Here, we describe two measures of pulmonary function – barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.
Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.
Gas washout techniques have been used for decades to provide important information about the structure and uniformity of the airway tree. The lung is classically described as having two compartments – a conducting zone that is comprised of the anatomic dead space and the respiratory zone where gas exchange occurs in the alveoli. The conducting airways are termed as “dead space” because they do not participate in the exchange of oxygen and carbon dioxide. In the single breath gas washout method, the concentration profile of an exhaled gas can be used to determine the volume of the anatomic dead space and to derive information about the uniformity of ventilation. Some methods rely on the breathing of inert gases to make these measures (N2, argon, He, SF6, etc.). The use of inert gas is well-established, supported by scientific consensus statements1, and there are available commercial equipment with user friendly interfaces. However, the exhaled profile of carbon dioxide (CO2) can be used to derive similar information. Evaluating the profile of CO2 as a function of the exhaled volume, or volumetric capnography, does not require the participant to breathe special gas mixtures and allows the investigator to gather additional information flexibly about metabolism and gas exchange with minimal adjustment to the technique.
During a controlled exhalation, the concentration of CO2 can be plotted against the total exhaled volume. At the beginning of an exhalation, the dead space is filled with atmospheric gas. This is reflected in Phase I of the exhaled CO2 profile where there is an undetectable amount of CO2 (Figure 1, top). Phase II marks the transition to the alveolar gas, where gas exchange occurs and CO2 is abundant. The volume at the midpoint of Phase II is the volume of the anatomic dead space (VD). Phase III contains alveolar gas. Because airways with different diameters empty at different rates, the slope (S) of Phase III provides information about airways uniformity. A steeper slope of Phase III suggests a less uniform airway tree proximal to the terminal bronchioles, or convection-dependent inhomogeneity2. In the case where a perturbation may change the rate of CO2 production, and to make comparisons between individuals, the slope can be divided by the area under the curve to normalize for differences in metabolism (NS or normalized slope). Volumetric capnography has been used previously to evaluate the changes in airways volume and uniformity following air pollutant exposure3,4,5,6.
Gas transport in the lung is governed by both convection and diffusion. Single breath washout measures are highly dependent on air flow and the measured value of VD occurs at the convection-diffusion boundary. Changing the flow rate of the exhalation or preceding inhalation changes the location of that boundary7. Capnography is also highly dependent on the volume of the lung immediately preceding the maneuver. Larger lung volumes distend the airways, resulting in larger values of VD8. One solution is to consistently make the measurement at the same lung volume – usually functional residual capacity (FRC). An alternative, described here, is to couple volumetric capnography with barometric plethysmography, in order to obtain the relationship between VD and lung volume. The participant then performs the maneuver at constant flow rates, while varying the lung volume. This still allows for classic capnographic measures to be made at FRC, but also for the relationship between the lung volume and dead space volume and between the lung volume and homogeneity to be derived. Indeed, the added value of coupling capnography with plethysmography comes from the ability to test hypotheses about the distensibility of the airways tree and the structure-function relationship of the lung. This may be a valuable tool for investigators aiming to quantify the influence of airways mechanics versus lung compliance and elastance on pulmonary function in healthy and diseased populations9,10,11. Furthermore, accounting for the absolute lung volume at which the volumetric capnographic measurements are being performed allows investigators to characterize the effects of conditions that can alter the inflation state of the lung, such as obesity, lung transplant, or interventions like chest wall strapping. Volumetric capnography may ultimately have clinical utility in the intensive care setting12,13.
This protocol has been previously approved by and follows the guidelines set by the University of Iowa Institutional Review Board. Data shown were collected as part of a project approved by the Institutional Review Board at the University of Iowa. Participants gave informed consent and the studies were performed in accordance with the Declaration of Helsinki.
1. Equipment
2. Plethysmography
NOTE: Barometric plethysmography is a well-described clinical tool and is performed using commercial equipment according to the consensus statements on standardizing lung volume measurements14,15. When necessary, lung flows and volumes are compared to predicted values from the NHANES data set and Goldman and Becklake16 that are included in the plethysmograph software.
3. Volumetric Capnography
NOTE: Steps 3.1 – 3.4 are performed before the arrival of the research subject.
4. Data Analysis
Representative plethysmography results are given in Figure 4. This participant required four attempts in order to collect three FRC values with <5% variability from the mean.%Ref reflects the percent of the predicted value for each variable based on population regression equations that take into account sex, age, race, height and weight
Figure 1 (top) shows a representative single capnogram used in analysis and Figure 1 (bottom) shows the raw data of the entire sequence of the maneuver. In Figure 1 (bottom), the capnogram and flow tracing are not aligned to account for the time delay. Data generated from running a sequence of breaths through the macro are shown at the end of Supplemental Figure 2. This individual had a dead space of 0.266 L, a slope of 0.523% CO2/L and a normalized slope of 0.0826 L-1. Quality information about the maneuver are also given in columns F, G, I, J, and K. Column F gives the average exhaled flow rate, with the standard deviation in column G. The exhaled tidal volume is given in column J and the R-squared value for the slope is in column K.
Dead space and slope plotted as a function of lung volume are given in Figure 5. In the left panels, dead space and slope are plotted versus lung volume relative to FRC, where FRC=0 L. In the right panels, lung volume and slope are plotted versus absolute lung volume. In both cases, dead space and slope are significantly correlated to lung volume (p<0.05 for all four regression analyses). This suggests that dead space and airways homogeneity increase as lung volume increases, although little is known about this relationship in populations with lung disease or with bronchodilator therapy. The investigator may also choose to use these data to describe the numerical value of dead space and slope at specific lung volumes (FRC, residual volume, 50% of total lung capacity, etc.)3.
Figure 1. Sample capnogram (top), with exhaled CO2 (%) plotted as a function of the exhaled volume. I, II, and III indicate the three phases of the capnogram. The dotted line indicates the volume of the dead space and the solid line represents the slope of the alveolar plateau (Phase III). The slope can be divided by the area under the capnogram (shaded grey, labeled A) to yield the normalized slope. The four breath sequence is shown in the bottom panel, followed by a sigh breath to determine functional residual capacity. Each pair of breaths is analyzed as a single maneuver. Please click here to view a larger version of this figure.
Figure 2. Equipment setup for capnographic measurements. Shown in this figure are the pneumotach and gas analyzer required for capnographic measurements. The left monitor and tracing are used by the participant as a guide in generating the flow pattern while data are observed on the right monitor by the investigator. Please click here to view a larger version of this figure.
Figure 3. Channel settings for the acquisition of the volumetric capnogram. Flow is collected in Channel 1, CO2 concentration (%) is collected in Channel 2, and the tidal volume is calculated in Channel 3. Please click here to view a larger version of this figure.
Figure 4. Representative plethysmograph data from a healthy, male subject. Particularly relevant to the protocol reported here are the total lung capacity (TLC), residual volume (RV) and functional residual capacity (FRC). Please click here to view a larger version of this figure.
Figure 5. Dead space and alveolar slope plotted as a function of absolute lung volume (right panels) and as the volume relative to the functional residual capacity (volume-FRC, left). Note the dependence of the airways volume and lung heterogeneity on lung volume. Lung volume may be expressed as a function of FRC or absolute volume, depending on the experimental design. Please click here to view a larger version of this figure.
Figure 6. Factors impacting data accuracy. Data are given as the mean ± 95% confidence interval. Relationship between the CO2 sampling rate and the time delay between the gas analyzer and pneumotach (top). The time delay should accurately be determined before beginning the experiment. Measuring eight total maneuvers allows for the measurement of the dead space at a single lung volume with <5% variability (bottom). Please click here to view a larger version of this figure.
Here, a protocol for the measurement of VD and airways homogeneity (slope) is provided. These measurements can be made at FRC, or as a function of lung volume. Measuring FRC before the start of the experiment and after a perturbation allows VD and slope to be plotted as a function of lung volume and may provide useful information about the structure-function relationship of the lung that is not obtained from capnography at FRC alone.
Airways volume and high-resolution structure can be obtained from computed tomographic imaging17,18, but this requires exposure to radiation and expertise in image processing. With volumetric capnography, repeated measures can be made without increasing risk to the participant. It also does not require expensive equipment or advanced data processing capabilities. Volumetric capnography is an ideal method for experiments with multiple time points and multiple lung volumes and in-patient populations whose radiation exposure should be minimized.
With regard to the barometric plethysmography, care should be taken to perform the measurement according to consensus statements. When it is important to compare participant values to predicted population values, weight should be measured with a scale and height should be verified with a stadiometer. As noted in the protocol, the most critical component to measure before beginning volumetric capnography is the time delay between the pneumotach and the gas analyzer. The time delay is highly dependent on the analyzer sampling rate (Figure 5, top) and small changes in the sampling rate can have large influences on measured values. The analyzer flow rate should be checked at the beginning and throughout the experiment. Calibration of the analyzer and pneumotach are also critical and care should be taken to ensure their accuracy before beginning an experiment.
We have also determined the accuracy of the measurement at a single lung volume in 3 participants. Figure 5 (bottom) demonstrates that it is necessary to complete four maneuvers (8 total breaths) at a single lung volume to measure dead space so that the variation is <5%. Investigators should take care to make a sufficient number of measurements when having data at a particular lung volume is important. In a subset of 36 maneuvers analyzed in duplicate by two investigators, intra-investigator analysis variability was less than 0.5%.
These methods also require a technician or investigator that is skilled in coaching the participant to make the ventilatory maneuvers. A limitation in pulmonary function studies can be the participant’s ability to perform the maneuver. However, participants that are able to perform clinical pulmonary function are typically able to perform the capnographic maneuvers. If the study is designed such that capnography follows plethysmography and spirometry, participants that are unable to perform a coached spirometric or plethysmographic maneuver can be excluded. In 60 previous studies, one participant who performed clinical spirometry was excluded because they could not follow the capnographic breathing pattern. There are currently no consensus guidelines defining acceptable capnographic measurement criteria. However, intersubject variability is 8±1% of the target flow rate in our 10 most recent participants. Intrasubject (between maneuver) variability is 4±2%.
Issues relating to data accuracy and reproducibility are the result of errors in the time delay or the analyzer and pneumotach calibration. Before each experiment, take care to calibrate the analyzer with a set of known gases and generate a multi-point standard curve to confirm the analyzer’s accuracy.
Beyond the scope of the information provided here, the macro contains two additional calculations that may be of interest. When the maneuvers are made at FRC, the FRC column provides an estimate of FRC based on the Farmery method19. Calculation of the peripheral bronchial cross sectional area is based on the method described by Scherer, et al.20. Finally, if desired, the end tidal CO2 and average expired CO2 concentration can be used to calculate the physiological dead space for comparison to the anatomic dead space21,22.
The authors have nothing to disclose.
This work was funded by the Departments of Health and Human Physiology and Internal Medicine at the University of Iowa. This work was also supported by the Old Gold Fellowship (Bates) and Grant IRG-15-176-40 from the American Cancer Society, administered through The Holden Comprehensive Cancer Center at The University of Iowa (Bates)
Computer with dual monitor | Dell Instruments | ||
PowerLab 8/35* | AD Instruments | PL3508 | |
LabChart Data Acquisition Software* | AD Instruments | Version 8 | |
Gemini Respiratory Gas Analyzer* (upgraded option) | CWE, Inc | GEMINI 14-10000 | *indicates that part is available in the Exercise Physiology package from AD Instruments |
Heated Pneumotach with Heater Controller* (upgraded option) | Hans Rudolph, Inc | MLT3813H-V | |
3L Calibration Syringe | Vitalograph | 36020 | |
Nose Clip* | VacuMed | Snuffer 1008 | |
Pulse Transducer* | AD Instruments | TN1012/ST | |
Barometer | Fischer Scientific | 15-078-198 | |
Flanged Mouthpiece* | AD Instruments | MLA1026 | |
Nafion drying tube with three-way stopcock* | AD Instruments | MLA0343 | |
Desiccant cartridge (optional for humid environments)* | AD Instruments | MLA6024 | |
Resistor | Hans Rudolph, Inc | 7100 R5 | |
Flow head adapters* | AD Instruments | MLA1081 | |
Modified Tubing Adapter (optional) | AD Instruments | SP0145 | |
Two way non-rebreather valve (optional)* | AD Instruments | SP0146 | |
Plethysmograph | Vyaire | V62J | |
High Purity Helium Gas | Praxair | He 4.8 | |
6% CO2 and 16% O2 Calibration Gas | Praxair | Custom | |
Microsoft Excel | Microsoft | Office 365 |