The study was funded by CIHR-NSERC Collaborative Health Research Projects (CWC), Pettit Block Term grant (CWC), The Lung Health Foundation, and Canadian Lung Association - Breathing as One: Allied Health Grant (JW). We thank the many participants of our oscillometry research studies who have allowed us to develop expertise in conduct of oscillometry.

The respiratory oscillometry studies were approved by the University Health Network Research Ethics Board (REB# 17-5373, 17-5652 and 19-5582). Written informed consent was obtained from participants prior to oscillometry test.
NOTE: This video outlines the standard operating procedure for oscillometry. Our laboratory uses a device manufactured by Thorasys Thoracic Medical Systems Inc but the technique is the same regardless of the manufacturer. The software programs are different for each manufacturer, in the same way that different commercial spirometers have unique proprietary software for data collection and display. The protocol below is applicable for all respiratory oscillometry devices. Readers are directed to manuals of their commercial devices and refer to specific instructions regarding software of their device.
1. Pre-test patient screening/preparation
<ol>
	<li>Ensure that the patient is free of any active or suspected transmissible respiratory infection, such as coronavirus or tuberculosis.</li>
	<li>Ensure that the patient has not had any recent dental or facial surgeries, such as tooth extractions, and can form a proper tight seal around the mouthpiece.</li>
	<li>Ensure that the patient is as relaxed as possible, is not wearing tight-fitted clothing, and withholds from tobacco use and vigorous exercise at least 1 h prior to testing.</li>
	<li>Perform oscillometry prior to conventional PFTs, such as spirometry, if requested by a referring physician. 
	NOTE: Refer to Supplemental Table 1 for contraindications for spirometry/PFTs.</li>
	<li>Ensure that the patient withholds bronchodilators&#160;prior to testing, unless instructed by a referring physician to continue bronchodilator medication. 
	NOTE: Refer to Supplemental Table 2 for bronchodilator withholding times for PFTs and Supplemental Table 3 for bronchodilator withholding times for the methacholine challenge test.</li>
</ol>
2. Equipment/materials preparation
<ol>
	<li>Equipment preparation
	<ol>
		<li>Verify the resistance load of the oscillometry device by using a valid factory calibrated mechanical test load prior to patient testing.</li>
		<li>Remove the dust caps at both ends of the mechanical test load and attach onto the oscillometry device.</li>
		<li>Select calibration from the oscillometry software menu and proceed with impedance test load verification. 
		NOTE: The recommended tolerance for the verification is &#8804; &#177;10% or &#177;0.1 cmH2O&#183;s/L, whichever is met first.</li>
		<li>Upon successful verification, save and proceed with testing.</li>
	</ol>
	</li>
	<li>Materials preparation
	<ol>
		<li>Have multiple &#39;single-patient-use-bacterial/viral&#39; filters and nose clips readily available.</li>
		<li>Have personal protection equipment (PPE), such as gloves and masks, and disinfectant wipes available. 
		&#8203;NOTE: Refer to laboratory policies for donning and doffing of PPE and infection control guidelines.</li>
	</ol>
	</li>
</ol>
3. Patient preparation
<ol>
	<li>Anthropometry
	<ol>
		<li>Verify the patient&#39;s information: first and last names, date of birth, birth sex, height, weight, and gender identity, if applicable.</li>
		<li>Measure the patient&#39;s height without shoes, with feet together, standing as tall as possible with the eyes level and looking straight ahead, and the back flush against a wall or flat surface. 
		NOTE: For patients unable to stand erect, height may be estimated using arm span. For patients aged 25 years or older, where height measurement has been made previously in the same laboratory, remeasuring height at subsequent visits within 1 year may not be necessary.</li>
		<li>Update weight measurement at each visit.</li>
		<li>Record patient&#39;s usage of bronchodilators, dosage, time/date of last administration, and any medication allergies, such as salbutamol.</li>
	</ol>
	</li>
	<li>Oscillometry test preparation
	<ol>
		<li>Ask the patient to sanitize their hands prior to entering the testing station.</li>
		<li>Outline the test duration of 30 s and the minimum requirement of three trials.</li>
		<li>Explain the sensation generated by the oscillations such as &#39;vibrations&#39; or &#39;fluttering.&#39;</li>
		<li>Ensure that the patient is seated properly in a slight &#39;chin-up&#39; position with both feet on the floor. Avoid slouching against the back of the chair or leg crossing.</li>
		<li>Instruct the patient to breathe normally while holding their cheeks with their palm and fingers and using their thumbs to support the soft tissue of the jaw during measurements. 
		NOTE: Cheek and floor of the mouth support is enforced to avoid upper airway shunt. If the cheeks and soft tissues of the mouth are not supported, flow measured at the mouth is lost in the motion of the upper airway wall.</li>
		<li>Explain to the patient that swallowing should be avoided and the tongue must be below the mouthpiece during the test. 
		&#8203;NOTE: The instructions above apply to both children and adults. Depending on the age of the child, holding a picture or other forms of&#160;visual distraction in front of the child can help ensure that the head posture is maintained during the oscillometry recording period. For adults with cognitive impairment, consider having an accompanying person nearby to coach and calm the patient to breath normally. For patients with physical impairment, some oscillometry devices are portable, and can be brought to the patient bedside or wheelchair. Also consider asking the accompanying or another person to provide cheek and jaw support during tests.</li>
	</ol>
	</li>
</ol>
4. Software setup
NOTE: Please refer to the manufacturer&#39;s instruction manual for individual instructions.
<ol>
	<li>New patient setup
	<ol>
		<li>Select New Patient and enter the patient&#39;s information such as first and last names, date of birth, birth sex, height, weight, ethnicity (if applicable), and smoking history.</li>
		<li>Check that all information entered is correct prior to selecting Standard Test.</li>
		<li>Ensure that the correct wavelength setup is selected. In this demonstration, select Airwave Oscillometry from the Template dropdown menu. The choice of specific wavelengths and wavelength combinations will be different amongst the different manufacturers. Follow the software instruction manual for the specific device.</li>
		<li>Ensure the appropriate set of reference values is selected: Oostveen et al.56 or Brown et al.57 for adults, and Nowowiejska et al.58 for children ages 3 to 17. 
		NOTE: The preferred and availability of reference values may differ depending on each laboratory&#39;s policy and oscillometry device manufacturer.</li>
	</ol>
	</li>
	<li>Existing patient setup
	<ol>
		<li>Click on Select Patient and choose the correct patient&#39;s file by verifying their information such as first and last names and date of birth.</li>
		<li>Ensure patient&#39;s weight and height (if applicable) are updated prior to the start of testing.</li>
		<li>Select Standard Test and choose Airwave Oscillometry from the Template dropdown menu. See also section 4.1.3.</li>
	</ol>
	</li>
</ol>
5. Testing procedure
<ol>
	<li>Oscillometry device setup
	<ol>
		<li>Attach a &#39;single-patient-use-bacterial/viral&#39; filter to the oscillometry device.</li>
		<li>Ensure the oscillometry device is ready in the testing mode.</li>
	</ol>
	</li>
	<li>Spectral measurement 
	NOTE: Airwave oscillometry 5-37 Hz in the device shown in the video.
	<ol>
		<li>Remind the patient of the 30 s test duration and the minimum requirement of three measurements.</li>
		<li>Instruct the patient to wear a nose clip and provide instructions described in step 3.2.4 and step 3.2.5.</li>
		<li>Adjust the oscillometry device to the patient&#39;s head level.</li>
		<li>Instruct the patient to wet his/her lips before wrapping them around the mouthpiece to form a proper, tight seal. Instruct the patient to begin to breathe normally. 
		NOTE: Inspect for potential air leaks around the mouthpiece and nose clip. Supplementary oxygen must be turned off during measurements to avoid any drift into the oscillometry device.</li>
		<li>Observe the patient&#39;s breathing pattern and start recording following a minimum of three stable tidal breaths. 
		NOTE: (Optional): During the test, inform the patient of the time remaining during each measurement.</li>
		<li>Provide adequate rest time in between each measurement and adjust accordingly, based on the patient. 
		NOTE: Patients on supplemental oxygen may require longer rest intervals. Provide supplemental oxygen as needed during the rest intervals.</li>
		<li>Following a minimum of three measurements, proceed to Step 6 to assess acceptability and reproducibility.</li>
	</ol>
	</li>
	<li>Post-bronchodilator response - optional
	<ol>
		<li>Administer bronchodilator (salbutamol or ipratropium bromide) via a spacer.</li>
		<li>Record the method and number of doses administered.</li>
		<li>Wait for 10 min post-salbutamol/albuterol and 20 min post-ipratropium bromide inhalation.</li>
		<li>Repeat Step 5.2 to assess post-bronchodilator response</li>
	</ol>
	</li>
	<li>10 Hz (intra-breath) measurement - optional
	<ol>
		<li>Remind the patient that each test duration is 30 s and a minimum of three measurements will be obtained.</li>
		<li>Ensure that the correct wavelength setup for intra-breath measurement is selected.</li>
		<li>Repeat Steps 5.2.2 to 5.2.6.</li>
	</ol>
	</li>
</ol>
6. Access acceptability and reproducibility
<ol>
	<li>Acceptability
	<ol>
		<li>Ensure that the measurements have a validity greater than 70%.</li>
		<li>Check that the symbol beside the measurements have obtained a checkmark. 
		NOTE: If a &#39;Caution&#39; symbol is present, the measurement is unacceptable.</li>
		<li>Inspect each measurement for anomalies or artefacts that may be caused by coughing, tongue obstruction, glottis closure, air leakage around the mouthpiece, attempting to talk, swallowing, and taking a deep breath. 
		NOTE: If the patient is observed taking a deep breath, reset the oscillometry device, as forceful breaths disrupt the motors and the quality of subsequent measurements. To reset, stop testing and then click Zero Channels.</li>
		<li>Review measurements automatically excluded by the software; these include anomalies or artefacts such as cough or glottis closure.</li>
		<li>Exclude any unacceptable measurement with anomalies outlined in Step 6.1.3 and repeat Step 5.2 to obtain additional measurements.</li>
	</ol>
	</li>
	<li>Reproducibility
	<ol>
		<li>Ensure that a minimum of three acceptable measurements are recorded.</li>
		<li>Ensure that the coefficient of variance (CoV) of Rrs (resistance of respiratory system) is &#8804;10% in adults and &#8804;15% in children.</li>
		<li>Repeat Step 5.2 to obtain additional measurements if the 3 acceptable measurements have CoV &#62;10% in adults and &#62;15% in children.</li>
		<li>Repeat 6.1 to determine acceptability and report three acceptable measurements with CoV &#8804;10% in adults and &#8804;15% in children.</li>
	</ol>
	</li>
</ol>
7. Disinfection
<ol>
	<li>Discard patient&#39;s mouthpiece and nose clip into the wastebin.</li>
	<li>Use disinfectant wipes to clean the oscillometry device and patient&#39;s chair.</li>
	<li>Doff gloves and sanitize hands.</li>
	<li>Place the red dust cap back onto the oscillometry device to avoid any contamination. 
	NOTE: Each laboratory&#39;s infection control policy may be different.</li>
</ol>
8. Reporting results
NOTE: Refer to Figure 3 for details.
<ol>
	<li>Include patient&#39;s first and last names, height, weight, age, birth sex, BMI, and smoking history.</li>
	<li>Include device name, model, software version, and manufacturer.</li>
	<li>Include input signal frequencies and duration of individual recordings.</li>
	<li>Report the mean of acceptable and reproducible measurements and the CoV for these reported measurements. 
	NOTE: If the CoV is higher than the specified upper limit, the results should be flagged so that the interpreting physician may interpret the results with caution.</li>
	<li>Select reference equations.</li>
	<li>Include impedance graph demonstrating Rrs and Xrs versus oscillation frequency.</li>
	<li>Include post-bronchodilator response with dosage and method of administration including z-scores and absolute percentage change - optional</li>
</ol>
9. Quality control/quality assurance
<ol>
	<li>Perform regular audits (weekly or monthly) depending on the volume of oscillometry testing in the laboratory.</li>
	<li>Assess each operator using a standardized checklist to ensure oscillometry tests are conducted accurately and professionally.</li>
	<li>Provide regular feedback to operators and hold quarterly quality assurance meetings to reflect on laboratory matters.</li>
	<li>Ensure biologic&#160;quality controls are conducted weekly with at least two healthy non-smoking subjects, and measurements are within &#177;2SD of their mean baseline. 
	NOTE: This is extremely important for validating testing equipment and procedures when there are multiple oscillometry devices in the laboratory.</li>
	<li>Conduct quarterly self-inspection and annual factory maintenance of oscillometry devices for calibration and quality checks.</li>
</ol>

CWC has received speaking fees for webinars supported by Thorasys Thoracic Medical Systems Inc. and consulting fees from Theravance Biopharma, Inc.

The critical steps in a high quality oscillometry measurement can be categorized into the domains of patient, equipment, and operator. Ensuring that the patient is relaxed and comfortable so that the measurements collected are at resting functional residual volume is key. The patient posture is very important; ensure that the patient is sitting upright with both feet on ground with no crossing of legs. The enforcement of cheek and jaw support, good placement of the nose clip, and ensuring lips are sealed around the mouthpiece will eliminate shunting and air leaks1,2,3. The equipment must be calibrated and verified prior to use. The operator must be able to recognize acceptable and unacceptable recordings and able to troubleshoot the underlying cause of unacceptable readings or artefacts to ensure reported measurements have CoV &#8804;10%1,2,3. Quality control and assurance must be maintained to not only ensure the oscillometry device is validated, but also the quality of tests.
Training of the operator to recognize the patterns produced by common artefacts such as swallowing, leaks, and shunting will allow for timely repeated measurements to obtain quality tests. There are instances when oscillometry is performed at different lung volumes, (e.g., in supine position). Under these circumstances, all the steps described in the protocol can still be applied.
While oscillometry is an easier and more rapid modality of pulmonary function testing, errors in measurements, and thus interpretation, will occur if deviations from the standardized protocol and quality control steps occur. Our protocol is based on the device used at our center. The conduct of oscillometry will be the same across devices. However, there will be differences in the technical aspect of calibration and software applications. Readers are advised to follow the manual for the different instruments.
Oscillometry is faster and easier to perform than spirometry. Moreover, young children and adults with language, physical, and/or cognitive impairment that impede the ability to perform the forced expiratory maneuvers needed for spirometry can still perform oscillometry as it is conducted during normal breathing. In some centers, oscillometry has supplanted spirometry as the initial screening tool for lung disease. Enhancing training in the conduct of oscillometry will facilitate its wider application as a diagnostic tool and ensure quality control of the tests conducted.
Although oscillometry is a fast and easy technique, quality controls are needed to ensure accurate and reproducible measurements. By following international guidelines, research and clinical oscillometry data can be interpreted appropriately so that findings can be applied across different patient populations.

Respiratory oscillometry measures the impedance of the lung and is exquisitely sensitive to changes in respiratory mechanics1, particularly to the peripheral lung and small airways, regions of the lung that are not well assessed by traditional pulmonary function tests.
Over the last several years, the availability of commercial devices and updated technical and quality control/assurance standards2,3 have led to increasing use of oscillometry for clinical and research purposes. However, to date it is not a routine test in the repertoire of pulmonary function modalities, but the technique is anticipated to become more widely used with increasing recognition of its clinical utility. The overall goal of respiratory oscillometry is to provide measurement of respiratory mechanics during normal breathing and assessment of lung function, that is not discernible by current methods of spirometry and plethysmography. Oscillometry offers other advantages over traditional pulmonary function tests as it can be performed in the very young, the elderly, or in patients with cognitive impairment where forced expiratory maneuvers needed for spirometry are impossible. Furthermore, oscillometry can be conducted in anyone who can breathe spontaneously while wearing a nose clip. Unlike standard pulmonary function tests, it is not contraindicated following cataract, intra-abdominal or cardiothoracic surgery, nor following acute myocardial infarction and heart failure. Lastly, several of the oscillometry devices currently available are portable, and can be used in settings outside of a diagnostic laboratory, including clinic and office settings, bedside, or in workplaces.
Oscillometry measures the total respiratory impedance (Zrs) to multi-frequency oscillatory pressure waves1,2,4,5,6. Impedance is composed of the complex sum of respiratory resistance (Rrs) and reactance (Xrs). Rrs reflects the resistance of airways and is largely frequency-independent in health4,7,8. In small airway diseases, Rrs becomes frequency-dependent and increases more in the lower frequencies5,9,10, so that a difference in Rrs&#160;at frequencies between 5 and 19 Hz (R5-19) or 5 and 20 Hz (R5-20) indicates small airway obstruction and heterogeneity of ventilation in different regions of the lung 10,11,12.&#160;Xrs measures the balance of elastic and inertial impedances of the respiratory system. At lower frequencies (e.g., 5 to 11 Hz), Xrs reflects the stiffness or elastance of the pulmonary and chest wall tissues13,14. At higher frequencies, Xrs is dominated by the inertia of the air column in the conducting airways. The resonance frequency (Fres) is the point at which the magnitudes of elastic and inertive reactance are equal. AX is an integrative index of Xrs and is calculated as the area under the Xrs versus frequency graph between 5 Hz and Fres. AX has the units of elastance and is inversely related to the volume of the lung in communication with ventilation. AX increases with restrictive processes and peripheral inhomogeneity. X5 becomes increasingly negative while AX and Fres are increased in both obstructive and restrictive lung diseases4,5. See Figure 1 for depiction of these metrics.
While initially focused on measurement of lung function in children, emerging data show that oscillometry provides useful clinical information in adults as well. It is increasingly used in the clinical setting15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45. Oscillometry has been most extensively studied in obstructive lung diseases where it has been found to offer better diagnostic information than spirometry with respect to asthma control31,32,33,34,35, better correlation with symptoms23,34, and earlier detection36,37,38 of chronic obstructive lung disease (COPD). Our group has shown oscillometry to be more sensitive than spirometry for tracking graft injury following lung transplant46. Several studies have shown that Xrs, specifically&#160;the difference in the mean inspiratory and expiratory reactance at 5 Hz, can distinguish restrictive defects in interstitial lung disease (ILD) from asthma and COPD47, and can differentiate combined pulmonary fibrosis and emphysema from ILD-only48,49. Figure 2 demonstrates the typical oscillometry patterns for normal, restrictive, and obstructive lung diseases. There has been increasing interest to implement oscillometry as another routine modality of pulmonary function testing to supplement and potentially replace some of the current testing modalities for lung function monitoring50,51.
We suggest that oscillometry is useful for screening of lung diseases, in follow-up of patients with known obstructive and restrictive lung diseases, and following lung transplant. The commercial devices are suitable for use in children as young as 2 years old. There is ongoing research with even younger populations52, and as the field grows it may be possible to evaluate infants and newborns.
The goal of the current manuscript is to provide a training manual for clinicians, technologists, and research personnel on the appropriate conduct of oscillometry, following international standard operating protocols and quality control guidelines. Due to the small footprint of most commercial oscillometers, oscillometry can be implemented in multiple settings. The protocol outlined is appropriate for pulmonary function laboratories, physician offices, clinic settings, and other outpatient settings such as workplace occupational health units.

<table><tbody><tr><td>Accel Prevention Disinfectant wipes - 160/canister</td><td>Diversey Care</td><td>100906721</td><td>https://diversey.com/en/</td></tr><tr><td>clearFlo F-100 - 100 Airwave Oscillometry filters</td><td>Thorasys</td><td>101635</td><td>https://www.thorasys.com/</td></tr><tr><td>Noseclip w/cushions, &quot;Snuffer&quot;, bx/1000</td><td>McArthur Medical Sales Inc.</td><td>785-1008BULK</td><td>https://mcarthurmedical.com/</td></tr><tr><td>Tremoflo C-100 Airwave Oscillometry System</td><td>Thorasys</td><td>101969</td><td>https://www.thorasys.com/&lt;br/&gt; Software verison: 1.0.43 build 43&lt;br/&gt; Signal Type: Pseudo-random, relative primes&lt;br/&gt; Frequencies (Hz): 5, 10, 11, 14, 17, 19, 23, 29, 31, 37</td></tr><tr><td>Tremoflo C-100 Calibrated Reference Load 15 cm H2O. s/L</td><td>Thorasys</td><td>101059</td><td>https://www.thorasys.com/</td></tr></tbody></table>

conducting respiratory oscillometry in an outpatient setting

Respiratory oscillometry is a different modality of pulmonary function testing that is increasingly used in a clinical and research setting to provide information regarding lung mechanics. Respiratory oscillometry is conducted through three acceptable measurements of tidal breathing and can be performed with minimal contraindications. Young children and patients who cannot perform spirometry due to cognitive or physical impairment can usually complete oscillometry. The main advantages of respiratory oscillometry are that it requires minimal patient cooperation and is more sensitive in detecting changes in small airways than conventional pulmonary function tests. Commercial devices are now available. Updated technical guidelines, standard operating protocols, and quality control/assurance guidelines have recently been published. Reference values are also available. 
We conducted oscillometry test audits before and after implementing a formal respiratory oscillometry training program and standard operating protocol. We observed improvement in the quality of tests completed, with a significant increase in the number of acceptable and reproducible measurements. 
The current paper outlines and demonstrates a standard operating protocol to conduct respiratory oscillometry in an outpatient setting. We highlight the key steps to ensuring acceptable and reproducible quality measurements according to the recommended European Respiratory Society (ERS) guidelines, as quality control is critical to measurement accuracies. Potential problems and pitfalls are also discussed with suggestions to resolve technical errors.

From October 17, 2017 to April 6, 2018, we conducted the first quality assurance/quality control (QA/QC) audit of the 197 oscillometry tests3. Although all of the operators were trained prior to testing patient with a one hour seminar and on-site testing, 10 (5.08%) unacceptable and/or irreproducible measurements were identified. These measurements were excluded due to cough, tongue obstruction, and CoV greater than 15% following the initial suggested ERS guidelines52. Biologic&#160;quality control (BioQC) was not conducted regularly. The research personnel underwent additional oscillometry training and developed a standard operating protocol to ensure proper ERS guidelines and medical professionalism were in place. The importance of BioQC, a tool to validate testing equipment and procedures, was highlighted to the research personnel, who were reminded to perform regular BioQC tests.3 Improvements were found in subsequent QA/QC audits. Out of the total of 1930 oscillometry tests conducted from April 9, 2018 to June 30, 2019, only three (0.0016%) tests were invalid measurements; these had CoV greater than 15%. Between July 2, 2019 and March 12, 2020, there were 1779 oscillometry tests performed and nine (0.005%) were considered unacceptable, including measurements that had glottis closure, air leakage, and CoV greater than 15%. Refer to Table 1 for additional information.
Since the reinforcement of BioQC in April 2018, research personnel conducted BioQC regularly. At our center, four healthy non-smoking individuals conducted oscillometry daily for the initial 2 weeks to gather a minimum of 10 measurements with the mean with the upper and lower limit (&#177;2SD or standard deviation) with coefficient of variation &#8804;10% between Rrs in the two oscillometry devices in our laboratory. On August 30, 2021, we observed a BioQC measurement that fell outside the individual&#39;s mean &#177;2SD. The individual&#39;s observed R5 was 3.36 cmH2O&#183;s/L (open circle), while R5 mean from the 20 most recent recordings was 4.95 cmH2O.s/L &#177;2SD (dotted line with lower limit at 4.03 and upper limit at 5.86; Figure 4). A second individual conducted their BioQC oscillometry on the same day with the same oscillometry device, and the observed R5 measurement was also outside the mean &#177;2SD. These findings indicate problems related to the instrument rather that than the procedure. Subsequently, the manufacturer was contacted and the device was sent for repair. Upon return of the device, BioQC was repeated on October 15, 2021 to ensure it was within the individual&#39;s R5 measurement range prior to redeployment of device in our laboratory.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63243/63243fig01.jpg" /> 
Figure 1: The impedance versus frequency oscillogram with the resistance curve (solid line) and reactance curves (dotted line), and frequencies at which the measurements are made (solid and open circles in each curve) shown. The area of reactance (AX, hatched area), resonant frequency (Fres. X), and resistance between 5 Hz to 19 Hz (R5-19; two-sided arrow) are illustrated. <a href="https://www.jove.com/files/ftp_upload/63243/63243fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63243/63243fig02.jpg" /> 
Figure 2: The typical oscillometry pattern differences between normal (A), restrictive (B) and obstructive (C) lung diseases. Note the rightward shift of the reactance curve (open circle, dotted line) in the restrictive disease (B), and the trumpet shaped pattern of the obstructive oscillogram (C) with upward shift of the resistance curve (solid circle and line), increased R5-19, and the downward and rightward shift of the resistance curve (broken line; open circles). <a href="https://www.jove.com/files/ftp_upload/63243/63243fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63243/63243fig03.jpg" /> 
Figure 3: The standard template for reporting of oscillometry in our institution. We display the oscillogram using a standardized X-Y axis, and highlight the relevant pre- and post-bronchodilator measurements in different colors to facilitate interpretation of the results. <a href="https://www.jove.com/files/ftp_upload/63243/63243fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63243/63243fig04v2.jpg" /> 
Figure 4: The biologic&#160;quality control (BioQC) summary of R5 measurements from one individual from May 2020 to November 2021. The measurement that fell outside (open circle) the individual&#39;s mean (solid grey line) &#177;2SD (dotted line) was observed in August 30, 2021. <a href="https://www.jove.com/files/ftp_upload/63243/63243fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td>First Audit</td>
			<td>Second Audit</td>
			<td>Third Audit</td>
		</tr>
		<tr>
			<td>October 17, 2017 to April 6, 2018</td>
			<td>April 9, 2018 to June 30, 2019</td>
			<td>July 2, 2019 to March 12, 2020</td>
		</tr>
		<tr>
			<td>Valid</td>
			<td>187</td>
			<td>1927</td>
			<td>1770</td>
		</tr>
		<tr>
			<td>Invalid</td>
			<td>10</td>
			<td>3</td>
			<td>9</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of oscillometry tests acceptability at three time-points 
Personnel underwent refresher training in conduct of oscillometry following the first audit. We also implemented a standard operating protocol for conduct of oscillometry in the pulmonary function laboratory. Significant improvements in the percentage of tests meeting acceptable quality control occurred and were sustained over time. These results demonstrate the effectiveness of developing and adhering to standard operating protocols and quality control guidelines.
Supplemental Table 1. Contraindications for Spirometry53,54 <a href="https://www.jove.com/files/ftp_upload/63243/STable1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Table 2. Bronchodilators Withholding Times for Pulmonary Function Tests53,54 <a href="https://www.jove.com/files/ftp_upload/63243/STable2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Table 3. Bronchodilators Withholding Times for Bronchial Challenge Test53,55 <a href="https://www.jove.com/files/ftp_upload/63243/STable3.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Conducting Respiratory Oscillometry in an Outpatient Setting at JoVE.com

Conducting Respiratory Oscillometry in an Outpatient Setting

We demonstrate a standard operating protocol to conduct respiratory oscillometry, highlighting key quality control and assurance procedures.

Respiratory oscillometry is a different modality of pulmonary function testing that is increasingly used in a clinical and research setting to provide ...

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6.8K Views.  University of Toronto. We demonstrate a standard operating protocol to conduct respiratory oscillometry, highlighting key quality control and assurance procedures. 

Video: Conducting Respiratory Oscillometry in an Outpatient Setting

Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and minute ventilation are routinely reported. Moreover, information can be collected regarding the neural output of breathing, including the existence of central apneas and augmented breaths. An important consideration for UBP is obtaining a breathing segment with a minimal impact of anxious or active behaviors, to elucidate the response to breathing challenges. Here, we present a protocol that allows for short, quiet baselines to be obtained in aged mice, comparable to waiting for longer bouts of quiet breathing. The use of shorter time segments is valuable, as some strains of mice may be increasingly excitable or anxious, and longer periods of quiet breathing may not be achieved within a reasonable timeframe. We placed 22 month-old mice in a UBP chamber and compared four 15 s quiet breathing segments between minutes 60&#8211;120 to a longer 10 min quiet breathing period that took&#160;2&#8211;3 h to acquire. We also obtained counts of central apneas and augmented breaths prior to the quiet breathing segments, following a 30 min familiarization period. We show that 10 min of quiet breathing is comparable to using a much shorter 15 s duration. Additionally, the time leading up to these 15 s quiet breathing segments can be used to gather data regarding apneas of central origin. This protocol allows investigators to collect pattern-of-breathing data in a set amount of time and makes quiet baseline measures feasible for mice that may exhibit increased amounts of excitable behavior. The UBP methodology itself provides a useful and noninvasive way to collect pattern-of-breathing data and allows for mice to be tested over several time points.

Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and ...

Behavior

Home-Based Prescribed Pulmonary Exercise in Patients with Stable Chronic Obstructive Pulmonary Disease

As a systemic disease, chronic obstructive pulmonary disease (COPD) affects the respiratory system, inducing restless and exercise dyspnea. It also impacts exercise capacity and forms a vicious circle in which it further aggravates the condition of patients and accelerates disease progression. As a functional holistic exercise, traditional Chinese exercises (TCE) play an important role in the rehabilitation of COPD on the basis of adjusting the breath and performing coordinated movements. This study investigates the effects of prescribed pulmonary exercises (which are modified from TCE) on exercise capacity of upper and lower limbs, endurance exercise capacity, and quality of life in stable COPD patients. The goal is to determine the accessibility of these prescribed exercises in COPD rehabilitation. Participants are randomly divided into a non-exercise control group (CG) or prescribed pulmonary exercise group (PG) at a ratio of 1: 1. The PG receives intervention for 60 min twice per day, 7 days a week, for a total of 3 months. The intensity is measured using the Borg category-ratio 10 scale and with a heart-rate monitor. Then, an exercise capacity test and quality of life questionnaire are scheduled at 1 week before and after the formal intervention. After 3 months of intervention, the 30 s arm curl test, 30 s sit-to-stand test, 6 min walking test, and quality of life show significant improvement in COPD patients (p &#60; 0.05). These findings indicate that prescribed pulmonary exercises can be applied as alternative, convenient, and effective home- and community-based exercises for stable COPD patients.

Presented here is a protocol to investigate the effects of home-based prescribed pulmonary exercise in stable chronic obstructive pulmonary disease (COPD) patients, which is modified based on traditional Chinese exercises according to dyspnea and limited exercise capacity observed in COPD patients.

As a systemic disease, chronic obstructive pulmonary disease (COPD) affects the respiratory system, inducing restless and exercise dyspnea. It also ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Biology

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Neuroscience

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ensure that both animal welfare and scientific insights advance with the availability of new technologies. This article demonstrates the use of Simplified Whole Body Plethysmography (sWBP) to non-invasively study respiratory failure in a model of lethal respiratory melioidosis. sWBP has the sensitivity to detect breathing in mice through the entirety of the course of the disease, allowing for the moribund-associated symptoms (bradypnea and hypopnea) to be measured and potentially used to develop humane endpoint criteria.
Some of the benefits of sWBP in the context of respiratory disease are that host breath monitoring comes the closest of any physiologic measurement at assessing dysfunction of the primary infected tissue, namely, the lung. In addition to biological significance, the use of sWBP is rapid and non-invasive, minimizing stress in research animals. This work demonstrates the use of in-house sWBP apparatus to monitor disease throughout the course of respiratory failure in the murine model of respiratory melioidosis.

This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively.

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ...

Immunology and Infection

Integration of Brain Tissue Saturation Monitoring in Cardiopulmonary Exercise Testing in Patients with Heart Failure

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction (HF). However, in clinical cardiopulmonary exercise testing (CPET), cerebral hemodynamics is not assessed. NIRS is used to measure cerebral tissue oxygen saturation (SctO2) in the frontal lobe. This method is reliable and valid and has been utilized in several studies. SctO2 is lower during both rest and peak exercise in patients with HF than in healthy controls (66.3 &#177; 13.3% and 63.4 &#177; 13.8% vs. 73.1 &#177; 2.8% and 72 &#177; 3.2%). SctO2 at rest is significantly linearly correlated with peak VO2 (r = 0.602), oxygen uptake efficiency slope (r = 0.501), and brain natriuretic peptide (r = -0.492), all of which are recognized prognostic and disease severity markers, indicating its potential prognostic value. SctO2 is determined mainly by end-tidal CO2 pressure, mean arterial pressure, and hemoglobin in the HF population. This article demonstrates a protocol that integrates SctO2 using NIRS into incremental CPET on a calibrated bicycle ergometer.

This protocol integrated near-infrared spectroscopy into conventional cardiopulmonary exercise testing to identify the involvement of the cerebral hemodynamic response in exercise intolerance in patients with heart failure.

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

Employing the Forced Oscillation Technique for the Assessment of Respiratory Mechanics in Adults

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and diseased individuals. FOT, a complementary method to traditional pulmonary function testing, utilizes a range of oscillatory frequencies superimposed on tidal breathing to measure the functional relationship between airway pressure and flow. This passive assessment provides an estimate of respiratory system resistance (Rrs) and reactance (Xrs) that reflect airway caliber and energy storage and dissipation, respectively. Despite the recent increase in popularity and updated Technical Standards, clinical adoption has been slow which relates, in part, to the lack of standardization regarding the acquisition and reporting of FOT data. The goal of this article is to address the lack of standardization across laboratories by providing a comprehensive written protocol for FOT and an accompanying video. To illustrate that this protocol can be utilized irrespective of a particular device, three separate FOT devices have been employed in the case examples and video demonstration. This effort is intended to standardize the use and interpretation of FOT, provide practical suggestions, as well as highlight future questions that need to be addressed.

As the use of forced oscillation technique (FOT) is increasingly utilized to characterize respiratory mechanics, there is a need to standardize methods with respect to nascent technical guidelines and various manufacturer's recommendations. A detailed protocol is provided including FOT assessment and interpretation for two cases to facilitate the standardization of methods.

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and ...

Electrical Impedance Tomography: Enhancing Pulmonary Health and Treatment

Monitoring Lung Function with Electrical Impedance Tomography in the Intensive Care Unit

Electrical Impedance Tomography (EIT) is a groundbreaking, non-invasive, and radiation-free imaging technique for continuous, real-time ventilation monitoring. It also has an application in pulmonary perfusion monitoring. EIT quantifies ventilation and perfusion patterns across the lung from the measurement and processing of impedance changes in the thorax. It is a powerful tool for clinicians to visualize breath-by-breath changes in pulmonary function. 
An innovative application of EIT is its ability to assess pulmonary perfusion using the kinetic analysis of a hypertonic solution injection during a breath-hold. The solution generates an impedance change in the thorax as it circulates through the pulmonary vasculature. This indirect method allows for the estimation of perfusion patterns, contributing significantly to our understanding of pulmonary blood flow dynamics at the bedside.
EIT is not just a tool for monitoring but also can be critical for the diagnosis of respiratory pathologies such as pneumothorax and bronchial intubation. It can help identify the etiology of ventilation/perfusion (V/Q) mismatch in patients receiving invasive mechanical ventilation, which is not possible with other diagnostic tools. Moreover, EIT can assist in the individual optimization of ventilator settings, such as Positive End-Expiratory Pressure (PEEP) titration and tidal volume improving oxygenation and lung health in critical care.
In summary, EIT represents a paradigm shift in bedside pulmonary monitoring and diagnostics. Its non-invasive nature and immediacy of data make EIT an indispensable tool in modern respiratory medicine. With its growing applications, EIT will be pivotal in advancing our understanding of and approach to respiratory care, particularly in intensive care settings.

Electrical Impedance Tomography is a non-invasive, radiation-free, real-time pulmonary ventilation monitoring tool. By measuring impedance changes in the thorax, it can visualize the distribution of air on a breath-by-breath basis. Initially intended for ventilation monitoring, electrical impedance tomography can also measure perfusion via intravenous injection of a saline solution.

Electrical Impedance Tomography (EIT) is a groundbreaking, non-invasive, and radiation-free imaging technique for continuous, real-time ventilation ...