethysmography and multi-breath helium dilution, varying by severity. The results are plotted as means ± SD; Differences between body plethysmography and helium dilution methods were analyzed with paired T-test. † p < 0.001, ‡ p < 0.05. he, helium dilution; pleth, plethysmography; TLC, total lung capacity; RV, residue volume.
additional parameters may help to exclude potential restrictive diseases from obstructive lung disease in patients with dyspnea. Secondly, lung volume is a useful tool to evaluate some COPD properties that is advantageous to FEV 1 . For example, some studies found that an increase of RV/TLC was an independent risk factor of all-cause  mortality and frequent exacerbations in COPD population 14,15 . Lung volume is also very sensitive to bronchodilators and lung-volume reduction surgery, and better associated with patient-centered outcomes such as dyspnea and exercise tolerance [16][17][18] . Washko et al. found that preoperative RV/TLC ratio but not FEV 1 is predictive of postoperative outcomes after lung-volume reduction surgery 16 . Thus, integrative analysis of lung functions including both spirometry and lung volume in COPD patients is warranted.
Currently, measurement of lung volumes lacks a gold standard. The guideline of ATS/ERS on lung volume measurements lists out several methods including WBP, MBHD, and imaging techniques 6 . However, the guideline does not make a clear recommendation on which specific technique is the best to use, especially in COPD patients, that lung volumes measured by WBP and MBHD are always not consistent. Consistent with other studies, our study found that TLC%pred, RV%pred and RV/TLC measured in COPD patients by WBP were significantly higher than those measured by MBHD method 8,9,11,19 . Coertjens et al. found that in 93 COPD patients (29 mild/moderate, 29 severe, 35 very severe), the differences of TLC between the WBP and MBHD method ranged from 30.5% of the predicted value to 38.2% of the predicted value, which is similar to our finding 8 . O'Donnell et al. showed that TLC measured by WBP may be overestimated in COPD patients, as WBP-derived TLC was significant greater than the values measured with MBHD method and CT (0.63 L and 0.87 L, respectively), especially among the patients with FEV 1 < 30% of predicted 9 . The variations of different methods may be due to the physical principles. Lung volume measured by WBP is based on the Boyle's law that the product of gas volume and pressure is constant under isothermal conditions. Thus, at any given moment, when a constant amount of gas is compressed or decompressed, the gas volume decreases or increases and gas pressure changes such that the product of volume and pressure 6 . In COPD patients, WBP may overestimate RV with inadequate equilibration of mouth and alveolar pressure. On the other hand, the method of MBHD is based on the equilibration of gas in the lung with a gas containing helium with known volume. As the proportion of poorly ventilated lung units increases, the smaller the values are measured by MBHD. Herein, based on different physical principles between WBP and MBHD, it is reasonable to test the differences of lung volumes by WBP and MBHD in COPD patients.
We further investigated the differences between these two different methods to evaluate the relation between the differences and COPD severity. We found that the differences measured by these two methods were negatively correlated with FEV 1 %pred, and can effectively differentiate severe COPD from non-severe patients. As previous mentioned, underestimation of lung volume by MBHD is associated with the degree of gas trapping. Our findings align with Jarenback et al. 19 in that with single breath or multiple breath helium dilution methods, the helium dilution-derived TLC did not increase based on GOLD classifications. Moreover, O'Donnell et al. found FEV 1 %pred < 30% is an independent factor for Δ TLC > 1L, which also indicates that the difference of lung volumes measured by different methods maybe associated with airflow limitation 9 . Thus, it is reasonable that high sensitivity and specificity of Δ TLC%pred for differentiating severe and very severe COPD from non-severe COPD patients were found in our study. However, the exact diagnostic efficacy of these variables needs further validation with a larger sample population. D L CO is a traditional physiologic marker to assess the potential of the lung for gas exchange. Destruction of alveolar walls in emphysema patients directly disrupts the integrity of alveolar capillary bed, reducing D L CO. A pathologic and radiographic correlate of decreased D L CO with emphysema was reported that a good correlation between low D L CO and decreased total lung tissue volume on chest computed tomography 20,21 . In this study we found that diffusing capacity measured with D L CO%pred significantly decreased with increased severity of COPD. Moreover, Δ TLC%pred was negatively correlated with D L CO, which indicated the potential relationship between airway obstruction and decreased D L CO. One study showed that the severity of diffusing capacity impairment is correlated with airway wall thickness, which may induce air trapping 22 . This is further confirmed that increased air trapping induced by metronome-paced tachypnea correlated well with D L CO 23 . These findings may raise additional interest in further evaluation of differences in lung capacity between different methods as an index for understanding the structure and function change of COPD.
In addition, our study had several limitations, which may lead to cautious interpretation of the results. First, the sample is small in our study, which may result in bias of our findings. Second, assessment of COPD severity was based purely on spirometry, lacking information regarding exacerbation history and comorbiditiest. Further prospective studies are needed to evaluate the relationship of lung volume with clinical endpoints. In summary, our study indicates that the differences of lung volumes measured by WBD and MBHD in COPD patients are associated with the level of airflow limitation and impaired diffusing capacity, which may be optimal substitute to evaluate the severity of COPD.

Methods and Materials
Participants. Outpatients   diagnostic criteria as following: (a) FEV 1 /FVC ratio < 0.7 after bronchodilation; (b) no acute exacerbation during previous 4 weeks; (c) stop the medications, which may influence pulmonary function testing for at least 3 days. Participants coexisting medical conditions that would interfere with pulmonary function testing were excluded. This study has been approved by the Institutional Review Board of West China Hospital of Sichuan University, and written informed consents were obtained from all subjects. The methods in this study were carried out in accordance with the approved guidelines.

Pulmonary function testing. WBP and MBHD method were performed in all enrolled patients by a full
MasterScreen PFT System (Jaeger Corp, Germany), which was equipped with a mixing fan, carbon dioxide (CO 2 ) absorber, oxygen (O 2 ) and helium supply, a gas inlet and outlet, and a water vapor absorber. WBP measured both lung airflow (FEV 1 , peak expiratory flow (PEF)) and volumes (RV and TLC). All test procedures complied with the standardizations recommended by ATS/ERS guideline 2,6 , which contained a series of gentle pants at a frequency between 0.5 and 1.0 Hz to calculate lung volumes, and three distinct phases to depict the flow-volume curves including: (1) maximal inspiration; (2) a "blast" of exhalation; and (3) continued complete exhalation until the volume-time curve showed no change in volume (< 0.025L) for ≥ 1s and the subject had tried to exhale for ≥ 6s.
Lung volumes (RV and TLC) were also measured by MBHD method according to the following steps: patients were instructed to breathe for 30-60 seconds to achieve a stable end-tidal expiratory level, then switched them to the helium gas (turn in) and noted the helium concentration every 15 seconds until the helium equilibration is complete (i.e. change of helium concentration is < 0.02% for 30 seconds), and finally disconnected them from the helium gas (turn out).
Airflow parameters were detected as predicted percentage of FEV 1 (FEV 1 %pred), PEF (PEF%pred), and maximal mid-expiratory flow (MMEF%pred); while lung volumes were displayed as predicted percentage of RV (RV%pred) and TLC (TLC%pred), and RV/TLC. The predictive equations were adjusted for Chinese subjects 24 . Differences in lung volumes between WBP and MBHD method were calculated as predicted percentage of RV (Δ RV%pred), TLC (Δ TLC%pred) and RV/TLC (Δ RV/TLC). D L CO was measured by Single-breath testing 25 . The subject unforced exhaled to RV, and then inhaled testing gas rapidly to TLC, keeping breath-hold for 10 seconds, and the expiratory gas was collecting for analysis. Statistics Analysis. The statistical analysis was performed with SPSS19.0 (SPSS, Inc., Chicago, USA).
Normally distributed data were described as mean ± standard deviation (SD). Groups were defined by GOLD classification severity, and differences of the lung volume indexes between WBP and MBHD methods were analyzed with paired T-test. Differences between groups were tested by analysis of variance (ANOVA) and Student's-Newman-Keuls tests were further used for multiple comparison tests when significant differences among all groups were found. The correlations among these indexes were performed by Pearson correlation analysis. We depicted receiver operating characteristic (ROC) curves and calculated area under the curve (AUC) to evaluate the accuracy of Δ RV%pred, Δ TLC%pred, and Δ RV/TLC in discriminating different COPD severities. Cutoff points were defined as the point when Youden's index (= sensitivity + specificity − 1) reached the maximum, and the sensitivity, specificity, as well as likelihood ratios (LRs) were also calculated in different severities. A p value < 0.05 was considered statistically significant.