Mauro A., Franchina M., Consonni D., Penagini R. Lower oesophageal sphincter identification for gastro-oesophageal reflux monitoring: The step-up method revisited with use of basal impedance / United European Gastroenterol J. 2019 Dec; 7(10): 13731379.


: Mauro A. / Franchina M. / Consonni D. / Penagini R.


Lower oesophageal sphincter identification for gastro-oesophageal reflux monitoring: The step-up method revisited with use of basal impedance

Aurelio Mauro1,2,3, Marianna Franchina1,2, Dario Consonni4 and Roberto Penagini1,2


1 Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy
2 Gastroenterology and Endoscopy Unit, Fondazione IRCCS Ca Granda, Ospedale Maggiore Policlinico, Milan, Italy
3 Endoscopy Unit, First Department of Internal Medicine, San Matteo Hospital Foundation, University of Pavia, Pavia, Italy
4 Epidemiology Unit, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Milan, Italy

Corresponding author:
Aurelio Mauro, Viale Camillo Golgi 19, 27100 Pavia, Italy. Email: a.mauro@ smatteo.pv.it

Abstract
Background: Oesophageal manometry is the gold standard for accurate positioning of multichannel intraluminal impedance pH (MII-pH) monitoring. The pH step-up method is not as accurate as oesophageal manometry and needs patients to be off proton pump inhibitor (PPI) therapy.

Objective: This study aimed to evaluate the feasibility of the impedance step-up method for lower oesophageal sphincter (LOS) localisation.

Methods: A total of 100 patients who underwent 24-hour MII-pH monitoring were enrolled. High-resolution manometry (HRM) was performed before MII-pH monitoring in order to locate the LOS by a different operator. The impedance step-up was defined as an increase of ⩾50% with respect to gastric baseline. Lins concordance correlation coefficient (ρc) with 95% BlandAltman limits of agreement (LOA) and Spearmans rho correlation coefficient were used when appropriate.

Results: The median impedance step-up was on average 0.8 cm caudal to the manometric upper border of the LOS. Agreement between two step-up impedance performances and inter-observer agreement were excellent (ρ=0.98 and 0.95), although the LOA ranges were wide (2.4 to 4.0 cm). Impedance step-up performances were similar between patients off and on PPI.

Conclusions: We have described an alternative new method for pH impedance probe positioning using impedance step-up. Although less accurate than HRM in locating the LOS, it has excellent intra- and inter-observer agreement.

Keywords
pH impedance monitoring, high-resolution manometry, gastroesophageal reflux disease, step up, PPI

Received: 23 March 2019; accepted: 24 May 2019
Introduction
Gastro-oesophageal reflux disease (GORD) is common in the general population. When endoscopy is negative and patients have poor response to proton pump inhibitors (PPI), ambulatory reflux monitoring is required.1 From recent International Consensus reports, multichannel intraluminal impedance pH (pH-MII) monitoring is preferred over traditional pH monitoring because impedance measurement allows detection of retrograde bolus (liquid, gas or mixed) flow in the oesophagus independently of pH. Moreover, in patients with an undefined GORD diagnosis (acid exposure time between 4% and 6%), impedance allows measurement of other impedance variables in order to confirm or refuse GORD diagnosis.2

Catheter-based pH monitoring is conventionally placed 5 cm above the lower oesophageal sphincter (LOS); manometric localisation of the LOS is the gold standard for electrode placement.3 However, trans-nasal passage of the manometric catheter may be annoying for the patients. Moreover, in patients without dysphagia or surgical indication for fundoplication, evaluation of oesophageal motor function has poor diagnostic value.

An alternative technique for LOS identification is the pH step-up method. Some authors have found an adequate correlation between the pH step-up method and manometric localisation,4,5 whereas others have not, using traditional manometry.6,7 Moreover, in patients where the 24-hour reflux study is indicated on PPI, the pH step-up method is not possible due to a weakly acidic or neutral intragastric pH.

Impedance could be a useful variable in order to localise the LOS. One of the first studies where impedance was evaluated showed that gastric impedance was significantly lower than oesophageal impedance.8 Moreover, impedance values are not affected by pH. Therefore, we hypothesised that a change in impedance from lower to higher values may allow the identification of the LOS. The aim of our study was to evaluate step-up impedance with a pull-through and its correlation with the manometric localisation of the LOS in patients off or on PPI.
Materials and methods
Consecutive patients referred to our centre for pH-MII from July 2017 to May 2018 were prospectively considered for inclusion in the study. Patients with achalasia, prior oesophageal or gastric surgery, Barretts oesophagus or a mean nocturnal baseline impedance (MNBI) <500 Ω were excluded.9

The presence and nature of presenting symptoms were assessed by standardised medical interview.10 Informed consent for oesophageal manometry and pH impedance monitoring were acquired from all patients. The study was designed and carried out in accordance with the Declaration of Helsinki (sixth revision, Seoul, 2008). No ethical review board approval was required according to Italian Legislation (AIFA Determination March 20th 2008; GU number 76 of 31 March 2008) considering the observational nature of the study.

Oesophageal high-resolution manometry

High-resolution manometry (HRM) was performed by investigators A.M. or R.P. using a 4 mm solid-state probe with 36 circumferential sensors at 1 cm intervals (Medtronic, Minneapolis, MN) in the right lateral position, following a previously described protocol.11,12 The upper and lower borders of the LOS were located at the start of the recording. Manometric pressure data were visualised as topographic contour plots and were stored for subsequent analysis using ManoView software (Medtronic). The oesophago-gastric junction (OGJ) type (IIII) was classified according to a recent classification.13

Impedance and pH step-up procedure and analysis

After manometry, an investigator blinded to the results of HRM (R.P. or A.M.) performed the procedure with the pH impedance catheter before starting 24-hour pH-MII.14 A single-use catheter with eight impedance recording segments and one antimony pH electrode (Z61A; MMS, Enschede, The Netherlands) was used and connected to portable digital recorders (Ohmega; MMS). After external calibration, the catheter was first passed into the stomach and then the patient was placed in the right lateral recumbent position. Passage in the stomach was confirmed by a stable low impedance value (i.e. <688 Ω)8 in the second distal impedance channel (at the same level of the pH sensor, 5 cm above the tip of the catheter) with concomitant acidic pH (<4) if the test was off PPI. The recording was started, and the depth of insertion of the catheter in centimetres was marked on a dedicated sheet by a research nurse blinded to the HRM result. The catheter was withdrawn gradually 1 cm every 15 seconds up to 5 cm above the step-up impedance point, and each centimetre was marked with the digital recorders symptom button. Entry into the oesophagus was deemed to have occurred when a sharp and stable impedance rise was seen, defined as an increase of >50% with respect to gastric baseline. Two examples of the impedance step-up are shown in Figure 1. The impedance value at the variation point was acquired. In patients off PPI, the variation of pH during step-up was also written down and defined as a sharp increase in pH >4. The step-up procedure was repeated twice. Finally, the catheter was positioned 5 cm above the upper border of the LOS as assessed by HRM.


Figure 1. Two examples of the impedance step-up in a patient off (a) and on (b) proton pump inhibitors (PPI).


A different operator (M.F.), blinded to the manometric and impedance results, reviewed all the pH impedance tracings obtained during the step-up procedures in order to evaluate the inter-observer agreement with the measurement obtained by R.P. or A.M.

pH impedance monitoring and analysis

After the step-up procedure, pH-MII monitoring started, and the patient left the clinic.

During the tests, patients were encouraged to carry out their usual activities. They were also asked to press the event button on the receiver whenever they experienced their dominant symptom. After 24 hours of recording, data were downloaded onto a computer using commercial software (MMS).

Oesophageal acid exposure was considered increased if pH was <4 more than 6% of the time.2 The correlations between the dominant symptom and acid and weakly acidic reflux events were evaluated as symptom index (SI) and symptom association probability (SAP), which were considered positive if they were >50% and >95%, respectively.2,15 The MNBI was measured 5 cm above the LOS according to previously published methods.16

The pH-MII tests were classified as: (a) GORD if oesophageal acid exposure was increased, irrespective of symptomreflux association results; (b) negative for GORD if acid exposure was normal and SI/SAP was negative; and (c) reflux hypersensitivity (RI) if acid exposure time was normal but SI and/or SAP were positive.

Statistical analysis

Concordance between both impedance and pH step-ups and HRM were evaluated by calculating Lins concordance correlation coefficient17c) and 95% BlandAltman limits of agreement (LOA).18 Impedance and pH step-up agreement, inter-observer agreement and concordance between the two step-up impedance performances were assessed using Spearmans rho correlation coefficient. Statistical analysis was performed with Stata v15 (StataCorp LLC, College Station, TX).
Results
A total of 115 patients were prospectively enrolled in the study. Fifteen patients were excluded: six patients with MNBI values <500 Ω, six with poor compliance during the step-up procedure and three with an unstable gastric baseline (i.e. presence of impedance peak >10,000 Ω suggestive of contact of the catheter with air). A total of 100 patients were included in the final analysis (59 males; median age 47 years; range 1884 years), 19 of whom were on PPI. Their symptoms were heartburn and acid regurgitation in 41 patients, cough in 16 patients, ENT symptoms in 16 patients, non-cardiac chest pain in 9 patients, dysphagia in 12 patients and troublesome belching in 6 patients. In 91 patients for whom previous upper gastrointestinal endoscopy results were available, erosive oesophagitis was present in 15 patients (10 grade A, three grade B and two grade C according to the Los Angeles classification), and 13 patients had eosinophilic oesophagitis. Final HRM diagnosis was normal motility in 42 patients, ineffective oesophageal motility in 45 patients, fragmented peristalsis in 10 patients and other major disorders in three patients (two with distal oesophageal spasm and one with aperistalsis). Types I, II and III OGJ were present in 68, 26 and 6 patients, respectively.

All patients completed pH-MII monitoring. The final pH-MII diagnosis was GORD in 23 patients, negative in 66 and RI in 11.

The 24-hour pH-MII results, divided according to off or on PPI test, are detailed in Table 1.

Table 1. Descriptive data of pH-MII parameters in patients off and on PPI.


Off PPI (n = 81) On PPI (n = 19) p
Acid exposure time, % 2.9 (0.84.2) 0.8 (00.5) 0.0007
Total refluxes 47 (3162) 47 (2865) 0.8
Acid refluxes 34 (2045) 15 (325) 0.0001
Weakly acid refluxes 14 (517) 30 (1641) 0.00001
Non-acid refluxes 5 (04) 5 (010) 0.17
Gastric impedance, Ω 326 (250400) 428 (340500) 0.0006
MNBI, Ω 1462 (9001900) 1709 (10002229) 0.17
Impedance at variation points, Ω 2123 (13622851) 2512 (15753090) 0.23
Data shown are the mean (interquartile range).
pH-MII: multichannel intraluminal impedance pH; PPI: proton pump inhibitors.

Figure 2. Concordance between the two step-up
impedance (left) and pH (right) performances
evaluated with Spearmans rho correlation coefficient.

Figure 3. Inter-observer agreement of impedance
(left) and pH variation point (right) evaluated with
Spearmans rho correlation coefficient.


Impedance step-up

The median impedance step-up was 0.8 cm caudal (95% LOA: 2.4 cm to 4.0 cm) to the manometric upper border of the LOS and 1.9 cm cranial (95% LOA: 5.1 cm to 1.3 cm) to the manometric lower border of the LOS (Lin concordance correlation coefficients 0.83 and 0.78, respectively). Agreement between the two step-up impedance performances was excellent (mean 44.8 cm for both measurements, ρ = 0.98; see Figure 2). In 62% of patients, the distance between the upper border of the LOS and the impedance step-up was in the range 1 cm, and in 84%, it was in the range 2 cm.

pH step-up and correlation with impedance step-up

In the 81 patients off PPI, the pH step-up method was also evaluated. The median pH step-up was 0.7 cm caudal (95% LOA: 2.3 cm to 3.7 cm) to the manometric upper border of the LOS and 1.9 cm cranial (95% LOA: 5.1 cm to 1.7 cm) to the manometric lower border of the LOS (Lin concordance correlation coefficients 0.72 for both measurement). Agreement between the two step-up pH performances was excellent (mean 44.8 cm for both measurements, ρ = 0.94; see Figure 2). Moreover, the correlation between the pH and the impedance variation point was optimal (Spearmans ρ = 0.90). The distance between the upper border of the LOS and the pH step-up was in the range 1 cm and 2 cm in 64% and 88% of patients, respectively.

Inter-observer agreement

When M.F. reviewed the impedance and the pH step-up, excellent agreement between the two operators for both measurement was observed (Spearmans ρ = 0.95 and 0.93 for impedance and pH variation point, respectively; see Figure 3).

Subgroup analysis

The differences between the impedance step-up and both the upper and lower borders of the LOS were similar when we divided the population according to PPI administration and OGJ type (type I vs. types II+III). There were also similar impedance step-up results between patients with and without oesophagitis (0.5 cm caudal to the upper border of the LOS; 95% LOA: 3.3 cm to 4.3 cm; and 0.9 cm caudal; 95% LOA: 2.0 cm to 3.7 cm, respectively).

No differences were seen when subgroup analysis were performed with pH step-up data in the patients off PPI.
Discussion
In this study, we have developed a novel and rapid impedance step-up method that identifies the LOS with good accuracy. Considering the withdrawal time of 15 s/cm, the procedure is accomplished within two and a half minutes.

It has been established that oesophageal manometry is the most accurate method for LOS identification,3 although the accuracy is known to decrease in the presence of hiatal hernia. The advent of HRM has also increased the accuracy in cases of hiatal hernia.19 However, HRM is not available ubiquitously, especially in non-tertiary centres. Except for cases of preoperative evaluation for laparoscopic fundoplication or cases of GORD symptoms associated with dysphagia, oesophageal manometry is not strictly necessary for the evaluation of patients with PPI-refractory GORD symptoms.1,13 In peripheral centres, where economic and human resources are strictly controlled, the pH step-up method is often used, but in some studies, its accuracy has been shown to be below excellent.6,7 In this study, we found that the impedance step-up could be an alternative option to HRM for LOS location where oesophageal manometry is not available and can be used for patients both off and on PPI. We found that it correlates better with the upper border of the LOS rather than the lower border: the former was medially located with the impedance step-up 0.7 cm caudal, whereas the latter was 1.9 cm cranially. Thus, our data indicate that pH impedance probes need to be positioned 6 cm above the sharp impedance rise. We obtained similar results with the pH step-up method, which correlated very well with the impedance step-up (Spearmans ρ = 0.90). Some studies4,5 where the pH step-up method was evaluated have found the position for the pH change 11.3 cm below the upper border of the LOS and therefore a little bit more caudal than our results. However, those studies were performed with traditional manometry, and the different recording technique could be responsible for the difference.

Other studies have concluded that the pH step-up method was not an accurate method to locate the LOS.6,7 It is difficult to explain the discrepancy among the various reports. However, the study by Marples et al. lacks details in the methods section and was performed in only 32 patients, including 10 with unspecified oesophageal conditions different from reflux.7 The study by Mattox et al. was conducted in an adequate cohort of patients (n = 71) and concluded that 58% of patients were outside the range of 3 cm with respect to the manometric measurement.6 We could argue that this high percentage of pH step-up inaccuracy could actually be related to inadequate identification of the LOS by manometry, as 44% of patients had hiatal hernia. Our study is the first conducted with HRM where a real gold standard for LOS location can be recorded.

We also investigated if factors that could alter both gastric and oesophageal physiology may influence the impedance step-up. First, we evaluated the effects of PPI which are known to neutralise the gastric contents but not alter mucosal impedance. We showed that in the 19 patients studied on PPI, the impedance step-up was similar to that of patients studied off PPI. Second, we characterised hiatal hernia with HRM.13 We found similar concordance of the impedance step-up with HRM data between patients with type I and types II+III OGJ. Concordance of the pH step-up results with HRM was also similar between patients with and without hiatal hernia, which is in disagreement with a previous study5 that showed that patients with hiatal hernia had weaker concordance. Third, we evaluated the effect of oesophagitis. It is known that inflammation of the oesophageal mucosa decreases baseline impedance of the oesophagus.20,21 In patients with erosive and eosinophilic oesophagitis, this effect is more evident,2224 and mucosal restoration to normal increases baseline impedance.25 In spite of this evidence, in our cohort, concordance between the impedance step-up and HRM was similar in patients with and without oesophagitis.

A limitation of the impedance step-up technique is that accuracy in the location of the LOS is not perfect, being 2 cm of the HRM gold standard in 84% of patients, a result similar to our pH step-up. However, accuracy was enough to allow positioning of the pH impedance probe always well within the distal oesophageal body in a spatial range which is unlikely to alter detection of pathological reflux significantly. Previous studies have looked at differences of oesophageal acid exposure along the oesophagus, showing differences when pH probes were more distant than our inaccuracy range (i.e. 0.5 cm vs. 5 cm and 5 cm vs. 10 cm above the LOS).26,27 Thus, the impedance step-up, although not being the method of choice, could be the best alternative for pH impedance probe positioning when HRM or traditional manometry is not available temporarily or is unavailable.

One methodological point needs comment: the definition of the impedance step-up. As there are no data on the behaviour of impedance at the level of the LOS, we decided to use the sharp increase of 50% from the gastric baseline values for LOS location rather than a fixed impedance value. This decision was driven by our observations of highly variable impedance values at varying points among subjects, ranging from 700 to 5000 Ω.

This study has some strengths, most notably the prospective nature and the size of the population, which to our knowledge is the highest reported in the literature for gastro-oesophageal step-up studies. Second, the use of HRM allowed us to define the location of the LOS accurately, irrespective of hiatal hernia (i.e. to have a sound gold standard, irrespective of anatomy). Third, all the step-up procedures were made by an operator blinded to the HRM results, and a third operator reviewed all the procedures in order to obtain data on inter-observer agreement, which proved excellent.

Some limitations need to be acknowledged. The first one is that real-time visualisation of impedance is possible with most but not all devices. MMS/Laborie equipment used in the present study allows real-time visualisation of impedance tracings on the computer monitor, and the Digitrapper pH-Z system (Medtronic) allows real-time visualisation of impedance values for each channel on its recorder. However, the impedance step-up is not feasible with Sandhill devices because they do not have the ability to record the impedance data in real time. A second limitation is that the impedance step-up cannot be used in Barretts oesophagus patients where basal impedance is known to be very low in the presence of columnar epithelium. However, it should be acknowledged that generally, the majority of patients undergoing reflux studies are not patients with markedly increased reflux, such as those with Barretts oesophagus, but endoscopy-negative patients with suspicion of GORD, as our series shows. Finally, the impedance step-up was not feasible in 15/115 patients, although it should be noted that failure was due to poor compliance in six patients, a proportion which may vary in different cohorts. Further minor limitations are the single-centre nature of the study, the small number of patients on PPI and those with erosive oesophagitis, even if these variables did not seem to impact the results.

In conclusion, we have described an alternative new method for pH impedance probe positioning using the impedance step-up method. The probe needs to be positioned 6 cm above the sharp increase of the impedance rise. Although it is less accurate than HRM in locating the LOS, it has excellent intra- and inter-observer agreement. It can be used independently of PPI treatment whenever manometry is not available or patients do not tolerate manometry catheters.


Declaration of conflicting interests
None declared.

Ethics approval
No ethical review board approval was required according to Italian Legislation (AIFA Determination March 20th 2008; GU number 76 of 31 March 2008) considering the observational nature of the study.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

Informed consent
Written, informed consent was obtained from each patient included in the study.
References
1. Roman S, Gyawali CP, Savarino E, et al. Ambulatory reflux monitoring for diagnosis of gastro-esophageal reflux disease: update of the Porto consensus and recommendations from an international consensus group. Neurogastroenterol Motil 2017; 29: 115.

2. Gyawali CP, Kahrilas PJ, Savarino E, et al. Modern diagnosis of GERD: the Lyon Consensus. Gut 2018; 67: 13511362.

3. Kahrilas PJ, Quigley EM. Clinical esophageal pH recording: a technical review for practice guideline development. Gastroenterology 1996; 110: 19821996.

4. Klauser AG, Schindlbeck NE, Muller-Lissner SA. Esophageal 24-h pH monitoring: is prior manometry necessary for correct positioning of the electrode? Am J Gastroenterol 1990; 85: 14631467.

5. Pehl C, Boccali I, Hennig M, et al. pH probe positioning for 24-hour pH-metry by manometry or pH step-up. Eur J Gastroenterol Hepatol 2004; 16: 375382.

6. Mattox HE, 3rd, Richter JE, Sinclair JW, et al. Gastroesophageal pH step-up inaccurately locates proximal border of lower esophageal sphincter. Dig Dis Sci 1992; 37: 11851191.

7. Marples M, Mughal M, Bancewicz J. Can esophageal pH electrode be accurately positioned without manometry? Diseases of the esophagus, Berlin: Springer, 1987, pp. 789791.

8. Nguyen HN, Domingues GR, Winograd R, et al. Impedance characteristics of normal oesophageal motor function. Eur J Gastroenterol Hepatol 2003; 15: 773780.

9. Heard R, Castell J, Castell DO, et al. Characterization of patients with low baseline impedance on multichannel intraluminal impedance-pH reflux testing. J Clin Gastroenterol 2012; 46: e5557.

10. Grigolon A, Consonni D, Bravi I, et al. Diagnostic yield of 96-h wireless pH monitoring and usefulness in patients management. Scand J Gastroenterol 2011; 46: 522530.

11. Mauro A, Savarino E, De Bortoli N, et al. Optimal number of multiple rapid swallows needed during high-resolution esophageal manometry for accurate prediction of contraction reserve. Neurogastroenterol Motil 2018; 30: e13253e13253.

12. Price LH, Li Y, Patel A, et al. Reproducibility patterns of multiple rapid swallows during high resolution esophageal manometry provide insights into esophageal pathophysiology. Neurogastroenterol Motil 2014; 26: 646653.

13. Gyawali CP, Roman S, Bredenoord AJ, et al. Classification of esophageal motor findings in gastro-esophageal reflux disease: Conclusions from an international consensus group. Neurogastroenterol Motil 2017; 29: e13104e13104.

14. Della Coletta M, Galeazzi F, Ioannou A, Torresan F, Marabotto E, Zentilin P, et al. Esophagogastric junction morphology variability during standard manometric protocol and after esophageal stimulation and body change position. United European Gastroenterol J 2016; 4: A312A312.

15. Weusten BL, Roelofs JM, Akkermans LM, et al. The symptom-association probability: an improved method for symptom analysis of 24-hour esophageal pH data. Gastroenterology 1994; 107: 17411745.

16. Martinucci I, de Bortoli N, Savarino E, et al. Esophageal baseline impedance levels in patients with pathophysiological characteristics of functional heartburn. Neurogastroenterol Motil 2014; 26: 546555.

17. Lin LI. A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989; 45: 255268.

18. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 1: 307310.

19. Tolone S, Savarino E, Zaninotto G, et al. High-resolution manometry is superior to endoscopy and radiology in assessing and grading sliding hiatal hernia: a comparison with surgical in vivo evaluation. United European Gastroenterol J 2018; 6: 981989.

20. Farre R, Blondeau K, Clement D, et al. Evaluation of oesophageal mucosa integrity by the intraluminal impedance technique. Gut 2011; 60: 885892.

21. Saritas Yuksel E, Higginbotham T, Slaughter JC, et al. Use of direct, endoscopic-guided measurements of mucosal impedance in diagnosis of gastroesophageal reflux disease. Clin Gastroenterol Hepatol 2012; 10: 11101116.

22. Katzka DA, Ravi K, Geno DM, et al. Endoscopic mucosal impedance measurements correlate with eosinophilia and dilation of intercellular spaces in patients with eosinophilic esophagitis. Clin Gastroenterol Hepatol 2015; 13: 12421248.e1.

23. Frazzoni M, Savarino E, de Bortoli N, et al. Analyses of the post-reflux swallow-induced peristaltic wave index and nocturnal baseline impedance parameters increase the diagnostic yield of impedance-pH monitoring of patients with reflux disease. Clin Gastroenterol Hepatol 2016; 14: 4046.

24. Kandulski A, Weigt J, Caro C, et al. Esophageal intraluminal baseline impedance differentiates gastroesophageal reflux disease from functional heartburn. Clin Gastroenterol Hepatol 2015; 13: 10751081.

25. Kessing BF, Bredenoord AJ, Weijenborg PW, et al. Esophageal acid exposure decreases intraluminal baseline impedance levels. Am J Gastroenterol 2011; 106: 20932097.

26. Fletcher J, Wirz A, Henry E, et al. Studies of acid exposure immediately above the gastro-oesophageal squamocolumnar junction: evidence of short segment reflux. Gut 2004; 53: 168173.

27. Anggiansah A, Sumboonnanonda K, Wang J, et al. Significantly reduced acid detection at 10 centimeters compared to 5 centimeters above lower esophageal sphincter in patients with acid reflux. Am J Gastroenterol 1993; 88: 842846.

""
,

.
 -