Cytosponge Protocol
Phase: NA
Conditions: Eosinophilic Esophagitis
Sponsor: Mayo Clinic

 
 
Cytosponge Protocol  
 
NCT01585103 
 
November 24, 2021 

 
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11/24/2021 
 
 
 
Title    
 
Cytosponge Protocol 
 
PI:  
 
 
Jeffrey Alexander, M.D. 
 
Collaborators: 
 
Rebecca Fitzgerald, MD 
Michael Henry, MD 
Kevin Halling, MD, PhD 
Thomas C. Smyrk, MD 
Benjamin L. Wright, MD 
 
 
Study Coordinator: Crystal Lavey, CCRP 
 
 
 
 
 
 
 
 Division of Gastroenterology & Hepatology, Mayo Clinic Rochester, MN 
  
 
 Eosinophilic esophagitis is a relatively new disease in which esophageal eosinophilia leading to 
inflammation and stricture formation results from exposure to food antigens.  Although it would 
seem logical that identification and removal of food antigens would be the definitive treatment, 
this is more easily said than done.  
 
There are several reasons for this: 
 
• Current available allergen testing, mostly through blood and skin, do not reliably reflect 
specific allergen sensitivity in the esophagus.  
 
• As there has not been a non-invasive test to determine the esophageal response to single 
food allergens, clinicians have had to choose between allergen avoidance therapy because 
it is too cumbersome or performing serial and at times numerous endoscopies and 
biopsies to fully evaluate a range of potential foods that may trigger the inflammatory 
response.  
 
• Treatment of eosinophilic esophagitis is also further hampered by a distinct lack of 
symptoms in adults due to what is typically a silent inflammatory response until stricture 
formation and subsequent dysphagia and impaction occur.   
 
• Furthermore, adult patients with this disease typically accommodate by careful chewing 
and slow eating such that an absence of symptoms commonly underestimates the severity 

 
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of disease.  As a result, an invasive test that reliably determines esophageal eosinophilic 
would be extremely valuable for monitoring disease activity and the response to food 
allergen withdrawal or introduction. 
 
 
Cytosponge is an ingestible gelatin capsule containing a compressed mesh attached to a string. 
The capsule is swallowed and once in the stomach, the gelatin dissolves and a spherical mesh of 
3 cm diameter is released. The mesh is withdrawn through the mouth by the attached string and a 
cytologic specimen is collected.   
 
 
Current cytosponge from University of Cambridge 
 
Covidien Cytosponge
 
 

 
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In a recent study evaluating the Cytosponge in patients with Barrett’s esophagus, excellent 
results were achieved with a the sensitivity and specificity of the test was 73.3% (95% 
confidence interval 44.9% to 92.2%) and 93.8% (91.3% to 95.8%) for 1 cm or more 
circumferential length and 90.0% (55.5% to 99.7%) and 93.5% (90.9% to 95.5%) for clinically 
relevant segments of 2 cm or more.   
 
There was also overall excellent tolerance by patients using the Cytosponge.  As a result, one 
might wonder if the Cytosponge would similarly be a reliable relatively non-invasive tool for 
evaluating patients with eosinophilic esophagitis. 
 
Specific Aim: To evaluate the sensitivity and specificity of Cytosponge when compared to upper 
endoscopy and biopsies/swab testing in patients undergoing clinically indicated endoscopy for 
evaluation of eosinophilic esophagitis and healthy controls 
 
 
 
Methods:   
• Patients with eosinophilic esophagitis and health controls, (patients without esophageal 
symptoms such as GERD or dysphagia), will be enrolled.   
• Patients with EoE will be having a clinically indicated upper endoscopy and biopsies 
either for initial diagnosis or for monitoring the activity of their disease.   
• All patients will be asked to be fast, no solid food 8 hours prior to swallowing the 
cytosponge, can drink clear liquids up to 2 hours prior to swallowing the cytosponge 
• The EoE subjects two hours prior to endoscopy will be asked to swallow the Cytosponge.  
Briefly, the capsule and bunched up string are swallowed with water. Two hours is 
chosen as the time interval because water will likely be needed to swallow the capsule 
and this is the standard time allowed for to last safely consume a clear liquid prior to 
endoscopy. 
• Controls will be will have the same fasting instructions but will not be required to be 
having and endoscopy. 
• The string is held without any tension to allow the capsule to move into the stomach. The 
patient holds onto the string for five minutes after ingestion to allow the capsule to 
dissolve in the proximal stomach, where a spherical mesh of 3 cm diameter is released.  
• The back of the throat is then sprayed with 1% lidocaine and the expanded mesh 
withdrawn by pulling on the string with the patient’s head in an extended position.  
• After retrieval the string is cut and the Cytosponge containing the cytological specimen 
placed in preservative fluid and kept at room temperature until transportation to the 
laboratory. 
•  The whole process of administering the Cytosponge, including instructing the patient, 
takes less than 10 minutes. 
• Following this, the clinically indicated endoscopy will be performed. The cytology 
specimens will be reviewed by one pathologist who will read them blindly without 
knowledge of the results of the biopsy specimens.    The cytology specimens will be 

 
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compared to the biopsy in terms of esophageal eosinophil presence and density.  A 
scoring system for cytology will be devised.  
• Samples from the University of North Carolina will be sent to the Mayo Clinic to be 
reviewed by the same pathologist. 
• After the Mayo pathologist have completed their analysis, slides and part of the uncut 
blocks will be sent to Dr. Rebecca Fitzgerald’s lab at the University of Cambridge 
looking at reproducibility of results, evaluation of the mucosal microbiome by 
sequencing and additional biomarker studies including RNA, methylation and protein 
analyses. 
• We will send samples from a cell block made from the cytology fluid from the 
cytosponge and a portion of the primary cytosponge placed in RNAlater solutionFor 
extra-cellular RNA signature analysis with possible detection of EoE specific lumen 
protein products proposed to Dr. Rodenberg’s lab at the University of Cincinnati. 
• FFPE RNA form the concurrent biopsies at the time of the EGD if available 
as biopsy tissue represents the source of the dislodged cells/tissue pieces by the 
cytosponge. Parallel comparison will provide key clue to validate the new diagnostic 
approach. 
• We will stain sections from esophageal biopsies and Cytosponge for the eosinophil 
secondary granule protein eosinophil peroxidase (EPX).  This may increase the 
diagnostic sensitivity of the Cytosponge because EPX staining accounts for eosinophil 
degranulation products in addition to intact eosinophils.  H+E stains and their matching 
EPX stains will be digitized using an Aperio slide scanner and Aperio ImageScope 
software will be used for quantification of EPX. 
• Modification 11/24/2021 – we would also like to utilize slides from IRB15-004741 on 
patients that had a cytosponge and biopsy. These will undergo the EPX staining as 
described above. Possible samples used will be cross checked in Ptrax to ensure the 
subjects consented to allowing their samples to be utilized in future research and only the 
applicable samples will be used. Total Mayo participants to use from this study will be up 
to 30.  
 
 
 
Recruitment:  We will recruit 130  participants at Mayo there will be 180 total subjects in this 
trial 
 
Inclusion Criteria:   
• Adult patients between the ages of 18 and 65 with eosinophilic esophagitis or health 
controls including lactating or pregnant women undergoing clinically indicated 
endoscopy and esophageal biopsy.   
 
 
Exclusion Criteria:  
• Patients for whom clinically indicated endoscopy is not safe.  Patients who have small 
caliber esophagus, an esophageal stricture that will not permit passage of the gelatin 
capsule or are unable to swallow the capsule.  Patients who have taken any 
antithrombotic medication within 10 days of the procedure. 

 
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• Vulnerable populations, such as those with diminished mental acuity, will be excluded.  
Patients with known Lidacaine/Acetylcysteine allergies  
Study flow: 
 
 
• The study coordinator will look for patients being seen in Esophageal clinic with the 
indication of EoE or healthy controls (patients without esophageal symptoms such as 
GERD or dysphagia) 
•  When the physician has an EoE patient scheduled for a clinically indicated upper 
endoscopy, to diagnose or monitor their disease, these patients will be approach by the 
study coordinator to see if they are interested in the cytosponge study? 
• Subjects willing to swallow the cytosponge that do not have esophageal symtoms will be 
approached by the study coordinator. 
• Patients with EoE will then be scheduled to come to Gonda 9s two hours prior to the 
endoscopy. Healthy controls will be scheduled to come to Gonda 9 at their convenience 
as long as they adhere to the fasting instructions. 
•  The physician will have the patient swallow the Cytosponge. 
•  The patient holds onto the string for five minutes after ingestion 
  
to allow the capsule to dissolve in the proximal stomach, where a spherical 
      mesh of 3 cm diameter is released. 
• The back of the throat is then sprayed with 1% lidocaine and the expanded mesh 
withdrawn by pulling on the string with the patient’s head in an extended position. 
• After retrieval the string is cut and the Cytosponge containing the cytological specimen 
placed in preservative fluid and kept at room temperature until transportation to the 
laboratory. 
•  The whole process of will take appoximately 10 minutes. 
• A throat swab for collection will also be performed at this time to be processed for 
bacterial population of the esophageal mucosa.   
• Following this, the clinically indicated endoscopy will be performed. Prior to biopsies we 
will do a brushing of the esophagus.   
• The cytology specimens will be reviewed by one Mayo pathologist who will read them 
blindly without knowledge of the results of the biopsy specimens.   
• The cytology specimens will be compared to the biopsy in terms of esophageal 
eosinophil presence and density.  A scoring system for cytology will be devised. 
• We will do further EoE/allergy analysis with FFPE slides including EDN and tryptase 
staining. 
• When the Mayo pathologist have completed their analysis, slides and part of the uncut 
blocks will be sent to Dr. Rebecca Fitzgerald’s lab looking at reproducibility of results, 
evaluation of the mucosal microbiome by sequencing and additional biomarker studies 
including RNA, methylation and protein analyses. 
• We will send samples from a cell block made from the cytology fluid from the 
cytosponge and a portion of the primary cytosponge placed in RNAlater solution. For 
extra-cellular RNA signature analysis with possible detection of EoE specific lumen 
protein products proposed to Dr. Rodenberg’s lab at the University of Cincinnati. 
• FFPE RNA form the concurrent biopsies at the time of the EGD if available 

 
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as biopsy tissue represents the source of the dislodged cells/tissue pieces by the 
cytosponge. Parallel comparison will provide key clue to validate the new diagnostic 
approach. 
• H+E sections will be digitized.  Cytosponge and esophageal biopsy sections will be 
stained for EPX and analyzed. 
 
 
 
Data Collection: 
 
Several types of data will be collected.   The first includes demographic data on patients 
including age, sex, allergy history, history of EoE.  Routine pathology data will be 
collected based on biopsy findings.   Cytology specimens will be analyzed both at  
Mayo Clinic, Rochester and Dr. Rebecca Fitzgerald’s lab at the University of Cambridge, 
looking at reproducibility. 
 
 Two center site for conduction of the Cytosponge and eosinophilic esophagitis trial, Dr. 
Evan Dellon at the University of North Carolina and I have agreed that we will pool our 
data for this study.  This is thought best as we will use the same methodology and we will 
each collect mutually needed data to perform the study.  We will not be using the exact 
same data collection forms, however, as both Mayo and UNC have their established data 
bases for eosinophilic esophagitis where there are subtle differences in some of the types 
of data collected.  On the other hand, the data that is needed for this two center study will 
be collected in an identical fashion. 
 
When the Cytosponge is taken out of the patients’ mouth, washed with sterile PBS, 
followed by centrifugation. The supernatant will be stored at -80oC for extra-cellular 
RNA signature analysis with possible detection of EoE specific lumen protein products 
proposed.   The pellet will also be subjected to total RNA extraction immediately after 
the supernatant aspiration. The resulting RNA will be used for microRNA array and for a 
qPCR based array on representative EoE mRNA signature transcripts. Both methods are 
mature approaches regularly performed in the Rothenberg laboratory1-3 and have been 
validated with multiple cohorts, forming the foundation for diagnostic purposes.  
 
We will extract RNA from the remaining of FFPE sponge, aiming to acquire the RNA 
from the residue tissue after histological examination. The RNA isolated this way will be 
subjected to the same experiments as the fresh wash pellet cells. 
 
In order to validate the proposed new method, we also aim to acquire FFPE RNA from 
the concurrent biopsy taken by EGD if available, as biopsy tissue represents the source of 
the dislodged cells/tissue pieces by the cytosponge. Parallel comparison will provide key 
clue to validate the new diagnostic approach. 
 
We will make parallel comparisons of EPX staining from the Cytosponge and esophageal 
biopsy specimens.  We will also assess correlations between EPX stains and peak 
eosinophil counts from the corresponding H+E stain. 

 
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Data Handling 
A study coordinator will be responsible for collating and handling the above data 
into a computer accessible only though password entry by the coordinator and 
principal investigator of this study. 
 
Data Analysis 
A scoring system will be devised for cytology scoring of eosinophil density.   
EPX will be quantified by a pixel count algorithm using Aperio ImageScope 
software and a previously validated scoring system. 
 
Feasibility and Time 
It is expected to take six months to complete this study.  For recruitment of 
patients, the Mayo Clinic has a long track record in studying patients with EoE 
and has over four hundred patients with this disease currently in their data base.    
 
Strengths 
The strengths of this study are the relatively small group of patients required, the 
non-invasive and well tolerated nature of this test relative to endoscopy and 
biopsy and the potential efficacy of this test.  Other strengths include the expertise 
of the investigators in eosinophilic esophagitis and the collaboration with Dr. 
Rebecca Fitzgerald, the inventor of the cytosponge, whose team has great 
familiarity with the administration and interpretation of the test and its results.   
 
Weaknesses 
The main weaknesses of this study will be the potential inability of a patient to 
swallow the capsule or risk of impacting (though the capsule will dissolve and it 
will be easily removable). 
 
References: 
 
1.  Kadri SR, Lao-Sirieix P, Maria O’Donovan M, et al.  Acceptability and accuracy of a non-
endoscopic screening test for Barrett’s esophagus in primary care: cohort study.  Brit Med J 
2010; 341:c4372 
2. Blanchard C, Wang N, Stringer KF, et al. Eotaxin-3 and a uniquely conserved gene-expression 
profile in eosinophilic esophagitis. J Clin Invest 2006;116:536-47. 
3. Collins MH, Blanchard C, Abonia JP, et al. Clinical, pathologic, and molecular 
characterization of familial eosinophilic esophagitis compared with sporadic cases. Clin 
Gastroenterol Hepatol 2008;6:621-9. 
4. Lu TX, Sherrill JD, Wen T, et al. MicroRNA signature in patients with eosinophilic 
esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers. J Allergy 
Clin Immunol 2012;129:1064-75 e9.