The authors would like to thank Restech for providing an educational grant.

The following study protocol was reviewed and approved by the Ethics Committee of the Medical Faculty of the University of Cologne.
NOTE: Ensure that the patient arrives NPO for the following gastrointestinal function testing. Perform a high-resolution manometry to determine the exact location of the lower esophageal sphincter (LES) and to rule out esophageal dysmotility disorders such as achalasia. The patient should be off antisecretory medication for at least 7 days prior to ensure a valid pH measurement.
1. Setting up the esophageal pH monitoring system
<ol>
	<li>Insert batteries into the esophageal monitoring system.</li>
	<li>Connect the esophageal pH catheter to the device.</li>
	<li>Start the device. Make sure date and time is accurate. Select Start study.</li>
	<li>Calibrate the catheter in solutions with pH 4 and 7 and rinse the probe in water. Set the probe aside for insertion.</li>
</ol>
2. Setting up laryngopharyngeal pH monitoring system by first formatting the SD card with patient&#8217;s data.
<ol>
	<li>Insert the SD card into the computer and open the software for laryngopharyngeal pH monitoring (e.g., DataView 4). Add a new patient by clicking on New and typing in all details about the patient. Click Save to save the data to the SD card. Eject the SD card and insert it into the recorder.</li>
	<li>Insert batteries into the transmitter using the supplied screwdriver to remove the cover of the transmitter case and install a new CR1632 lithium coin battery.</li>
	<li>Insert two AA batteries into the recorder.</li>
	<li>Turn the recorder on. Use the up/down keys to select Setup. Press any round key to select the Setup mode. Modify the time and date if needed by using the up/down keys to change the digits and any round key to select the respective value. If time and date is correct, select Yes and continue.</li>
	<li>Select No when the Recorder asks &#8220;Tst PSG Adapter?&#8221;.</li>
	<li>Select Study in the main menu.</li>
	<li>The transmitter is now detected automatically. Confirm that the correct transmitter is detected and press any round key for confirmation. The transmitter displays the serial number that is paired with the recorder.</li>
	<li>Attach the probe to the transmitter. 
	NOTE: When connected to the transmitter, the red light-emitting diode (LED) at the probe tip will flash once per second. The LED stops flashing after four hours to conserve battery life.</li>
	<li>Select Hydrate for the calibration process. Hydrate the probe with the provided clear water solution by placing the probe tip in the clear water solution and agitating briefly. Press any round key to start the hydration process. 
	NOTE: The Recorder will display &#8220;Hydrating&#8221; and will count down from &#8220;300 seconds&#8221; to zero or will transition to 15 seconds countdown when the recorder detects that the probe is hydrated. Set the probe aside for insertion.</li>
</ol>
3. Placing the esophageal pH catheter first
<ol>
	<li>Ensure that the patient is sitting upright, looks straight and swallows periodically. Provide a small glass of water with a straw to assist swallowing.</li>
	<li>Ask the patient which side of their nose is clearer and easier to breathe through.</li>
	<li>Apply a topical gel that contains a local anesthetic to the probe shaft for ease of insertion.</li>
	<li>Insert the Probe through the patient&#8217;s nose, straight in and not up. A measuring scale on the probe shaft helps to determine the correct position. 
	NOTE: The correct position is 5 cm above the LES as previously determined by high resolution manometry.</li>
</ol>
4. Securing the esophageal pH catheter
<ol>
	<li>Secure the probe as closely to the nares as possible to ensure that it does not move during the study using surgical tape.</li>
	<li>Attach the probe to the cheek, near the nose.</li>
	<li>Loop the probe over the ear and use tape to affix the probe to the neck, behind and below the ear.</li>
</ol>
5. Placing the laryngopharyngeal pH catheter
<ol>
	<li>Ensure that the patient is sitting upright, looks straight and swallows periodically.</li>
	<li>Apply a topical gel that contains a local anesthetic to the distal end of the probe shaft for ease of insertion. 
	Caution: DO NOT apply topical gel to the probe tip where the sensor is located.</li>
	<li>Insert the probe through the patient&#8217;s opposite nostril of the esophageal catheter, straight posteriorly and not up. 
	NOTE: The round probe tip will help it curve around and through the velopharyngeal port into the oropharynx. The distance from the patient&#8217;s nares to the oropharynx can often be estimated by using the distance from the nares to the earlobe.</li>
	<li>Confirm that the red light is clearly visible, lateral to or slightly below the uvula. Use a tongue depressor for clear visibility if needed. 
	NOTE: If the patient&#8217;s gag reflex is triggered, retract the probe to a higher position in the oropharynx.</li>
</ol>
6. Securing the probe and transmitter
<ol>
	<li>Secure the probe as closely to the nares as possible to ensure that it does not move during the study using surgical tape.</li>
	<li>Attach the probe to the cheek, near the nose.</li>
	<li>Loop the probe over the ear and use tape to affix the probe to the neck, behind and below the ear.</li>
	<li>Attach the transmitter to the patient&#8217;s clothing with the clip-on transmitter carrying case.</li>
</ol>
7. Ensuring that simultaneous pH measurements are performed
<ol>
	<li>Make sure the internal clocks of both pH measurement devices are synchronized. Manually adjust the times if needed.</li>
	<li>Start esophageal and laryngopharyngeal pH studies concurrently. Select a 24-hour study for the laryngopharyngeal pH measuring device.</li>
</ol>
8. Patient diary and instructions
<ol>
	<li>Ask the patient to fill out a detailed diary that includes mealtimes, exact time of supine periods and symptoms they experience during the time of the study.</li>
	<li>Provide the following instructions for the 24-hour measurement period to the patient to ensure a successful data recording: 
	Three mealtimes on day 1 of the study, one mealtime on day 2 of the study. 
	No supine periods before 9pm. 
	Eat, drink, and behave as usual (daily activities can be done). 
	Caution: Devices are NOT waterproof.</li>
</ol>
9. Removing the esophageal and laryngopharyngeal probe after the 24-hour study period is completed
<ol>
	<li>Concurrently end the study on both devices.
	<ol>
		<li>End the esophageal pH study by pressing the two buttons located in the middle simultaneously until &#8220;upload later&#8221; appears on the display.</li>
		<li>End the laryngopharyngeal pH study by simultaneously pressing the cough, ESC and heartburn button until &#8220;complete&#8221; appears on the display.</li>
	</ol>
	</li>
	<li>Remove tape and gently pull out both catheters.</li>
</ol>
10. Interpreting the results obtained by the esophageal pH study
<ol>
	<li>Connect the device to a computer. Open the software used for analyzing results obtained from the esophageal pH measuring device and upload the patient&#8217;s study.</li>
	<li>Manually type in the patient&#8217;s data and the information obtained by the patient&#8217;s diary, such as mealtimes, upright and supine periods and symptoms experienced during the study period. Delete any button presses that might have accidentally been performed by the patient during the study period.</li>
	<li>Exclude mealtimes from the data analysis.</li>
	<li>On the report, look at important parameters and thresholds provided to determine an abnormal esophageal acid exposure such as the composite score, the total % pH below baseline (upright and supine) and total number of events.</li>
	<li>Calculate a composite score (DeMeester score) for the study period using % time pH &#60; 4 total, upright and supine, total number of events, number of events lasting longer than 5 minutes and the duration of the longest event14. Make sure the report shows the composite score. 
	NOTE: A DeMeester score &#62; 14.72 displays an abnormal esophageal acid exposure.</li>
</ol>
11. Interpreting results obtained by laryngopharyngeal pH monitoring
<ol>
	<li>Insert the SD card into the computer and open the software used for analyzation of results obtained by the laryngopharyngeal pH measuring system. To upload the study, click on Retrieve.</li>
	<li>Manually add the information obtained by the patient&#8217;s diary, such as mealtimes, upright and supine periods and symptoms experienced during the study period. Left click on the graph and drag and highlight the area on which you want to add the event and choose the respective event. Delete any button presses that might have accidentally been performed by the patient during the study period.</li>
	<li>Exclude mealtimes from the data analysis.</li>
	<li>Click on Report to see a graph displaying acid exposure in the oropharynx during the study period, including supine period, symptoms and mealtimes as well as a study summary.</li>
	<li>On the report, look at important parameters and thresholds to determine an abnormal oropharyngeal acid exposure such as the composite score, the total % pH below baseline (pH &#60; 5.5 upright and pH &#60; 5 supine) and total number of events.</li>
	<li>Calculate a composite score (RYAN Score) for both the upright and supine period using the % time pH below 5.5 upright and 5 supine, total number of events and the duration of the longest event8. Make sure, a composite score for the upright and supine period is shown in the report. 
	NOTE: A RYAN Score of &#62; 9.4 upright and &#62; 6.8 supine shows a severely abnormal oropharyngeal acid exposure.</li>
</ol>

<ol>
	<li>Dent, J., El-Serag, H. B., Wallander, M. A., Johansson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+gastro-oesophageal+reflux+disease:+a+systematic+review.">Epidemiology of gastro-oesophageal reflux disease: a systematic review.</a> Gut. 54 (5), 710-717 (2005).</li><li>Koop, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S2k+guideline:+gastroesophageal+reflux+disease+guided+by+the+German+Society+of+Gastroenterology:+AWMF+register+no.+021-013.">S2k guideline: gastroesophageal reflux disease guided by the German Society of Gastroenterology: AWMF register no. 021-013.</a> Zeitschrift f&#252;r Gastroenterologie. 52 (11), 1299-1346 (2014).</li><li>Becker, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+aspects+in+the+pathomechanism+and+diagnosis+of+the+laryngopharyngeal+reflux-clinical+impact+of+laryngeal+proton+pumps+and+pharyngeal+pH+metry+in+extraesophageal+gastroesophageal+reflux+disease.">New aspects in the pathomechanism and diagnosis of the laryngopharyngeal reflux-clinical impact of laryngeal proton pumps and pharyngeal pH metry in extraesophageal gastroesophageal reflux disease.</a> World Journal of Gastroenterology. 21 (3), 982-987 (2015).</li><li>Vakil, N., van Zanten, S. V., Kahrilas, P., Dent, J., Jones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Montreal+definition+and+classification+of+gastroesophageal+reflux+disease:+a+global+evidence-based+consensus.">The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus.</a> American Journal of Gastroenterology. 101 (8), 1900-1920 (2006).</li><li>Issing, W. J., Karkos, P. D., Perreas, K., Folwaczny, C., Reichel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-probe+24-hour+ambulatory+pH+monitoring+for+diagnosis+of+laryngopharyngeal+reflux.">Dual-probe 24-hour ambulatory pH monitoring for diagnosis of laryngopharyngeal reflux.</a> Journal of Laryngology &amp; Otology. 118 (11), 845-848 (2004).</li><li>Smit, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+pH+measurements+at+the+upper+esophageal+sphincter.">Ambulatory pH measurements at the upper esophageal sphincter.</a> Laryngoscope. 108 (2), 299-302 (1998).</li><li>Worrell, S. G., DeMeester, S. R., Greene, C. L., Oh, D. S., Hagen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+pH+monitoring+better+predicts+a+successful+outcome+for+extraesophageal+reflux+symptoms+after+antireflux+surgery.">Pharyngeal pH monitoring better predicts a successful outcome for extraesophageal reflux symptoms after antireflux surgery.</a> Surgical Endoscopy. 27 (11), 4113-4118 (2013).</li><li>Ayazi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+measurement+of+pharyngeal+pH:+normal+values+and+discriminating+pH+threshold.">A new technique for measurement of pharyngeal pH: normal values and discriminating pH threshold.</a> Journal of Gastrointestinal Surgery. 13 (8), 1422-1429 (2009).</li><li>Becker, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+agreement+analysis+and+day-to-day+comparison+of+pharyngeal+pH+monitoring+with+pH/impedance+monitoring+in+patients+with+suspected+laryngopharyngeal+reflux.">First agreement analysis and day-to-day comparison of pharyngeal pH monitoring with pH/impedance monitoring in patients with suspected laryngopharyngeal reflux.</a> Journal of Gastrointestinal Surgery. 16 (6), 1096-1101 (2012).</li><li>Wilhelm, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+pH+monitoring+in+gastrectomy+patients+-+what+do+we+really+measure.">Pharyngeal pH monitoring in gastrectomy patients - what do we really measure.</a> United European Gastroenterology Journal. 4 (4), 541-545 (2016).</li><li>Mazzoleni, G., Vailati, C., Lisma, D. G., Testoni, P. A., Passaretti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+oropharyngeal+pH-monitoring+and+esophageal+pH-impedance+monitoring+in+patients+with+suspected+GERD-related+extra-esophageal+symptoms.">Correlation between oropharyngeal pH-monitoring and esophageal pH-impedance monitoring in patients with suspected GERD-related extra-esophageal symptoms.</a> Neurogastroenterology &amp; Motility. 26 (11), 1557-1564 (2014).</li><li>Fuchs, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+laryngopharyngeal+pH+monitoring+(Restech)+and+conventional+esophageal+pH+monitoring-correlation+using+a+large+patient+cohort+of+more+than+100+patients+with+suspected+gastroesophageal+reflux+disease.">Simultaneous laryngopharyngeal pH monitoring (Restech) and conventional esophageal pH monitoring-correlation using a large patient cohort of more than 100 patients with suspected gastroesophageal reflux disease.</a> Dis Esophagus. 31 (10), (2018).</li><li>Fuchs, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refluxassoziierte+Ver&#228;nderungen+des+Rest&#246;sophagus+nach+&#214;sophagektomie+und+Magenhochzug+-+Funktionsdiagnostik+im+Langzeitverlauf+bei+Patienten+mit+&#214;sophaguskarzinom+mit+einem+neuen+Instrument.">Refluxassoziierte Ver&#228;nderungen des Rest&#246;sophagus nach &#214;sophagektomie und Magenhochzug - Funktionsdiagnostik im Langzeitverlauf bei Patienten mit &#214;sophaguskarzinom mit einem neuen Instrument.</a> Zeitschrift f&#252;r Gastroenterologie. 54 (08), 436 (2016).</li><li>Jamieson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+24-h+esophageal+pH+monitoring:+normal+values,+optimal+thresholds,+specificity,+sensitivity,+and+reproducibility.">Ambulatory 24-h esophageal pH monitoring: normal values, optimal thresholds, specificity, sensitivity, and reproducibility.</a> American Journal of Gastroenterology. 87 (9), 1102-1111 (1992).</li><li>M&#252;ller, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+improvement+for+evaluation+of+laryngopharyngeal+pH+testing+(Restech)+-+a+comparison+between+DataView+3+and+4.">Software improvement for evaluation of laryngopharyngeal pH testing (Restech) - a comparison between DataView 3 and 4.</a> World Journal of Gastrointestinal Surgery. 12 (5), 236-246 (2020).</li><li>Weitzendorfer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pepsin+and+oropharyngeal+pH+monitoring+to+diagnose+patients+with+laryngopharyngeal+reflux.">Pepsin and oropharyngeal pH monitoring to diagnose patients with laryngopharyngeal reflux.</a> Laryngoscope. 130 (7), 1780-1786 (2020).</li><li>Neto, R. M. L., Herbella, F. A. M., Schlottmann, F., Patti, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+DeMeester+score+still+define+GERD.">Does DeMeester score still define GERD.</a> Dis Esophagus. 32 (5), (2019).</li><li>Anandasabapathy, S., Jaffin, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+intraluminal+impedance+in+the+evaluation+of+patients+with+persistent+globus+on+proton+pump+inhibitor+therapy.">Multichannel intraluminal impedance in the evaluation of patients with persistent globus on proton pump inhibitor therapy.</a> Annals of Otology, Rhinology &amp; Laryngology. 115 (8), 563-570 (2006).</li></ol>

Dolores M&#252;ller received an educational grant from Restech. All other authors have nothing to disclose.

Esophageal pH monitoring is commonly used to confirm the diagnosis of GERD in patients with typical reflux symptoms. However, many patients present, in addition to typical reflux symptoms, with atypical reflux symptoms such as cough or hoarseness most likely related to LPR. Current guidelines suggest an empiric trial with PPIs to proof a reflux related origin of those symptoms and a standardized objective measurement is lacking2. Laryngopharyngeal pH testing, due to its location above the upper esophageal sphincter, provides a new objective diagnostic method to measure abnormal oropharyngeal acid exposure and may lead to more reliable results in those patients.
Previous studies tried to analyze the correlation between laryngopharyngeal and esophageal pH monitoring9,10,11. However, those studies did not establish a standardized protocol, included only a small number of patients (n &#8804; 36) or did not perform both measurements simultaneously. Hence, a valid correlation between both techniques could not be found. Weitzendorfer et al. recently published a larger series of 70 patients, that underwent simultaneous esophageal and laryngopharyngeal pH testing in addition to a thorough assessment of symptoms, quality of life and saliva sampling16. The authors state that no correlation between a positive oropharyngeal acid exposure and any other objective or subjective test result could be established. Wilhelm et al. previously tried to establish a reliable reference group for the validation of laryngopharyngeal pH monitoring in patients after total gastrectomy10. Unfortunately, only a small number of patients (n=10) was included in the study and since upright and supine periods were not entered correctly, no valid results could be obtained. In addition, no esophageal pH monitoring was performed to investigate the correlation between both methods, or the origin of positive laryngopharyngeal test results. We recently published data on the correlation between simultaneous laryngopharyngeal and esophageal pH measurement of 101 patients with suspected GERD and extraesophageal symptoms and showed that results of both measurements do not necessarily need to correspond12. Due to different thresholds, different probe location and pathophysiology, comparison of both methods can be challenging and misleading. We believe that more than one reflux scenario exists, and esophageal and laryngopharyngeal pH monitoring rather complement each other leading to a better evaluation of this challenging patient cohort. We agree with previous studies, that as of now, laryngopharyngeal pH monitoring alone is not suitable as a screening device for GERD or LPR and further studies are needed to clarify the origin of non-correlating test results. However, our previous study has shown that this standardized protocol presents as a great addition to the diagnostic pathway of this challenging patient cohort especially for surgical decision making. Furthermore, we developed a human reflux model with patients following esophagectomy and reconstruction with a gastric interposition. Those patients often suffer from severe reflux symptoms postoperatively and present with no reflux barrier and a limited esophageal motility, concepts that are important contributors to the pathophysiology of GERD. We used this cohort as a control group to further validate our standardized protocol. Final numbers are unpublished, but early work showed 100% correlation between both pH monitoring methods in this volume-reflux model13.
In addition, previous studies did not consider that some reflux episodes may not reach the oropharynx leading to a discrepancy that can, however, be physiologically explained. In addition, different thresholds for laryngopharyngeal and esophageal pH lead to a different calculation of composite acid exposure scores for both pH monitoring methods. Whereas a pH of &#60; 5.5 upright or &#60; 5.0 supine is considered abnormal in the oropharynx a pH of &#60; 4 is abnormal for the distal esophagus8,14. Therefore, a reflux episode with a pH of 5 is considered normal in the distal esophagus but measured as an abnormal reflux episode in the oropharynx. Furthermore, a DeMeester Score of &#62; 14.72 shows abnormal esophageal acid exposure and a negative score states that the patient has no pathological reflux17. However, only severe oropharyngeal reflux leads to an abnormal RYAN Score of &#62; 9.4 upright and &#62; 6.8 supine. A patient with mild or moderate abnormal oropharyngeal acid exposure might still have a normal RYAN Score.
This standardized protocol ensures simultaneous performance of esophageal and laryngopharyngeal pH measurement. Both measurements are started concurrently, and internal clocks are synchronized to ensure a truly simultaneous measurement. In addition, patients are asked to maintain a detailed diary throughout the measurement for mealtimes, symptoms, upright and recumbent phases. Diary entries are then entered manually for both devices, ensuring the right timing and accuracy. Moreover, this ensures that the same lifestyle and day-to-day changes are displayed in both measurements.
We recommend that patients are off antisecretory medication for at least 7 days prior to this standardized protocol to ensure a valid pH measurement. Both, laryngopharyngeal and esophageal pH monitoring, show the highest sensitivity and specificity when performed off PPIs. If the patient cannot pause this medication due to an unbearable symptom load or other reasons, the same protocol may still be performed. However, instead of the DeMeester and the RYAN score, the graphic presentation of the data as well as impedance can be helpful tools to interpret the test results. Furthermore, this protocol includes a hand-written diary. However, instead of a detailed written diary, patients could also use button presses on both devices to record their symptoms and supine periods. Our clinical experience has shown, that keeping a written diary is easier for most patients and less error prone. In addition, both pH measurements can be performed separately if needed, however, no correlation of results can be obtained in that case. We usually place the esophageal probe first, as shown in the video manual. It does not matter from a scientific standpoint which probe is placed first, but the esophageal probe is usually more difficult to place making it easier for the patient to start with the most uncomfortable part of the study and having both nostrils to choose from. If the probe curls or does not seem to be able to pass down to the lower esophagus, changing the patient&#8217;s head position and overall posture may help. Often times, using the other nostril may lead to a successful placement of the probe. If the red light of the laryngopharyngeal measurement probe stops blinking, before the probe has been successfully placed, the battery of the transmitter can be reinstalled to make it blink again.
Critical steps of the protocol include the exact determination of the LES, ensuring a correct placement of the esophageal pH probe 5 cm above the LES and correct placement of the oropharyngeal pH probe lateral to or slightly below the uvula. Only if both probes are placed correctly, a valid correlation between results can be obtained. In addition, securing the probes to maintain exact position is most important and challenging. Especially heavy make-up, facial hair and hot days can complicate securing the probe. Furthermore, ensuring that the patients keeps a detailed and honest diary with exact times is crucial to later correlate supine periods, symptoms, and mealtimes with measured reflux episodes. Overall, all patients in our cohort tolerated both pH probes.
Limitations of the method lay especially in the location of the probes. Esophageal pH monitoring has shown to present reliable results determining abnormal acid exposure in patients with GERD14. However, due to its location 5 cm above the LES, an evaluation of acid exposure in the oropharynx cannot be performed. We typically use a long probe impedance pH monitoring system at our institution to not only measure distal esophageal pH but in addition, bolus movement and non-acid reflux events. Particularly, impedance has shown to increase the sensitivity of the measurement in patients with atypical symptoms18. The protocol, however, can also be used for a regular distal esophageal pH monitoring system. Wireless distal esophageal pH monitoring can also be used instead of a conventional measuring system. Laryngopharyngeal pH testing provides a validated measurement of acid exposure above the upper esophageal sphincter, measuring liquid and vaporized acid, however, does not measure reflux episodes that do not reach the oropharynx8. Furthermore, day-to-day lifestyle changes challenge a 24-hour pH measurement as the patient can have a normal test result for the measured time period, however, complain of reflux symptoms on another days, that has not been measured. The laryngopharyngeal pH monitoring system provides an option for a 48-hour study period which may lead to more reliable results. In addition, a new software for evaluation of results obtained by laryngopharyngeal pH monitoring was released recently. A study comparing both software versions showed that results obtained by either software cannot be compared to each other15. Results from this study were evaluated using the new and improved software version, however, retrospective comparison of data may be limited by the use of different software versions.
Future studies need to address if laryngopharyngeal pH monitoring can be used as screening tool for LPR and if this new technique is able to predict a successful surgical outcome in patients with mainly atypical reflux symptoms. However, the use of the protocol ensures a comprehensive evaluation of acid exposure in this challenging patient cohort is maintained and provides guidance on complementary laryngopharyngeal and esophageal pH monitoring.

Gastroesophageal reflux disease (GERD) is one of the most common benign diseases affecting up to 20% of people in western countries1. In addition to typical symptoms such as heartburn or regurgitation, some patients may suffer from atypical symptoms such as cough, hoarseness, or asthma2,3. Despite the agreement that chronic cough, chronic laryngitis, and asthma can have a reflux related origin and are significantly associated with laryngopharyngeal reflux (LPR), the exact pathomechanism still remains unclear. As these symptoms are usually part of a multifactorial process, they depict a great diagnostic and therapeutic challenge4.
Distal esophageal pH monitoring 5 cm above the lower esophageal sphincter is commonly used to determine abnormal esophageal acid exposure in patients with suspected GERD2. In an attempt to use the same technique, proximal conventional pH monitoring was introduced in the late 1990s as a diagnostic device to measure abnormal acid exposure at the upper esophageal sphincter (UES) as the probe is placed at or slightly above the UES. However, this method does not always provide valid and accurate results as the probe is not designed for an oropharyngeal environment leading to the measurement of invalid artifacts such as pseudoreflux events caused by drying out of the probe5,6.
Lately, laryngopharyngeal pH monitoring was introduced as a new diagnostic device specifically designed to measure acid exposure in the oropharynx, as the probe is placed above the UES slightly lateral to the uvula (Figure 1). Since previous research has shown it to have a positive predictive value of 80% for a successful outcome after antireflux surgery in patients with primarily atypical symptoms, this new tool has been a valuable addition to the diagnostic pathway in selected patients. Its tear drop sensor is equipped with an antimony technology that detects liquid and aerosolized acid and does not need direct mucosal contact to measure valid results. In addition, the sensor can, in contrast to proximal pH monitoring, resist drying out which may lead to more reliable results7,8.
Current literature on the correlation of concomitant conventional esophageal and laryngopharyngeal pH measurement is sparse. Previous studies either included only a small number of patients or did not perform both measurements simultaneously9,10,11. We recently published data on the correlation between both pH measurements in a large cohort of 101 patients with suspected GERD. We concluded that laryngopharyngeal and esophageal pH measurement do not necessarily need to correspond due to the existence of a variety of different reflux scenarios12. We furthermore developed a human reflux model with patients following esophagectomy and reconstruction with a gastric interposition showing 100% correlation between both pH monitoring methods in volume-refluxers13.
Here, we aim to provide instructions for simultaneous pH measurement using distal esophageal and laryngopharyngeal pH monitoring. In addition, guidance on analysis of composite acid exposure scores and correlation between results obtained by both methods is given. We furthermore present the newest data of a large patient cohort evaluated using simultaneous esophageal and laryngopharyngeal pH monitoring.

<table><tbody><tr><td>AA Battery</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Calibration Solutions pH 4 and 7</td><td>Medtronic</td><td></td><td>part of the Digitrapper Reflux Testing system</td></tr><tr><td>CR 1632 Lithium coin cell battery</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Digitrapper pH &amp;amp; Impedance catheter</td><td>Medtronic</td><td></td><td></td></tr><tr><td>Digitrapper Recorder</td><td>Medtronic</td><td></td><td></td></tr><tr><td>Gelicain</td><td>PUREN</td><td></td><td>topical gel</td></tr><tr><td>Hydration vials with clear water</td><td>Respiratory Technology Corporation</td><td></td><td>part of the Restech Dx pH system</td></tr><tr><td>Leukoplast</td><td>BSN medical GmbH</td><td></td><td>surgical tape</td></tr><tr><td>Restech Dx pH probe</td><td>Respiratory Technology Corporation</td><td></td><td>part of the Restech Dx pH system</td></tr><tr><td>Restech Recorder</td><td>Respiratory Technology Corporation</td><td></td><td>part of the Restech Dx pH system</td></tr><tr><td>Restech Transmitter</td><td>Respiratory Technology Corporation</td><td></td><td>part of the Restech Dx pH system</td></tr><tr><td>Screwdriver</td><td>Respiratory Technology Corporation</td><td></td><td>part of the Restech Dx pH system</td></tr><tr><td>SD Card plus Adapter</td><td>Respiratory Technology Corporation</td><td></td><td>part of the Restech Dx pH system</td></tr><tr><td>tongue depressor</td><td>NOBAMED</td><td></td><td>wooden</td></tr></tbody></table>

simultaneous laryngopharyngeal and conventional esophageal ph monitoring

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, hoarseness or asthma, which can be caused by laryngopharyngeal reflux (LPR). Due to their multifactorial origin, those symptoms can be a great diagnostic and therapeutic challenge. Esophageal pH-monitoring is commonly used to determine abnormal esophageal acid exposure and confirm the diagnosis of GERD. However, for better evaluation of acid exposure above the upper esophageal sphincter, a new laryngopharyngeal pH measurement system is now available and may lead to more reliable results in patients with predominantly extraesophageal symptoms. This article aims to present a standardized protocol for simultaneous pH measurement using esophageal and laryngopharyngeal pH probes in order to obtain acid exposure scores from both measurements.

A total of 181 patients were evaluated using the previously described standardized protocol. Results of the first 101 patients have been previously published12. The following data depicts an extension of the previously published cohort, however, evaluated using the new software for analyzation of results obtained by laryngopharyngeal pH testing15. Demographic data is depicted in Table 1. All patients presented with atypical symptoms including chronic cough, hoarseness, sore throat, or pharyngeal burning. In addition, most patients suffered from typical GERD symptoms such as heartburn, regurgitation, or dysphagia.
A normal oropharyngeal acid exposure obtained by laryngopharyngeal pH testing was defined as a RYAN Score &#60; 9.4 upright and/or &#60; 6.8 supine. A normal esophageal pH test result was defined as a DeMeester Score of &#60; 14.72. Detailed data on results obtained by both pH measurements is depicted in Table 2. Of patients with a normal laryngopharyngeal test result, 58 patients (55.2%) also showed a corresponding normal esophageal pH test with a mean DeMeester Score of 5.3 (range 0.3 &#8211; 14.1).
Abnormal oropharyngeal acid exposure was more frequently seen in a upright position, with 72 patients showing an abnormal RYAN Score upright compared to 9 patients in a supine position, p value &#60; 0.0001. Correlation with abnormal distal esophageal acid exposure was shown in 49 patients (64.5%) with a mean DeMeester score of 64.7 (range 15.5 &#8211; 285.4).
Overall, correlation between esophageal pH monitoring and laryngopharyngeal pH monitoring, defined as both tests showing corresponding abnormal or normal results, was seen in 107 patients (59%).
Abnormal oropharyngeal acid exposure was seen in 76 patients. A non-corresponding, normal distal esophageal acid exposure was seen in 27 of those patients (35.5%). Interestingly, 11 patients with an abnormal oropharyngeal acid exposure but a normal esophageal test result showed reflux associated mucosal changes during upper gastrointestinal endoscopy. A normal oropharyngeal test result was found in 105 patients. A non-corresponding, abnormal distal esophageal acid exposure was seen in 47 of those patients (44.8%). Reflux associated mucosal changes were found in 25 patients with an abnormal esophageal acid exposure and a normal oropharyngeal test result.
Twenty-six patients underwent laparoscopic antireflux surgery after simultaneous pH measurement. Abnormal oropharyngeal acid exposure was shown in 16 patients (61.5%) with a mean RYAN score upright of 59.9 (range 4.3 &#8211; 153.9). Correlation with an abnormal distal esophageal acid exposure was seen in 13 patients with a mean DeMeester Score of 90.4 (range 20.4 &#8211; 283.5). Correlation of an abnormal laryngopharyngeal pH test results with abnormal esophageal acid exposure increased to 81.2% in surgical candidates.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>N</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Total patients</td>
			<td>181</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Females</td>
			<td>110</td>
			<td>60.8</td>
		</tr>
		<tr>
			<td>Age (mean; range), years</td>
			<td>52</td>
			<td>22 &#8211; 86</td>
		</tr>
		<tr>
			<td>BMI (mean; range), kg/m2</td>
			<td>25.2</td>
			<td>18.4 - 46.8</td>
		</tr>
		<tr>
			<td>Atypical reflux symptoms</td>
			<td>181</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Regurgitation</td>
			<td>116</td>
			<td>64.1</td>
		</tr>
		<tr>
			<td>Heartburn</td>
			<td>127</td>
			<td>70.2</td>
		</tr>
		<tr>
			<td>Dysphagia</td>
			<td>80</td>
			<td>44.2</td>
		</tr>
		<tr>
			<td colspan="3">Surgical Therapy</td>
		</tr>
		<tr>
			<td>Nissen</td>
			<td>15</td>
			<td>8.3</td>
		</tr>
		<tr>
			<td>Toupet</td>
			<td>4</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>LINX</td>
			<td>4</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>Endostim</td>
			<td>2</td>
			<td>1.1</td>
		</tr>
	</tbody>
</table>
Table 1: Demographic information, symptoms, and surgical therapy. Detailed demographic information and symptom distribution of our study cohort as total number of patients and percentage from study cohort is shown. Cough, hoarseness and globus sensation were summarized under atypical symptoms. In addition, this figure shows how many patients underwent surgical therapy for treatment of GERD and which method was chosen.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="3"></td>
			<td colspan="5">laryngopharyngeal pH testing</td>
			<td colspan="3">esophageal pH testing</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">RYAN upright</td>
			<td colspan="2">RYAN supine</td>
			<td></td>
			<td colspan="2">DeMeester</td>
		</tr>
		<tr>
			<td>n (%)</td>
			<td>mean</td>
			<td>range</td>
			<td>mean</td>
			<td>range</td>
			<td>n (%)</td>
			<td>mean</td>
			<td>range</td>
		</tr>
		<tr>
			<td>normal</td>
			<td>105 (58)</td>
			<td>1.12</td>
			<td>0 - 8.41</td>
			<td>0.33</td>
			<td>0 - 6.43</td>
			<td>85 (47)</td>
			<td>6.31</td>
			<td>0.3 - 14.2</td>
		</tr>
		<tr>
			<td>abnormal</td>
			<td>76 (42)</td>
			<td>52.8</td>
			<td>0 - 228.9</td>
			<td>2.92</td>
			<td>0 - 48.41</td>
			<td>96 (53)</td>
			<td>59.53</td>
			<td>14.8 - 285.4</td>
		</tr>
	</tbody>
</table>
Table 2: Results obtained by laryngopharyngeal and esophageal pH testing (n = 181). A summary of results obtained by laryngopharyngeal and esophageal pH testing is shown. A normal laryngopharyngeal pH test was defined as a RYAN score &#60; 9.4 upright and &#60; 6.8 supine. A normal esophageal pH test was defined as a DeMeester score &#60; 14.72. The total number of patients with respective results, the percentage from the study cohort, mean scores and ranges of RYAN or DeMeester scores are displayed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61641/61641fig01.jpg" /> 
Figure 1: Laryngopharyngeal pH testing system.&#160;This figure shows the laryngopharyngeal pH monitoring system, consisting of a recorder and a transmitter with an attached measuring probe. In addition, the calibration vials as well as the water vial for the hydration process are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61641/61641fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring at JoVE.com

Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring

Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously.

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, ...

simultaneous-laryngopharyngeal-conventional-esophageal-ph

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Medicine

Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

2.7K 조회수.  University of Cologne. Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously.

비디오: Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

1 차 인간 기도 상피 세포의 기도 표면 액체 pH의 실시간 반 자동화 형광 측정

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

연구

생화학

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

빠르고 정확한 날숨 암모니아 측정

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

의학

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or non-acidic refluxate. This paper describes a method for the development, manufacturing, and implantation of a miniature wireless-enabled pH sensor. The sensor is designed to be implanted endoscopically with a single hemostatic clip. A fully passive rectenna-based receiver based on a zero-bias Schottky diode is also constructed and tested. To construct the device, a two-layer printed circuit board and off-the-shelf components were used. A miniature microcontroller with integrated analog peripherals is used as an analog front end for the ion-sensitive field-effect transistor (ISFET) sensor and to generate a digital signal which is transmitted with an amplitude shift keying transmitter chip. The device is powered by two primary alkaline cells. The implantable device has a total volume of 0.6 cm3 and a weight of 1.2 grams, and its performance was verified in an ex vivo model (porcine esophagus and stomach). Next, a small footprint passive rectenna-based receiver which can be easily integrated either into an external receiver or the implantable neurostimulator, was constructed and proven to receive the RF signal from the implant when in proximity (20 cm) to it. The small size of the sensor provides continuous pH monitoring with minimal obstruction of the esophagus. The sensor could be used in routine clinical practice for 24/96 h esophageal pH monitoring without the need to insert a nasal catheter. The &#34;zero-power&#34; nature of the receiver also enables the use of the sensor for automatic in-vivo calibration of miniature lower esophageal sphincter neurostimulation devices. An active sensor-based control enables the development of advanced algorithms to minimize the used energy to achieve a desirable clinical outcome. One of the examples of such an algorithm would be a closed-loop system for on-demand neurostimulation therapy of gastroesophageal reflux disease (GERD).

The manuscript presents a miniature implantable pH sensor with ASK modulated wireless output together with a fully passive receiver circuit based on zero-bias Schottky diodes. This solution can be used as a basis in the development of in vivo calibrated electrostimulation therapy devices and for ambulatory pH monitoring.

제로 바이어스 쇼트키 다이오드 기반 수신기를 사용하여 pH 모니터링을 위한 무선 지원 내시경 이식형 센서 구축

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or ...

생체공학

Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere with breathing. In recent years, intraoperative neural monitoring (IONM) has been widely adapted as an adjunct technique to localize the RLN, detect RLN injury, and predict vocal cord function during the operations. Many studies have also used animal models to investigate new applications of IONM technology and to develop reliable strategies for preventing intraoperative RLN injury. The aim of this article is to introduce a standard protocol for using a porcine model in IONM research. The article demonstrates the procedures for inducing general anesthesia, performing tracheal intubation, and experimental design to investigate the electrophysiological characteristics of RLN injuries. Applications of this protocol can improve overall efficacy in implementing the 3R principle (replacement, reduction and refinement) in porcine IONM studies.

This study aims to develop a standard protocol of intra-operative neural monitoring of thyroid surgery in a porcine model. Here, we present a protocol to demonstrate general anesthesia, to compare different types of electrodes, and to investigate the electrophysiological characteristics of the normal and injured recurrent laryngeal nerves.

돼지 모델에서 갑 상선 수술의 내부 작용 신경 모니터링

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

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.

Here, we describe two measures of pulmonary function &#8211; 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.

체적 Capnography와 기 Plethysmography 폐 구조-기능 관계를 측정 하

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

공 초점 현미경을 사용하여 살아있는 세포에서 세포 내 이입과 세포질 구획 동시 pH 측정

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

생물학

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: 폐 기능 및 대사 평가

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Endoscopic Approach for Studying Laryngeal Dysfunction in Rodents

Minimally Invasive Murine Laryngoscopy for Close&#45;Up Imaging of Laryngeal Motion During Breathing and Swallowing

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty&#160;breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions.
To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification).
Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

This protocol describes a serial transoral laryngoscopy approach for mice and rats that permits close-up, unobstructed video imaging of the larynx during breathing and swallowing using an optimized anesthetic regimen and finely tuned endoscopic manipulation techniques.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to ...

신경과학

미세전극을 이용한 3D 위 오가노이드의 정밀한 발광 pH 측정

Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This paper presents a highly reproducible method for the precise measurement of pH within the lumina of 3D human gastric organoids via micromanipulator-controlled microelectrodes. The pH microelectrodes are commercially available and consist of beveled glass tips of 25 &#181;m in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (&gt;200 &#181;m) that is suspended in Matrigel, while a reference electrode rests submerged in the surrounding medium in the culture plate. 
Using such microelectrodes to profile organoids derived from the human gastric body, we demonstrate that luminal pH is relatively consistent within each culture well at ~7.7 &plusmn; 0.037 and that continuous measurements can be obtained for a minimum of 15 min. In some larger organoids, the measurements revealed a pH gradient between the epithelial surface and the lumen, suggesting that pH measurements in organoids can be achieved with high spatial resolution. In a previous study, microelectrodes were successfully used to measure luminal oxygen concentrations in organoids, demonstrating the versatility of this method for organoid analyses. In summary, this protocol describes an important tool for the functional characterization of the complex luminal space within 3D organoids.

저자 스포트라이트: 헬리코박터 파일로리 감염에서의 위 상피 세포 반응

The present protocol describes pH measurements in human tissue-derived gastric organoids using microelectrodes for spatiotemporal characterization of intraluminal physiology.

미세전극을 사용한 3차원 위장 오가노이드의 루미날 pH 프로파일링

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This ...

Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring

요약

더 많은 비디오 탐색

이 비디오의 챕터

1 차 인간 기도 상피 세포의 기도 표면 액체 pH의 실시간 반 자동화 형광 측정

빠르고 정확한 날숨 암모니아 측정

제로 바이어스 쇼트키 다이오드 기반 수신기를 사용하여 pH 모니터링을 위한 무선 지원 내시경 이식형 센서 구축

돼지 모델에서 갑 상선 수술의 내부 작용 신경 모니터링

체적 Capnography와 기 Plethysmography 폐 구조-기능 관계를 측정 하

공 초점 현미경을 사용하여 살아있는 세포에서 세포 내 이입과 세포질 구획 동시 pH 측정

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: 폐 기능 및 대사 평가

Minimally Invasive Murine Laryngoscopy for Close-Up Imaging of Laryngeal Motion During Breathing and Swallowing

미세전극을 사용한 3차원 위장 오가노이드의 루미날 pH 프로파일링

미세전극을 사용한 3차원 위장 오가노이드의 루미날 pH 프로파일링