Barrett's esophagus is a condition that can lead to adenocarcinoma. Proper diagnosis and follow up treatment is very important for patients diagnosed with Barrett’s esophagus. It can sometimes be difficult to diagnose. The Alcian Blue – H&E – Metanil Yellow (AB-H&E-MY) stain is very effective in distinguishing between Barrett’s and other gastrointestinal disorders. The stain is simple to perform and the results can easily be duplicated from one staining batch to another. The AB - H&E - MY stain is a very comprehensive stain that can be used for the diagnosis of Barrett’s esophagus.
The GI tract is composed of four distinct layers, the mucosa, submucosa, muscularis propia, and adventia. The epithelium of the GI tract varies distinctly as you transition from one area of the tract to the next, from the esophagus through the stomach and the intestine. The epithelium is very specific to the properties and purpose of each area of the tract. The esophagus is composed of stratified squamous epithelium that provides protection from the contact with the food we swallow. The point in which the esophagus and the stomach meet is called the gastro-esophageal (GE) junction and there is a distinct change from the stratified squamous epithelium of the esophagus to the absorptive tubular epithelium of the stomach. The epithelium of the stomach contains mucus-secreting glands that contain neutral mucins. The intestine is comprised of tubular epithelium that contains mucin-secreting goblet cells.
Barrett's esophagus is a condition of the esophagus that occurs when the normal squamous epithelium of the esophagus is replaced by intestinal columnar epithelium. This is arises from the chronic irritation caused by gastroesophageal reflux disease (GERD). Acid from the stomach causes the cells of the esophagus to be damaged. When they are regenerated, they are replaced with the columnar epithelium similar to that found in the intestine. This is called metaplasia. An individual with the presence of intestinal metaplasia will be monitored with repeat biopsies approximately every two years if no dysplasia is found and every six months if dysplasia is present.
The basis for this stain is to help save time and money by combining multiple stains to demonstrate many structures on an H&E, rather than ordering additional special stains after the initial H&E. Endoscopic biopsies comprise a large percentage of the specimens we process in the lab, and among these biopsies, a large number are esophageal biopsies. These biopsies are generally very small fragments of tissue that routinely are cut with two or three levels per block. By picking up a section from each level for the special stain at the same time that the sections for the H&E are cut, it allows the pathologist the opportunity to evaluate both slides at the same time.
The H&E is the most used stain in histology and is used routinely in all histology labs. Hematoxylin and eosin stain a vast array of tissue structures, providing a simple and comprehensive starting point for the pathologist to make a diagnosis. The hematoxylin is used to stain the nuclear chromatin giving the results of blue nuclei. The eosin is used to stain the connective tissue in usually three shades of pink, the red blood cells staining the darkest, the muscle slightly lighter and the collagen the lightest shade of pink.
The Alcian Blue stain is primarily used to stain for acid mucopolysaccharides. The alcian blue stain at a pH of 2.5 will stain for both sulfated (sulphomucins) and carboxylated (sialomucins) mucopolysaccharides which are found in the goblet cells located in the intestine. The alcian blue dye is a positively charged, very large, molecule that will bind to the negatively charged low density mucin found in the goblet cells staining it blue in color. The alcian blue can be modified to distinguish between the acid mucins by modifying the pH of the staining solution. For the AB – H&E – MY stain the alcian blue pH 2.5 is used. the gastric mucosa of the stomach is comprised of mucus-secreting cells. These cells contain neutral mucopolysaccharides and typically do not stain with alcian blue, however in some circumstances, there can be some variable weakly positive staining seen with alcian blue, yielding a very pale blue.
Metanil yellow is a counterstain that stains collagen yellow. Some procedures call for the use of saffron, for economical purposes Metanil yellow has been substituted for the saffron. When the metanil yellow is combined with the alcian blue, the mucin in Barrett’s esophagus stains a brilliant turquoise color.
The AB – H&E – MY stain can be very useful in making the cells of Barrett’s esophagus more visible. In some situations, it can become difficult to determine if the glands present in the biopsy are gastric mucosa or intestinal metaplasia as seen in Barrett’s esophagus.
The following are three case studies that represent the clinical significance of the stain. In all three cases the pathologist was presented with an H&E at three levels and an AB – H&E – MY of consecutive sections of the same three levels.
Tissue Preparation and Sectioning:
Hollandes fixative is the preferred fixative for gastrointestinal biopsies. Hollandes is a picric acid based fixative that is very effective in preserving the mucin in the tissue. However, since mucin does not break down as quickly as other carbohydrates, neutral buffered formalin can be used. Routinely processed paraffin sections are cut at 4 mm, and sections for the routine H&E and the AB-H&E-MY are cut at the same time.
3% Acetic Acid
Acetic acid 3.0 ml
Distilled water 97.0 ml
Stir together. Stable at room temperature for months.
Alcian Blue, pH 2.5>
Alcian blue 1.0 g
3% acetic acid 100.0 ml
Dissolve alcian blue in acetic acid. Check pH; adjust the pH as needed using acetic acid to pH 2.5. Add a few crystals of thymol to prevent mold growth. Solution is stable at room temperature for months and may be reused until weak.
Mayer Hematoxylin: Commercially made
0.25% Hydrochloric Acid
Hydrochloric acid 2.5 ml
Distilled water 997.5 ml
Carefully add hydrochloric acid to the distilled water slowly. Stable at room temperature for months.
0.25% Ammonia Water
Ammonium hydroxide 1.0 ml
Distilled water 250.0 ml
Slowly add ammonium hydroxide to distilled water. Use for one day only.
Eosin: Commercially made>
0.25% Metanil Yellow
Metanil yellow 0.25 g
Distilled water 100.0 ml
Glacial acetic acid 0.25 ml
Mix together well. Stable at room temperature for up to one year.
*For automated stainers, run a program on the stainer that takes the slides from water, through your routine H&E and stops at the second change of absolute ethanol.
** Timing of the Metanil Yellow is critical. If stained for too long, increased background staining will occur.
When the three stains used in the AB – H&E –MY are combined, the stains yield dark blue nuclei, pinkish-red cytoplasm, turquoise mucin in Barrett's esophagus vs. non staining/pale blue mucin of herniated tissue, yellow collagen and salmon smooth muscle. This stain gives the pathologist a colorful demonstration of the various components of the gastrointestinal tract with which to make a diagnosis. It is very useful in diagnosing Barrett's esophagus and distinguishing it from other GI conditions.