Dye solution optimizing staining conditions for in vivo endocytoscopy for normal villi and superficial epithelial tumors in the duodenum

Patients

The present study was conducted at the Jikei University Hospital from February 2011 to April 2013. We recruited 9 and 20 patients who had an endoscopically normal duodenum and SNADETs, respectively. This study was approved by the institutional review board of the Jikei University School of Medicine (No. 22-204 6381) and conducted in accordance with the Helsinki Declaration (as revised in 2000). Written informed consent was obtained from all patients.

Patients were eligible if they met the following inclusion criteria: (i) age ≥20 years; (ii) an endoscopically normal duodenum (study for normal villi); or (iii) a SNADET lesion destined for endoscopic resection (study for SNADETs). Exclusion criteria were: (i) serious underlying diseases; (ii) current pregnancy; and (iii) a recent history of chemotherapy for any malignancy. Patients with a recent history of chemotherapy were excluded because duodenal mucosal damage would preclude ECS evaluation.

Endoscopy equipment, setting, and performance

We used the prototype of ECS (GIF-Y0002; Olympus Corporation, Tokyo, Japan) connected to a video-endoscopy system (CV260, EVIS LUCERA SPECTRUM; Olympus). The GIF-Y0002 is an integrated type of endoscope with one lens that enables a progressive increase in magnification to ×380 (tissue field of view, 700 µm × 600 µm, 0.42 mm2) using a hand lever. Up to ×600 ultra-high magnification is available when the electronic zoom function (×1.6) is activated by pushing the button [24]. The consecutive zoom function allows us to find the target area through non-magnifying observation, while maintaining the center of the endoscopic view. The ECS system can visualize not only the gland duct lumens and villi, but also the shape of epithelial cells and nuclei at a focus depth of about 50 µm.

ECS procedure

All ECS exams were performed by a single endoscopist (NY) with ECS experience of more than 30 cases of superficial epithelial neoplasm in the upper GI tract. All patients underwent deep sedation through intravenous administration of pethidine hydrochloride (35-70 mg, Opystan®; Mitsubishi Tanabe Pharma, Osaka, Japan) and flunitrazepam (0.2-0.8 mg, Rohypnol®; Chugai Pharmaceutical, Tokyo, Japan). We administered butylscopolamine bromide (20-40 mg, Buscopan®; Boehringer Ingelheim GmbH, Ingelheim, Germany) or glucagon (Glucagon G Novo®; Eisai, Tokyo, Japan) as a peristalsis depressant just before ECS.

Prior to dye staining for ECS, any mucus on the region of interest was carefully flushed with water containing pronase (Pronase MS®; Kaken Pharmaceutical Products Inc., Tokyo, Japan) and dimethicone (Gascon®; Kissei Pharmaceutical Products Inc., Tokyo, Japan). The region of interest was sprayed with 2 mL dye solution and excess dye was removed by suction. The tip of an ECS scope was placed in contact with the surface and then focused.

First, the staining examination was completed in the 9 patients with normal villi. Then, based on these results, we used 2 dye solutions (1% MB and 0.5% TB) for the examination of the 20 patients with SNADETs.

Staining examination of normal villi

The study of normal duodenal villi used 3 clinically available staining dyes: MB, TB, and CV. The concentration for testing was determined using 2×, 4× or 5× dilution of these dyes (5%, 2.5%, and 1% MB; 1%, 0.5%, and 0.25% TB; and 1%, 0.5%, and 0.25% CV), based on an ex vivo animal study [20].

The 9 patients with normal villi were randomly and evenly allocated 1:1:1 to 3 staining groups (MB, TB, and CV) by the envelope method (Table 1). Before staining, we chose 3 duodenal areas per patient in each of the first, second and third portions, and allocated each duodenal portion to a concentration of a dye solution. We started each staining examination from the most distal portion (i.e., the third portion) to prevent regurgitation and staining of a mucosal area with 2 or 3 concentrations of a dye solution. For instance, in a patient allocated to MB staining, we started staining with 1% MB (2 mL) and took ECS images in the third portion, followed by staining with 2.5% MB (2 mL) and images in the second portion, and finally by staining with 5% MB (2 mL), taking photos in the first portion.

Table 1 Patient characteristics in the staining examination of normal duodenal villi

Staining examination of SNADETs

We decided to use just 2 dye solutions for this examination because SNADETs are rare and it would be difficult to examine many dye solutions and concentrations. According to the results of the examinations of normal villi, dye solutions of 0.5% TB and 1% MB yielded the best and second-best staining conditions. Furthermore, 1% MB has been most commonly used in previous studies [18,19,22,23]. Therefore, 0.5% TB and 1% MB were used for the staining examination of SNADETs.

The 20 patients were randomly and evenly allocated 1:1 to the 2 staining groups (MB and TB) by the envelope method. Ten SNADETs were stained by 1% MB and 10 by 0.5% TB. The staining solution was applied to the SNADET lesions with a spray catheter and excess dye was removed through suction. This staining procedure was repeated up to 3 times when the lesions could not be stained adequately.

ECS findings

Fig. 1 demonstrates typical normal villi on ECS images and microphotograph, respectively. Fig. 2 shows representative ECS images and microphotographs of SNADETs, tubulovillous and tubular adenocarcinoma in situ (Category 4.2), respectively.

Figure 1 (A) A typical endocytoscopy (ECS) image of duodenal normal villi stained by 0.5% toluidine blue. (B) A microphotograph shows a horizontal section view of normal duodenal mucosa with hematoxylin-eosin (H&E) staining; the 2 images are substantially identical in demonstrating villous structures covered with microvilli (transparent marginal zone in ECS and fine pinkish line in H&E section) and foveolar epithelial cells having palisading fusiform nuclei with belt-like formation. The microvilli can be seen along with broken lines and the palisading nuclei between the dotted lines in the ECS image and microphotograph

Figure 2 (A) A protruded lesion with a rough surface is seen in the second portion of the duodenum. (B) Endocytoscopy images after spraying 1% methylene blue solution for the small lesion area (black square in A). At maximum magnification, endocytoscopy visualizes irregularly-formed tubular structures with a slit-like lumen and enlarged fusiform nuclei, or distorted nuclei with a loss of polarity. Histology of an endoscopic resection specimen shows tubulovillous adenocarcinoma confined to the mucosal layer (noninvasive carcinoma, Category 4.2). (C) A slightly erythematous lesion with marginal elevation (arrowheads) is seen in the second portion. (D) An endocytoscopy image stained by 1% methylene blue solution. Maximum magnification of the small lesion area indicated by the black square in (C), a glandular structure with a small lumen (arrows) surrounded by distorted nuclei (arrowheads) appears as an onion ring-like formation. Histology of the endoscopic resection specimen showed a tubular adenocarcinoma in situ (Category 4.2)

Evaluation of ECS images

Rating system (Fig. 3)

Prior to the present study, 3 endoscopists (KG, NY, and AD) and one pathologist (MI) reviewed 5 ECS images of duodenal normal mucosa and 10 images of SNADETs, excluding all images from the image evaluation phase. They defined representative ECS findings of normal villi and SNADETs. Based on the representative ECS findings, they developed criteria for evaluating staining conditions.

Evaluation categories: 1. general quality, 2. structure, and 3. nuclei

Evaluation criteria and scoring method (Fig. 3)

Figure 3 Representative endocytoscopy images corresponding to each score and category for reviewer’s evaluation

1. “General quality” was evaluated based upon focus, optical noise, degree of staining, evenness of dyeing, and inappropriate dyeing, such as foreign matter staining.

2. “Structure” was evaluated based upon the visibility of cytoplasm of villi and microvilli, and abnormal structure (tubular/glandular, irregular, or disrupted pattern) in SNADET lesions.

3. “Nuclei” were evaluated based upon the visibility of palisading fusiform nuclei or punctiform nuclei in the evaluation of normal villi and abnormal nuclear shape (enlarged and/or distorted form) in SNADET lesions.

A 3-point visual analog scale for reviewers’ evaluations and scoring was rated as follows: 1, “Poor” (not interpretable or invisible); 2, “Moderate”; 3, “Good” (interpretable or visible). These corresponded to evaluation scores of 1, 2, and 3 points, respectively.

Preparation of image files

The endoscopist (NY) who performed all the ECS inspections took several images of each normal duodenal site and SNADET lesion. The endoscopist created 2 image catalogs for evaluating normal villi and SNADETs as follows. The ECS images were all stored and divided into 2 image groups: a normal villi group and a SNADET group. Each of the 2-image groups was subdivided into image files corresponding to the respective staining procedure (e.g., an image file of normal villi stained by 0.5% TB). Thus, image groups contained 27 (each dye × 3 concentrations × 9 patients) and 20 (2 dyes × 10 lesions) image files for normal villi and SNADETs, respectively. Each file that included several images was numbered separately for blinded evaluation, using randomly assigned numbers from a randomization table created by a computer program (Excel 2010, Microsoft Corp., Redmond, USA). No information about the dye solution and its concentration was displayed on the images and files.

Evaluation of staining conditions

In the review of the image files of normal villi, 2 endoscopists (KG, and AD) and a pathologist (MI) evaluated the staining conditions. In the evaluation of the image files of SNADETs, an endoscopist (KG) and a pathologist (MI) assessed the staining conditions. One endoscopist (AD) was excluded from reviewing the image file of SNADETs because he was considered to have inadequate knowledge of the microscopic morphologic features of SNADETs.

Three reviewers (KG, AD, and MI) and 2 reviewers (KG and MI) assigned scores to the 3 evaluation categories of 27 (sites of normal villi) and 20 (SNADETs) ECS image files, respectively. In evaluating an image file of normal villi, each of the 3 reviewers gave an evaluation score to each of the 3 evaluation categories for each of the 3 sites stained by a dye solution. Each of the 9 dye solutions consequently received 27 evaluations in total (Table 2). Evaluation of SNADETs was more complicated than that for normal villi. Hence, the 2 expert reviewers (KG and MI) mutually agreed upon and gave only one evaluation score to each of 3 evaluation categories for each of 10 SNADETs. Each of the 2 dye solutions consequently received 30 evaluations in total (Table 4).

Table 2 Evaluation results for normal duodenal villi

Table 3 Comparison of evaluation results for normal duodenal villi using 3 staining dyes

Table 4 Evaluation results of 20 superficial non-ampullary duodenal epithelial tumors

Histologic diagnosis

Histologic diagnosis of normal ECS sites or SNADET lesions was established by an expert GI pathologist (MI) using biopsied tissues or endoscopically resected specimens. The pathologist was blinded to the information about the ECS findings. SNADET lesions were histologically graded as follows: LGD, HGD, or adenocarcinoma, corresponding to category 3, category 4.1, and category 4.2 to 5, respectively, in the modified Vienna classification [25]. SNADET was defined as a combination of LGD, HGD, and adenocarcinoma, in which invasion was confined to the submucosal layer [6].

Statistical analysis

We calculated the frequencies and proportions of the individual scores of evaluation categories for each dye solution. In the statistical analysis, we divided the evaluation scores into 2 groups, “1 or 2” and 3, to avoid small data pools for some evaluation categories. Quantitative parameters were compared using Student’s t-test, while qualitative parameters were compared using Fisher’s exact test or one-way ANOVA. Statistical significance was defined as a 2-tailed P-value of <0.05. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).

Normal villi

Table 1 lists the characteristics of patients. There were no significant differences among the MB, TB, and CV groups as regards age or sex. Typical ECS images of normal duodenal villi stained by 9 dye solutions are shown in Fig. 4. Table 2 shows that the highest score of 3 (Good) was most frequently given to 0.5% TB for general quality of image and visibility of nuclei (n=7 and 8, respectively) and to 1% MB for structure (n=9). Of the 3 staining regimens, 0.5% TB obtained the most top scores of 3 (Good) (22/27, 81%), followed by 1% MB (19/27, 70%). In the CV group, no reviewer gave the highest score of 3 (Good) for 2 of the evaluation categories, general quality and nuclei. MB and TB showed significantly higher scores compared with CV in the overall reviewer’s evaluation (P<0.001 and P<0.001, respectively, Table 3).

Figure 4 Typical endocytoscopy images of normal duodenal villi stained by 9 dye solutions

SNADET

No significant difference in the baseline data of patients with SNADETs (age, sex, location, diameter, macroscopic type, and histology) was found between the 1% MB and 0.5% TB staining groups. Table 4 indicates that 1% MB achieved a significantly higher score compared with 0.5% TB (P=0.035) in all evaluation categories (general quality/structure/nuclei).

No patients experienced complications or adverse reactions related to the ECS examination.