Hematology & Histology

Alcian Blue


Alcian Blue - H&E - Metanil Yellow Stain for Diagnosing Barrett's Esophagus


Wendi Edgett, BS, HTL (ASCP)
Genesys Regional Medical Center, Health Park Campus
Grand Blanc, MI 48439
This article has been reprinted with permission from HistoLogic, vol 37 no. 2, 2004


On this page:
Introduction
Discussion
Materials and Method
Results
Conclusion
References


Introduction:
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.

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Discussion:
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 (Figur.e 1). 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. (Figure 2) 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.

Case Studies:
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.

1.  A 77-year-old Caucasian male who suffers from chronic reflux and has a history of Barrett’s esophagus. He also has a small hiatal hernia. The patient was scheduled for a follow-up endoscopic biopsy to determine if dysplasia was present. The biopsy was obtained from the long segment of Barrett’s esophagus, 28-40 cm from the incisors. The H&E revealed glandular mucosa with infiltration of plasma cells and lymphocytes and the biopsy was negative for dysplasia. Fragments showed ulceration and erosion with the presence of granulation tissue. The presence of specialized intestinal metaplasia was demonstrated by the AB – H&E – MY. (Figure 3a & 3b) The patient was diagnosed with ulceration with erosion and granulation tissue and chronically inflamed, non-dysplastic Barrett’s esophagus. The patient is recommended to undergo a repeat biopsy in two years.

2.  A 56-year-old Caucasian male with a history of Barrett’s esophagus. His last endoscopic evaluation was two years previous indicating Barrett’s esophagus and showing no dysplasia. The patient was scheduled for a follow-up endoscopic biopsy to determine if dysplasia was present. The biopsy was obtained from the long segment of Barrett’s esophagus, 25-40 cm from the incisors. The H&E revealed chronically inflamed glandular tissue, with numerous plasma cells and lymphocytes in the lamina propria and the biopsy was negative for dysplasia. The AB – H&E – MY demonstrated a large number of goblet cells present. (Figure 4a & 4b) The patient was diagnosed with chronic active inflammation of non-dysplastic Barrett’s esophagus. The patient is recommended to undergo a repeat biopsy in two years.

3.  A 65-year-old Caucasian female came to the emergency room presenting symptoms of diarrhea, bloody stool and tenderness in the lower abdominal area; the symptoms have been present for ten days and worsen after she eats. She had a history of dysphagia (difficulty swallowing), esophagitis (inflammation of the esophagus) and gastroesophageal reflux disease (GERD). The patient was scheduled for an endoscopic biopsy to rule out Barrett’s esophagus, and the biopsy was taken from the region of the GE junction. The H&E revealed esophageal mucosa of squamous epithelium that was negative for dysplasia. The gastric mucosa has a large amount of lymphatic infiltrate in the lamina propria. The AB – H&E – MY confirms the presence of focalized intestinal metaplasia. (Figure 5a & 5b) The patient was diagnosed with chronically inflamed, non-dysplastic Barrett’s esophagus.

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Materials and Method
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.

Solutions:
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
   Thymol crystals
   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.


Method:  
1.   Deparaffinize and bring sections to water  
2.   Stain with Alcian Blue, pH 2.5 solution 15 minutes
3.   Wash well with water  
4.   Stain in Mayer Hematoxylin * 4 minutes
5.  Rinse in running water, several changes  
6.   Differentiate in 0.25% hydrochloric acid 2-3 seconds
7.   Rinse in running water, several changes  
8.   Blue in 0.25% ammonia water 2-3 seconds
9.   Rinse well in running water, several changes  
10.   Place in 70% ethanol 1 minute
11.  Stain with Eosin Solution 1 minute
12.  Dehydrate in 95% ethanol 30-60 seconds
13.  Dehydrate in 100% ethanol, two changes 30 seconds each
14.  Place in Metanil Yellow solution** 1 minute
15.  Rinse with ethanol, 2 changes 10 dips each
16.  Clear with xylene, 3 changes 2 minutes each
17.  Mount in a resinous medium  

*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.

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Results:
Nuclei - blue
Cytoplasm - pink-red
Mucin – Turquoise for Barrett’s Esophagus Goblet Cells (some gastric mucin will stain a faint blue)
Collagen - yellow
Smooth muscle - salmon

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Conclusion:
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.

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References:
Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 4th edition, New York, NY, Churchill Livingston, 1996.

Blount PL, Cowan DS, Reid BJ, Fred Hutchinson Cancer Research Center and the University of Washington: Barrettsinfo.com, An in depth information source about Barrett’s esophagus, http://barrettsinfo.com/index.htm, 2003.

Burkitt HG, Young B, Heath JW: Wheater’s Functional Histology: A Text and Colour Atlas, Churchill Livingston, 1993.

Genesys Regional Medical Center Procedure Manual, Alcian Blue/H&E/Metanil Yellow Stain, Grand Blanc, Michigan, 2000.

Johns Hopkins University: Johns Hopkins Pathology, Barrett’s Esophagus: Understanding Barrett’s, http://pathology2.jhu.edu/beweb/menu_understanding.cfm, 2003.

Levine DS: Barrett's Esophagus. Scientific American Science & Medicine 1:16-25, 1994 (Nov-Dec).

Reid BJ, Blount PL, Rubin CE, Levine DS, Haggitt RC, Rabinovitch PS: Flow cytometric and histologic progression to malignancy in Barrett's esophagus: Prospective endoscopic surveillance of a cohort. Gastroenterology 102: 1212-1219, 1992.

Staples, T. C., 1996. Alcian blue-H&E-saffron stain for gastrointestinal biopsies, Histo-Logic, vol. 26, No1, March, 1996.

University of Washington Medical Center Procedure Manual, Alcian blue-H&E-saffron stain, GI Biopsy Laboratory University of Washington Medical Center, Seattle, Washington, 2003.

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