Two healthy, human CPET trainers participated as test subjects in the present study after providing informed consent. The testing protocol is consistent with the principles of the Declaration of Helsinki and was approved by Vyaire Medical Affairs. Experiments were carried out in a diagnostic training lab at Vyaire Medical in Mettawa, IL, a leased space in a commercial office building. The building air conditioning (A/C) system complies with the International Mechanical Code, ASHRAE Standard 90.1, and the International Energy Conservation Code as mandated by the State of Illinois.
The experimental setup is shown in Figure 1 (top). A gantry was built to hold a flashlamp-pumped, Q-switched double-cavity pulsed Nd:YAG laser (Quantel, Bozeman, MT) above the subject. A 90-degree beam-steering mirror and a variable-beam-waist optic (LaVision USA, Ypsilanti, MI) were utilized to direct and convert the laser beam into a light sheet to illuminate aerosol particles in the region of interest (ROI). A 5.5-megapixel CMOS camera (LaVision USA, Ypsilanti, MI) with global shutter and double-frame mode was combined with a 60mm f2.8 macro lens (Nikon USA, Melville, NY). Image acquisition was synchronized with the laser pulses using a PTU-X Synchronizer (LaVision USA, Ypsilanti, MI). The imaging ROI was located immediately downstream of the CPET interface to capture particles generated on a breath-by-breath basis.
Particle imaging data were captured and processed using commercial image-processing software (DaVis 10, LaVision USA, Ypsilanti, MI). Image pairs were captured at 15 Hz for durations selected to match the test being performed. Particle-image velocimetry (PIV) was used to measure particle velocities. Velocities of the flows downstream of the CPET patient-interface were utilized to assign particle images to either inhalation or exhalation. Particle counts were calculated for each image using a multi-step smoothing and pixel-threshold algorithm. Figure 1 (bottom) shows an example raw particle image prior to processing (left) and a processed image (right).
CPET Breath-by-Breath Measurements
CPET with breath-by-breath aerosol particle counting was performed using two different cycle-based CPET systems and associated patient interfaces. The first system consisted of a Vyntus™ CPX Metabolic Cart (Vyaire Medical, Mettawa, IL) using a Digital Volume Transducer (DVT) flow sensor (Vyaire Medical, Mettawa, IL) with head attachment of a 7450 Silicone Oro-Nasal mask (Hans Rudolph Inc, Shawnee, KS). The second system consisted of a Vmax™ Metabolic Cart (Vyaire Medical, Mettawa, IL) using a hot-wire mass flow sensor (MFS, Vyaire Medical, Mettawa, IL), and mouthpiece and nose clip (VacuMed, Ventura, CA) secured to the subject with headgear (Vyaire Medical, Mettawa, IL). An upright cycle ergometer (Ergoline GmbH, Bitz, Germany) and Cardiosoft 12-lead ECG (GE Healthcare, Chicago, IL) were common to both systems. The DVT and MFS were secured in place such that the laser sheet could be aligned precisely with the outlet of each sensor. The subjects breathed through an extension tube placed between the flow sensor and the mask or mouthpiece. Figure 2 provides a schematic and a camera image of the experimental setup and particle counting ROI for the CPET experiments.
Two healthy trained subjects exercised using a 3-phase incremental ramp protocol (2 minutes resting breathing, 30-watt ramp exercise, 2-minute active recovery) until volitional stoppage due to exhaustion when the subject reached respiratory compensation. Particle images were taken in sequences of 200 image pairs at 90 seconds resting breathing, at 2-minute intervals during exercise, and at reaching peak work-rate intensity. Following this, images were captured at 30 seconds and 2 minutes during recovery.
Estimation of endogenous aerosol particle generation during the testing followed a process of assigning particle counts to either inhalation or exhalation using measured velocities, followed by integration over each breath to determine the aerosol particles per unit volume generated at each point in the CPET regimen. Figure 3 contains a representative raw particle count (blue) and mean velocity plot (orange) from the breath-by-breath CPET measurements at peak exercise. Particle count and mean velocity are plotted as a function of time. Inhalation and exhalation phases can be clearly observed as the particle count rises to a maximum during exhalation and returns to a baseline value during inhalation. The transition from inhalation to exhalation occurs as the mean velocity changes from positive (inhalation) to negative (exhalation).
Measured particle counts were integrated over each breath (area under the curve, AUC) to calculate the aerosol concentration in the interrogation region for both inhalation and exhalation. Each exhalation of particles includes both endogenously generated aerosol particles plus particles inhaled from the surrounding ambient region on the prior inhalation. Therefore, the net amount of aerosol generated per breath was determined by subtracting the particle count of the prior inhalation from each measured exhalation count. The thickness of the laser sheet was measured optically such that the illuminated volume could be calculated as the area of the rectangular ROI shown in Figure 2 multiplied by the laser sheet thickness. This volume was 2.4 cm3 and enabled scaling of all aerosol particle counts to a per ml basis.
A qualitative comparison of aerosol particles generated by the test subjects during CPET with both side-stream tobacco cigarette smoke particles and jet nebulizer droplets enabled estimation of aerosol size; Figure 4 compares images of these aerosols. Aerosol droplets generated with a jet nebulizer (MistyFast, Vyaire Medical, Mettawa, IL) have a known size range of 2-3 microns and are observably much larger than the CPET aerosol particles. On the other hand, the tobacco smoke particles (with known size range of 0.1-0.3 microns) appear very similar in size to the CPET-generated particles. Based on this similarity, we estimate that the aerosols generated in this study during CPET are primarily in the lower sub-micron range (< 0.3 microns) which is consistent with other published literature.[24-28]