The Laryngoscope
C 2011 The American Laryngological,
V
Rhinological and Otological Society, Inc.
Immunohistochemical Detection of Pepsin in Laryngeal Mucosa for
Diagnosing Laryngopharyngeal Reflux
Aiyun Jiang, PhD; Maojin Liang, MM; Zhenzhong Su, MB; Liping Chai, MB; Wenbin Lei, PhD;
Zhangfeng Wang, MM; Anjiang Wang, PhD; Weiping Wen, PhD; Minhu Chen, PhD
Objectives/Hypothesis: To investigate whether the pepsin immunohistochemical (IHC) staining of the laryngeal mucosa
epithelia is an available test for diagnosing laryngopharyngeal reflux (LPR) in clinic.
Study Design: Prospective case series.
Methods: Biopsy specimens from interarytenoid mucosa of LPR patients (seven acid LPR and eight nonacid LPR) and
21 sex- and age-matched normal controls were obtained for pepsin IHC staining. The diagnosis of LPR was based on 24-hour
combined multichannel intraluminal esophageal impedance pH monitoring. The results of IHC staining were semiquantitatively analyzed and scored as negative (), weakly positive (þ), moderately positive (þþ), and strongly positive (þþþ).
Results: Six of seven acid LPR (85.7%) and six of eight nonacid LPR (75%) mucosa samples were moderate to strongly
positive for intracellular pepsin. By contrast, only three of 21 normal controls (14.3%) were moderately positive. The difference in intracellular pepsin between LPR and the normal laryngeal mucosa was statistically significant (P < .01). No significant difference in intracellular pepsin was observed between the acid and nonacid LPR mucosal samples (P ¼ .453). Using
weak positivity (þ) as a cutpoint, the presence of intracellular pepsin in the laryngeal mucosa had a sensitivity of 100% and
a specificity of 47.6% in detecting LPR (P < .05). However, using the moderate positivity (þþ) as the cutpoint, the pepsin
had a slightly decreased sensitivity of 80% but a sharply increased specificity of 85.7% (P < .05) in the detection of LPR.
Conclusions: Pepsin IHC staining of the laryngeal mucosa appears to be a sensitive and specific test for diagnosing LPR
in a clinical application.
Key Words: Laryngopharyngeal reflux, multichannel intraluminal impedance, pH monitoring, pepsin, immunohistochemical
staining.
Level of Evidence: 3b.
Laryngoscope, 121:1426–1430, 2011
INTRODUCTION
Laryngopharyngeal reflux (LPR) has been reported
to occur in more than 10% of the otorhinolaryngology
outpatient population1 and has a close relationship with
many laryngopharyngeal disorders, such as primary
subglottic stenosis,2 laryngeal cancer,1,3 vocal cord contact ulcers, and granuloma.4 Unfortunately, because of
the atypical symptoms and signs, most LPR patients
cannot be diagnosed and treated at the early stage. Currently, ambulatory 24-hour double-probe pH monitoring
(pH metry), which was developed and used for diagnosing gastroesophageal reflux disease, is considered to be
the gold standard for the diagnosis of LPR. However, it
is often difficult for patients to tolerate this pH metry
test, and the test itself has a variable sensitivity ranging
From the Department of Otolaryngology (A.J., M.L., Z.S., L.C., W.L., Z.W.,
and Department of Gastroenterology (A.W., M.C.), First Affiliated
Hospital, Sun Yat-Sen University, Guangzhou, China; and the Department
of Otolaryngology, Sun Yat-sen Memorial Hospital (M.L.), Sun Yat-sen
University, Guangzhou, China.
Editor’s Note: This Manuscript was accepted for publication March
2, 2011.
The authors have no funding, financial relationships, or conflicts
of interest to disclose.
Send correspondence to Weiping Wen, Department of Otolaryngology, First Affiliated Hospital, Sun Yat-Sen University, No. 58, Zhongshan
er Road, Guangzhou 510080, China. E-mail: [email protected]
W.W.)
DOI: 10.1002/lary.21809
Laryngoscope 121: July 2011
1426
from 50% to 80% in detecting LPR.1,5 In addition, pH
metry is expensive, making it impractical for routine use
in a clinical application.
Pepsin is an enzyme converted from pepsinogen,
which is produced by the chief cell of the stomach, and
plays an important role in digestion. Normally, pepsin is
only found in the stomach contents. However, if LPR happens, the stomach contents reflux to the larynx, and then
pepsin can be detected in the laryngopharyngeal areas.
Recently, Johnston et al.6 and Knight et al.7 reported that
pepsin was detected in laryngeal mucosa and sputum in
pH metry–confirmed LPR patients and concluded that the
presence of pepsin in the larynx was sensitive and specific
for the detection of LPR. However, the pH metry test used
by these investigators can only detect acid reflux (pH <
4); it is important to note that any reflux of the stomach
contents may contain pepsin but can be without acid. So
can pepsin be detected in laryngeal mucosa in nonacid
reflux? With the development of 24-hour multichannel intraluminal impedance and pH monitoring (MII-pH), not
only acid LPR (pH < 4) but also nonacid LPR (pH 4) can
be detected and effectively separated. In fact, most cases
of pharyngeal reflux are nonacid.8 It is believed that the
laryngeal squamous mucosa could uptake the pepsin present in nonacid reflux, which is reactive at pH < 6.5,9,10
and that the uptake of pepsin by laryngeal mucosa is
partly responsible for LPR disease.6,11–15 Therefore, we
Jiang et al.: Pepsin IHC Staining of Laryngeal Mucosa
assumed that pepsin can also be detected in the nonacid
LPR patients.
To further confirm the sensitivity and specificity of
pepsin immunohistochemical (IHC) staining for the diagnosis of LPR based on results from the MII-pH
monitoring, we prospectively recruited acid and nonacid
LPR patients diagnosed by MII-pH to evaluate whether
pepsin expressing in the laryngeal mucosa can be a
marker for LPR.
MATERIALS AND METHODS
Study Designs
From August 2007 to August 2008, patients complaining
of one or more of the following symptoms that were persistent
for more than 2 months were recruited: chronic hoarseness,
throat pain, foreign body sensation in the throat, excess throat
mucus, frequent throat clearing, chronic cough, and difficulty in
swallowing. Sex- and age-matched healthy volunteers without
complaints of laryngeal discomfort or MII-pH confirmed reflux
were used as controls. Both the patients and controls declared
no histories of respiratory diseases or use of proton pump inhibitors within 3 months and no smoking habit or smoking within
3 months. Electronic laryngoscopy was used for collecting interarytenoid mucosal biopsies. All specimens were formalin-fixed
immediately following the biopsies. After completion of sample
collection, all specimens were subjected to IHC staining for pepsin. The investigators had obtained institutional review board
approval from the First Affiliated Hospital of Sun Yat-sen University before the start of the study.
LPR Detected by 24-Hour Combined MII-pH
The Sleuth system (Sandhill Scientific, Highlands Ranch,
CO) was used for MII-pH monitoring. The system consisted of
two monitoring catheters (six impedance channels and two pH
channels in total), one recorder, and analysis software. One pH
channel was fixed at 5 cm above the lower esophageal sphincter, and another was located at about 0.5 cm above the upper
esophageal sphincter through the AirFlow Sphincter Locator
(Fig. 1). The participants were asked to fast for at least 8 hours
before the test. During the test, the subjects were asked to
maintain activities as usual and press the corresponding button
to record their eating time and position changes; the data were
recorded and analyzed by software (Bioview, Sandhill
Scientific).
The diagnosis of LPR was based on the entire six impedance channels impedance retrograde. The lowest impedance
channel should be dropped to at least 50% from baseline (less
than 2 seconds between two consecutive impedance channels),
and the highest impedance channel should show that it takes
more than 2 seconds to completely remove the reflux debris.
Pharyngeal pH is used to classify different types of LPR. Acid
LPR has pH values less than 4, and nonacid LPR has pH values
equal to or greater than 4. The schematic representation of
MII-pH is shown in Figure 1.
Electronic Laryngoscopy and Biopsy
An electronic laryngoscope (BF260; Olympus, Japan) was
used for laryngoscopy and biopsy in the electronic laryngoscopy
room of the Department of Otolaryngology; 1% dicaine was used
for superficial infiltration anesthesia of the larynx. Laryngoscopy showed no laryngeal malformation in either patients or
controls. Only very light or no edema or hyperemia was seen in
the laryngeal mucosa of normal controls. Erythema and edema
Laryngoscope 121: July 2011
Fig. 1. Schematic representation of 24-hour combined multichannel intraluminal esophageal impedance-pH monitoring. UES ¼
upper esophageal sphincter; LES ¼ lower esophageal sphincter.
were seen in the posterior cricoid wall, interarytenoid bar,
arytenoids complex, and posterior commissure of the laryngopharyngeal areas in all patients. Granuloma was seen in one
acid LPR patient, and vocal nodules were present in two acid
LPR and one nonacid LPR patient; one nonacid LPR patient
had false vocal sulcus.
Biopsy specimens were then obtained at the interarytenoid
mucosa of LPR patients detected by MII-pH and in controls. All
the specimens were fixed in 10% formalin at 4 C and embedded
in paraffin within 18 hours; then paraffin sections were made.
IHC Staining for Pepsin
Paraffin sections of interarytenoid mucosa were deparaffined and blocked by 10% horse serum and then were incubated
with rabbit antihuman polyclonal pepsin (product no. P3635Rbh, 1:100 dilutions. Uscnlife, USA). Antirabbit/mouse Immunohistochemistry Assay Kit (Envision system; DAKO, GK500705.
Uscnlife, USA) was used as a visualization kit. Phosphate-buffered saline was used to replace the primary antibody as
negative control, and chief cells in normal gastric mucosa were
used as positive control. Three randomly selected images at a
magnification of 400 were taken and preserved. All IHC staining results were evaluated independently by two experienced
pathologists who were blinded to the MII-pH monitoring results.
Statistical Analysis
All data were analyzed using the SPSS 13.0 program
(SPSS Inc., Chicago, IL). Kruskal-Wallis H test and Mann-Whitney U test were used for multigroup comparison and betweengroup comparison, respectively. A Fisher exact test was used to
Jiang et al.: Pepsin IHC Staining of Laryngeal Mucosa
1427
Fig. 2. Immunohistochemical (IHC) detection of pepsin (magnification 400). (A) Positive control (pepsin was detected as brown granules in the
chief cells of gastric mucosa); (B) negative control (phosphate-buffered saline in place of primary antibody against pepsin); (C) negative IHC reaction for pepsin (no cytoplasmic brown granules); (D) weakly positive IHC reaction for pepsin (scattered cells with cytoplasmic brown granules); (E)
moderately positive IHC reaction for pepsin (diffuse cytoplasmic brown granules of moderate staining intensity); (F) strongly positive IHC reaction
for pepsin (diffuse cytoplasmic brown granules of strong staining intensity). In cases with strong IHC reaction for pepsin, the pepsin was also
detected in the interstitial connective tissues. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
calculate the specificity and sensitivity. P < .05 was considered
statistically significant.
RESULTS
According to MII-pH monitoring, there were seven
acid LPR patients in this study, four males and three
females, with a mean age of 43.6 (613.6 standard deviation [SD]) years. Among eight nonacid LPR patients,
there were four males and four females, with a mean
age of 36.9 years (614.1 SD). The 21 healthy individuals, eight male and 13 female with a mean age of 37.8
years (617.0 SD), were controls. There were no significant differences in age and sex among the three groups
(acid LPR, nonacid LPR, and normal controls) (P > .05).
Laryngoscope 121: July 2011
1428
The positive IHC staining reaction for pepsin was
presented as intracytoplasmic brown granules as seen in
the chief cells from gastric mucosa (positive control; Fig.
2A). The negative IHC staining control applies the same
IHC staining procedure to the laryngeal mucosa, except
that the primary antipepsin antibody was replaced with
phosphate-buffered saline (Fig. 2B). No brown intracytoplasmic granules were seen in the negative IHC staining
control (Fig. 2B). For the study subjects, pepsin protein
was detected interstitially and in the cytoplasm of laryngeal squamous epithelial cells. The IHC staining
intensities were scored as negative () (Fig. 2C), weakly
positive (þ) (Fig. 2D), moderately positive (þþ) (Fig.
2E), and strongly positive (þþþ) (Fig. 2F).
Jiang et al.: Pepsin IHC Staining of Laryngeal Mucosa
Among the interarytenoid mucosa of seven acid
LPR patients, IHC staining for pepsin was strongly positive in four (57.1%), moderately positive in two (28.6%),
and weakly positive in one (14.3%) sample. Among the
interarytenoid mucosa of eight nonacid LPR patients,
three (37.5%) were strongly positive, three (37.5%) were
moderately positive, and two (25.0%) were weakly positive for pepsin protein. Among 21 normal healthy
controls, three (14.3%) were moderately positive, eight
(38.1%) were weakly positive, and 10 (47.6%) were completely negative for the pepsin protein. The difference in
the percentages of positive pepsin IHC staining was statistically significant among three groups (chi-squared
test, v2 ¼ 18.6, P < .01). Although both acid LPR and
nonacid LPR patients demonstrated higher levels of pepsin protein in their laryngeal mucosa compared with the
healthy controls (Z ¼ 3.54, P < .01; and Z ¼ 3.35, P <
.01), no significant difference in protein expression level
of pepsin was found between acid LPR and nonacid LPR
patients (Z ¼ 0.75, P ¼ .453).
Using weak positivity as a cutpoint, the positive
pepsin IHC staining reaction in the laryngeal epithelia
had a sensitivity of 100% and a specificity of 47.6% in diagnosis of LPR (P ¼ .01). However, when moderate
positivity was used as a cutpoint, the positive pepsin
IHC reaction in the laryngeal mucosa had a slightly
decreased sensitivity of 80% (85.7% and 75% for acid
and nonacid LPR, respectively) and a significantly
increased specificity to 85.7% in diagnosis of LPR (P <
.01; Table I).
DISCUSSION
Johnston et al.9 reported that pepsin was biologically active when pH was less than 6.5 and retained up
to 79% of the original biologic activity when the pH was
lowered to 3 or increased to 7 at 37 C for 24 hours.
Therefore, pepsin molecules can be very stable on the
surface of the laryngeal mucosa (pH ¼ 6.8) for a long
time. Further, they9,10 showed that laryngeal epithelial
cells can uptake pepsin via receptor-mediated endocytosis by using electron microscopy. Thus, if there is any
reflux (acid or nonacid) occurring in the larynx, pepsin
in the reflux content might be transferred into the laryngeal mucosa. Therefore, IHC detection of pepsin in the
laryngeal mucosa can be a sensitive way to detect any
laryngeal reflux but not specific for acid reflux. When
designating pepsin immunoreactivity of moderate to
strong intensity as positive cases, the positive pepsin
IHC stain had a sensitivity of 80% (85.7% and 75% for
acid and nonacid LPR, respectively) and a specificity of
85.7%. These results were consistent with those obtained
by Knight et al.,7 who reported that the presence of pepsin detected by Western blot and IHC in the laryngeal
mucosa had a sensitivity of 88.9% and a specificity of
100% in the diagnosis of acid LPR confirmed by pH
metry. Our study further showed that pepsin also presented in laryngeal mucosa of nonacid reflux patients.
In the present study, weak and moderate IHC staining for pepsin were detected in the laryngeal mucosa in
eight (38.1%) and three (14.3%), respectively, of 21
Laryngoscope 121: July 2011
TABLE I.
Difference of Pepsin Levels Between Laryngopharyngeal Reflux
Patients and Controls.
LPR (n ¼ 15)
Control (n ¼ 21)
Total
P*
.01
a
Pepsin
þ
Pepsinb
þ
Total
15
11
26
0
10
10
12
3
15
3
15
18
21
21
36
<.01
*Fisher exact test; in pepsina, (þ) was defined as any cases that are
positive for pepsin by IHC staining (weakly to strongly positive IHC reaction); in pepsinb, (þ) was defined as moderate to strongly positive IHC
reaction for pepsin.
LPR ¼ laryngopharyngeal reflux.
healthy volunteers without LPR following MII-pH monitoring. No laryngeal mucosa was strongly positive for
pepsin in healthy volunteers. There are several possible
reasons for low levels of pepsin present in normal laryngeal mucosa. First, our method of 24-hour MII-pH
monitoring might underestimate the true frequency of
LPR.14 It has been reported that up to 22% of patients
with gastroesophageal reflux disease symptoms who had
normal 24-hour esophageal pH monitoring would eventually show abnormal esophageal pH if the monitoring
was extended to 48 hours.16 Thus, this might also happen in the MII-pH monitoring, as the frequency of LPR
was very low. Second, dietary modifications may lead to
false-negative studies.16 Third, there are some mild and
asymptomatic cases of reflux that occasionally will
escape any form of monitoring.17 It is possible that low
levels of pepsin present in some of our normal controls
may represent an extremely mild form of LPR that
escaped our 24-hour MII-pH monitoring. We therefore
recommend that cases with focal and weak IHC staining
for pepsin be considered as negative and those with
moderate to strong pepsin immunoreactivity be considered as positive. With these criteria, we are able to
perform pepsin IHC staining in laryngeal mucosa with
rates of sensitivity and specificity that are very similar
to those obtained by others.6 Further research would be
warranted to compare the severity of LPR symptoms
with the levels of cytoplasmic pepsin, to extend pH monitoring to 48 hours in an attempt to reduce falsenegative rate for the diagnosis of LPR, and to determine
how long the cytoplasmic pepsin lasts in the laryngeal
mucosa.
Knight et al.7 suggest that enzyme-linked immunosorbent assay of pepsin in the sputum may provide a
noninvasive way to detect patients with LPR. However,
enzyme-linked immunosorbent assay is associated with
an increased false-negative rate, and the timing for sampling sputum is difficult to determine, making it not
practical for routine use in a clinical setting. IHC staining is one of the most commonly used methods in
clinical histopathologic laboratory analysis for disease
diagnosis.
Jiang et al.: Pepsin IHC Staining of Laryngeal Mucosa
1429
Johnston et al.6,14 reported that pepsin could be
detected by IHC in the mucosal samples collected from
the interarytenoid area, ventricular band, and vocal
folds. In our study, we took the mucosal biopsies from
the interarytenoid area with reproducible pepsin IHC
staining results. We selected the interarytenoid area as
the biopsy of choice because this area represents the
lowest point of the laryngeal inlet, through which the
reflux materials will always pass into the larynx. Also,
biopsy at the interarytenoid area poses the least risk of
damaging laryngeal function, such as vocal cord damage,
with relatively easier operation by otolaryngologists and
better tolerance by the patients. Therefore, biopsy of the
interarytenoid area is effective and more appropriate in
clinical work.
Furthermore, compared to MII-pH, which costs
more than 1,500 RMB and takes 24 hours (making most
patients intolerant), electronic laryngoscopy and IHC
cost less than 500 RMB total and most subjects can tolerate the biopsy. Although IHC requires a minimally
invasive procedure, such as mucosal biopsy, it is not that
expensive and is easy to carry out. It is cost-effective,
and most patients can tolerate it well with very minor
morbidity, which is amenable to the detection by IHC
staining method following biopsy.
CONCLUSION
Pepsin can be a marker for gastric content reflux to
the laryngeal mucosa. IHC detection of pepsin in the laryngeal mucosa of the interarytenoid area appears to
provide a sensitive and specific way to diagnose LPR in
clinical applications.
Acknowledgment
The authors thank Jinfen Du and Xiaojiao Wu for
their contribution to laryngoscopic exam and biopsy.
Laryngoscope 121: July 2011
1430
BIBLIOGRAPHY
1. Koufman JA. The otolaryngologic manifestations of gastroesophageal
reflux disease (GERD): a clinical investigation of 225 patients using ambulatory 24-hour pH monitoring and an experimental investigation of
the role of acid and pepsin in the development of laryngeal injury. Laryngoscope 1991;101:1–78.
2. Maronian NC, Azadeh H, Waugh P, et al. Association of laryngopharyngeal
reflux disease and subglottic stenosis. Ann Otol Rhinol Laryngol 2001;
110:606–612.
3. Chen MY, Ott DJ, Casolo BJ, et al. Correlation of laryngeal and pharyngeal carcinomas and 24-hour pH monitoring of the esophagus and pharynx. Otolaryngol Head Neck Surg 1998;119:460–462.
4. Ylitalo R, Ramel S. Extraesophageal reflux in patients with contact granuloma: a prospective controlled study. Ann Otol Rhinol Laryngol 2002;
111:441–446.
5. Vaezi MF. Gastroesophageal reflux disease and the larynx. J Clin Gastroenterol 2003;36:198–203.
6. Johnston N, Knight J, Dettmar PW, et al. Pepsin and carbonic anhydrase
isoenzyme III as diagnostic markers for laryngopharyngeal reflux disease. Laryngoscope 2004;114:2129–2134.
7. Knight J, Lively MO, Johnston N, et al. Sensitive pepsin immunoassay for
detection of laryngopharyngeal reflux. Laryngoscope 2005;115:
1473–1478.
8. Tutuian R, Castell DO. Use of multichannel intraluminal impedance to
document proximal esophageal and pharyngeal nonacidic reflux episodes. Am J Med 2003;115(suppl 3A):S119–S123.
9. Johnston N, Dettmar PW, Bishwokarma B, et al. Activity/stability of
human pepsin: implications for reflux attributed laryngeal disease. Laryngoscope 2007;117:1036–1039.
10. Johnston N, Wells CW, Blumin JH, et al. Receptor-mediated uptake of
pepsin by laryngeal epithelial cells. Ann Otol Rhinol Laryngol 2007;116:
934–938.
11. Axford SE, Sharp N, Ross PE, et al. Cell biology of laryngeal epithelial
defenses in health and disease: preliminary studies. Ann Otol Rhinol
Laryngol 2001;110:1099–1108.
12. Franchi A, Brogelli B, Massi D, et al. Dilation of intercellular spaces is
associated with laryngo-pharyngeal reflux: an ultrastructural morphometric analysis of laryngeal epithelium. Eur Arch Otorhinolaryngol
2007;264:907–911.
13. Gill GA, Johnston N, Buda A, et al. Laryngeal epithelial defenses against
laryngopharyngeal reflux: investigations of E-cadherin, carbonic anhydrase isoenzyme III, and pepsin. Ann Otol Rhinol Laryngol 2005;114:
913–921.
14. Johnston N, Dettmar PW, Lively MO, et al. Effect of pepsin on laryngeal
stress protein (Sep70, Sep53, and Hsp70) response: role in laryngopharyngeal reflux disease. Ann Otol Rhinol Laryngol 2006;115:47–58.
15. Remacle M, Lawson G. Diagnosis and management of laryngopharyngeal
reflux disease. Curr Opin Otolaryngol Head Neck Surg 2006;14:
143–149.
16. Vincent DA Jr, Garrett JD, Radionoff SL, Reussner LA, Stasney CR. The
proximal probe in esophageal pH monitoring: development of a normative database. J Voice 2000;14:247–254.
17. Ylitalo R, Lindestad PA, Ramel S. Symptoms, laryngeal findings, and 24hour pH monitoring in patients with suspected gastroesophago-pharyngeal reflux. Laryngoscope 2001;111:1735–1741.
Jiang et al.: Pepsin IHC Staining of Laryngeal Mucosa