Materials and Methods
We performed a prospective cohort study to evaluate the eosinophil density in the oesophagi of EoE patients. Under IRB approval (#0044645, approved 12/2010), patients were enrolled at the University of Utah and the Salt Lake Veterans Affairs Medical Center in Salt Lake City, UT. Patients with known EoE were eligible to enrol. Patients categorised as EoE had prior oesophageal biopsies with at least 15 eos/HPF (×400) in the area of greatest density (i.e. the peak within a biopsy) of at least one biopsy while on high dose proton pump inhibitor therapy. Exclusion criteria included inability to pass the endoscope through the oesophagus to obtain biopsies, bleeding diathesis prohibiting biopsies, chronic anticoagulation preventing biopsies at time of endoscopy or patients with systemic disease requiring chronic steroids or immunosuppression.
Populations
Subjects were categorised as EoE (n =10) if they had prior biopsies ≥15 eos/HPF on high dose acid suppression with recurring symptoms of food impaction, chest pain or dysphagia as per guidelines (two patients were biopsied twice in this study) or resolved EoE (Rx-EoE) (n =6) if they had prior biopsies ≥15 eos/HPF and subsequently underwent therapy with elimination diets and reported symptomatic resolution. Ten patients with EoE were chosen to provide a variety of endoscopic appearances from which to take biopsies. Two patients were subsequently biopsied twice to confirm stability of disease and findings. Thus, a total of 12 endoscopies were utilised in the outcomes.
Questionnaire
Prior to undergoing endoscopy, patients were asked to complete questionnaires including Reflux Disease Questionnaire and allergy questionnaires. Dysphagia scores were obtained as well on all subjects. Dysphagia was assessed based on the following scoring system: 0 = None, 1 = Solid food dysphagia once in 3–12 months, 2 = Solid food dysphagia once in 1–3 months, 3 = Solid food dysphagia once every 2–4 weeks, 4 = Solid food dysphagia once every 1–2 weeks, 5 = Solid food dysphagia once every 1–7 days, 6 = Solid food dysphagia with every meal and 7 = Dysphagia to solid AND liquid food (see Table 1).
Endoscopy
Upper gastrointestinal (GI) endoscopy was performed using midazolam or propofol with fentanyl for sedation. Biopsies were taken using a standard video gastroscope (Pentax, Tokyo, Japan or Olympus, Alexandria, VA, USA) and RJ3 biopsy forceps (Boston Scientific Marlborough, MA, USA). Biopsies were obtained beginning 1 cm proximal to the cardia. Thereafter, biopsies were collected 1 cm above the cardia at 0° (anterior) and 180° (posterior), and then 1 cm proximal to that at 90° and 270°. Thereafter, pairs of biopsies were performed each centimetre for 4 cm followed by a 2 cm break until the proximal oesophagus was reached with each pair of biopsy sites rotating by 90° or patient sedation limits or tolerance exceeded (Figure 1). Each specimen was submitted in a separate container and identified individually by depth, angle (i.e. 36 cm, 180°), and local mucosal appearance (e.g. rings, furrows, plaques, lines, normal, etc.) in the area at the level of the biopsy site in a datasheet. No identifiers were transmitted to the pathologist who remains blinded to endoscopic findings and location of the biopsies.
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Figure 1.
Schematic of the pattern of biopsies performed in the distal oesophagus. Initial biopsies were performed at the gastroesophageal junction/cardia. Subsequent biopsies were performed every centimetre starting at 0° (anterior) and 180° (posterior) alternating every centimetre with 90° and 270°. After 4 cm, a 2 cm break was applied and biopsies then resumed following the same pattern. The pattern of biopsies continues in to the proximal oesophagus.
If the biopsies were taken within 10 cm of the cardia, they were labelled 'distal oesophagus'. If they were taken proximal to 10 cm of the cardia, they were labelled 'proximal oesophagus'. By the nature of the design of the study, distal samples exceeded proximal samples in number.
Because mucosal manifestations often occur concurrently with each other (e.g. rings with furrows, etc.), phenotypic risk for disease was categorised. Classic endoscopic findings for EoE were defined as rings, furrows and white spots. Rings were defined as fixed circumferential rings within the oesophagus that were not flattened by insufflation. Furrows were defined as linear lines proceeding proximally distally with associated depth and contraction of surrounding tissue. Lines were defined as running proximally distally without associated depth or contractions. Plaques were defined as white exudates seen on the luminal surface (Figure 2). Areas populated with both white spots and furrows were ranked as most likely to have eosinophils followed by areas with only white spots, only furrows, or only linear markings or rings respectively. Normal-appearing (i.e. lacking lines, rings, furrows and plaques) tissue was thought to not likely contain eosinophils.
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Figure 2.
Endoscopic findings evaluated. (a) Furrows with linear marking associated with contraction of surrounding tissue. (b) Plaques seen as white exudates on the surface of the tissue. (c) Lines seen as subtle linear markings without surround contraction of the tissue. (d) Rings seen as fixed strictures unyielding to insufflation.
Pathology
Biopsy specimens were submitted in formaldehyde for routine processing and pathology evaluation. One pathologist blinded to the location and endoscopic appearances of the sites read all biopsies. All biopsies were read once identifying the area of highest density within each individual biopsy and determining the peak eosinophils/HPF. Peak eosinophil count was recorded as eos/HPF seen in the area of highest density (Olympus; 40× ocular 10×; area of field 0.55 m) and correlated with endoscopic findings at the site of the biopsy. Determinations of proximal vs. distal eosinophilia were made regardless of endoscopic findings. The peak values for each biopsy were recorded and then averaged and correlated with each endoscopic findings regardless of position in the oesophagus. Spongiosis and papillary elongation (PE) were graded based upon prior definitions.
Statistics
sas 9.2 software (Cary, NC, USA) was used for analysis when applicable. anova testing was performed to determine whether true differences existed in eosinophil distribution according to endoscopic findings. Eosinophil densities were compared via paired t-test, t-test or Mann–Whitney U when applicable to evaluate the differences in the mean peak eosinophil counts. Chi-squared analyses were used to determine sensitivity of biopsies. Spearman correlation was used to compare eosinophil counts histological changes.