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 The Esophageal
 Esophagogastric  Junction

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Volume: Barrett's Esophagus
Chapter: Diagnosis

What is the value of methylene blue staining to direct biopsies in Barrett's mucosa?

M.I.F. Canto (Baltimore)

Methylene blue is a vital stain taken up by actively absorbing tissues such as small intestinal and colonic epithelium. It had been used to highlight subtle mucosal changes in the small intestine (e.g. celiac disease) and colon (flat adenomas and carcinomas). It is does not stain non-absorptive epithelia such as squamous or gastric mucosa. Hence, it can positively stain metaplastic absorptive epithelium, such as intestinal-type metaplasia in the stomach [1] or not stain non-absorptive epithelium, such as ectopic gastric metaplasia in a background of positive staining duodenal mucosa [2]. The technique of methylene blue staining was originally described by Japanese investigators for improving the diagnosis of early gastric cancer.

Barrett's esophagus is defined as the metaplastic replacement of squamous epithelium in the esophagus by columnar epithelium. There are three types of metaplastic epithelial cells that have been described in patients with Barrett's esophagus, which resemble cells in the gastric fundus, gastric cardia, or intestine. It is the intestinal-type metaplasia or specialized columnar epithelium, which is considered pathognomonic of Barrett's esophagus. It is also the most clinically relevant type of epithelium due to its association with adenocarcinoma of the esophagus and esophagogastric junction. Specialized columnar epithelium has characteristic crypts and villi lined by mucus-secreting columnar cells and goblet cells. The similarity between specialized columnar epithelium in Barrett's esophagus and the incomplete intestinal metaplasia in the stomach resulted in the use of methylene blue for selective staining of specialized columnar epithelium.

The technique for methylene blue staining is simple and easy

Chromoendoscopy requires a spray catheter for delivery of stain to the mucosa. The most commonly used catheter is the Olympus washing catheter (PW-5L, Olympus America, Inc.), which delivers a fine mist and minimizes the amount of reagent lost into the lumen. Place the tip of the washing catheter just outside the biopsy channel so that the metal tip is just within view to minimize trauma to the mucosa during the spraying maneuver. The assistant must use a substantial amount of force (usually requiring both hands) to produce a good spray. Two to three of these reusable catheters can last several years even with routine use in a busy endoscopy unit. When spraying, the endoscopist needs to direct the endoscope and catheter tip towards the esophageal mucosa and use a combination of rotational clockwise-counterclockwise movements with simultaneous withdrawal of the endoscope tip. This will maximize the amount of reagent applied to the epithelium.

To minimize the amount of stain that can leak out and cause a mess, always ask for a new biopsy channel cap when performing chromoendoscopy.

If staining is being performed for cancer surveillance in a patient with biopsy-proven Barrett's esophagus, use a large-channel/therapeutic upper endoscope, which can accommodate the "jumbo" biopsy forceps.

Methylene blue staining involves application of a mucolytic, followed by dye, followed by washing off excess dye. Surface mucus must be removed to increase the uptake of dye into epithelial cells. This can be accomplished by spraying a 10% solution N-acetylcysteine (Mucosil or Mucomyst), which is a mucolytic agent used by pulmonologists. Mucomyst can be diluted with tap water. The volume of mucolytic agent required varies according to the length of the columnar mucosa being stained. In Barrett's esophagus, approximately 20 ml (milliliters) is required for every 5 cm of circumferential columnar mucosa. Wait two minutes before applying methylene blue.

Staining is performed with a 0.5% solution of methylene blue (American Reagent Lab, Shirley, N.Y.). This can be easily prepared by diluting the dye with tap water at a ratio of 1:1; i.e. mix 10 ml of water with the 10 ml of 1% methylene blue (10 ml/vial). As with Nacetylcysteine, the volume of methylene blue varies according to the length of columnar epithelium being stained. Approximately 20 ml is required for every 5 cm of Barrett's mucosa. Wait two minutes before the washing step.

Wash off excess dye with tap water applied from a 60 ml syringe attached to the washing catheter. I use an average of about 2-3 syringes (or 120-180 ml) per staining session. Long segments of Barrett's esophagus may require up to 240 ml or 4 syringes. The endpoint of staining is somewhat subjective and is the most difficult part to learn when learning the technique of methylene blue staining. Positive staining is defined as the presence of blue-stained non-eroded mucosa that persists despite vigorous water irrigation. Stain begins to fade after 20 minutes.

Methylene blue staining is a relatively inexpensive and simple endoscopic method for accurately diagnosing specialized columnar epithelium in Barrett's esophagus. It adds approximately 7 minutes to the procedure time and less than $ 9 to the procedure cost (minus the cost of the catheters).

Interpretation of methylene blue staining

Methylene blue staining of nondysplastic Barrett's esophagus may be either focal or diffuse (i.e. > 75% of Barrett's mucosa stains blue) [3]. Nearly all patients with long segment Barrett's esophagus (LSBE) have diffuse staining because specialized columnar epithelium comprises majority of the columnar mucosa [3]. In contrast, majority of short Barrett's esophagus exhibit focal staining due to the presence of gastric-type metaplasia along with specialized intestinal metaplasia.

Methylene blue selectively stains specialized columnar epithelium in Barrett's esophagus

In a pilot study, we showed that methylene blue staining is highly sensitive and specific for diagnosing specialized columnar epithelium in Barrett's esophagus [3]. It is nontoxic and without associated side effects. However, patients may notice blue discoloration of urine and stools after staining. Like other tissue stains, false positives may occur from nonspecific uptake of dye by erosions or ulcers. Hence, methylene blue staining is best accomplished after esophagitis has healed.

In a larger series of 52 patients undergoing endoscopic surveillance, we confirmed the high accuracy of methylene blue staining in patients with short, limited, and LSBE [4]. In a randomized sequential trial, methylene blue-directed biopsies led to increased detection of specialized columnar epithelium in limited- and LSBE, compared to 4-quadrant large particle random biopsies obtained every 2 cm of Barrett's esophagus. In patients with Barrett's esophagus 3-6 cm long, the average proportion of specialized columnar epithelium in biopsies was 87.8% versus 48.2% for methylene blue-directed biopsies and random biopsies, respectively (p = 0.002). In patients with LSBE more than 6 cm long, methylene blue-directed biopsies led to a significant but smaller difference compared to random biopsies (90.9% versus 73.3%, respectively, p = 0.0001).

Significance of methylene blue staining

Methylene blue staining can confirm the presence of distinctive Barrett's esophagus in short segments of Barrett's esophagus. It can direct biopsies to positively stained intestinal metaplasia and therefore improve the accuracy of diagnosis. Methylene blue staining is reproducible and it can map the areas of distinctive Barrett's esophagus within columnarlined esophagus. Therefore, methylene blue staining can potentially increase our understanding of the natural history of Barrett's esophagus over time.

In conclusion, methylene blue-directed biopsies is a more accurate technique than random biopsy for diagnosing specialized columnar epithelium, particularly in > 3 cm of Barrett's esophagus.


1. Fennerty MB, Sampliner RE, McGee DL, Hixon LJ, Garewal HS. Intestinal metaplasia of the stomach identification by a selective mucosal staining technique. Gastrointest Endosc 1992;38:696-698.

2. Fennerty MB. Tissue staining. Gastrointest Endosc Clin North Am 1994;4:297-311.

3. Canto MI, Setrakian S, Petras RE, Blades E, Chak A, Sivak MV Jr. Methylene blue selectively stains intestinal metaplasia in Barrett's esophagus. Gastrointest Endosc 1996;44:1-7.

4. Canto M, Setrakian S, Willis J, Chak A, Petras RE, Powe NR, Sivak MV Jr. Methylene blue-directed biopsy for improved detection of intestinal metaplasia and dysplasia in Barrett's esophagus: a controlled sequential trial. Gastrointest Endosc 2000;51(5):560-568.

Publication date: August 2003 OESO©2015