Carbohydrate staining is confusing isn’t it? Lets try and simplify it!
(Staining procedures and methods will not be discussed, only the interpretation)
PAS and Alcian blue staining will be discussed
1.TYPES OF CARBOHYDRATES
Group 1: Neural Polysaccharides or non-ionic homoglycans: This group has glucose containing glycogen, starch and cellulose as well as N-acetyl glucosamine containing chitin.
Group 2: Acid mucopolysaccharides or anionic heteroglycans: Hyaluronic acid and Chondroitin sulfate belong to this group.
Group 3: Neutral mucins, Carboxylated mucins (sialomucins) as well as sulfated mucins (sulfomucins) are present in this category.
| NEUTRAL MUCINS | CARBOXYLATED MUCINS | SULFATED MUCINS |
| | | Bronchial mucus glands |
Remember: Carboxylated mucins or sialomucins (also called simple mucins) are weak acids whereas sulfated mucins (also called complex mucins) are strong acids
Group 4: Glycolipids and Phosphatides belong to this group.
WHICH OF THESE GROUPS IS PAS (PERIODIC ACID SCHIFF) POSITIVE?
Group 1 is always PAS positive
Group 2 is always PAS negative.
Group 3 and 4 is also PAS positive.
Remember: Mesenchymal polysaccharides such as Hyaluronic acid and Connective tissues polysaccharides such as Chondroitin sulfate are always negative for PAS.
PAS Positive cell and tissue components
1.Glycogen
2.Starch
3.Mucins (Neutral mucins>sialomucins>sulfomucins)
4. Basement membranes
5. α-antitrypsin
6.Reticulin
7.Fungi (capsules)
8.Pancreatic zymogen granules
9.Thyroid colloid
10.Corpora amylacea
11. Russell bodies
Diseases in which PAS staining can be used for diagnosis
- Glycogen storage disease
- Adenocarcinoma
- Paget’s disease of the breast
- Staining macrophages in Whipple’s disease
- Fungal infection
- Alveolar soft part sarcoma
- Erythroleukemia
- α1-antitrypsin deficiency
- Ewing sarcoma
- Pulmonary alveolar proteinosis
3. LET’S MOVE ON TO ALCIAN BLUE STAINING
Group 1 is always Alcian blue negative
Group 2 is always Alcian blue positive. Hyaluronic acid is positive at a pH of 2.5 and Chondroitin sulfate is positive at a pH of 0.5.
Group 3 is the confusing part, we need to divide it into three parts for better understanding
1.Neutral mucins– Always alcian blue negative.
2. Sialomucins or carboxylated mucins– Alcian blue positive at a pH of 2.5
3. Sulfated mucins– Alcian blue positive at a pH of 1.
Group 4 is Alcian blue negative.
Alcian blue stain is frequently combined with PAS and High iron diamine for diagnostic purposes (discussed below).
4. COMBINED PAS AND ALCIAN BLUE STAINING
Primary use of PAS+Alcian blue combined staining is to distinguish neutral and acidic especially sialomucns.
Diagnostic utility: 1. To distinguish eccentric gastro-esophageal junction from Barret’s mucosa
2. Gastric intestinal metaplasia
Remember: Key finding an diagnostic feature intestinal metaplasia is the presence of goblet cells. Goblet cells and surface epithelial cells of stomach may be difficult to distinguish in routine Hematoxylin and Eosin stained sections.
Goblet cells are positive with both PAS and Alcian blue but gastric epithelial cells are positive with PAS alone. When combined PAS+Alcian blue staining is performed, Goblet cells stain PURPLE and Gastric foveolar cells stain MAGENTA.
5. COMBINED ALCIAN BLUE AND HIGH IRON DIAMINE
Combined PAS + High iron diamine is used to distinguish between sulfomucins and sialomucins.
Diagnostic utility: Large bowel metaplasia can be identified, since it contains both sulfomucins and sialomucins, in contrast to small bowel metaplasia (sialomucins only)
With Alcian blue + High iron diamine staining- Sulfomucins: BROWN COLOR and Sialomucins: BLUE COLOR
6. SUMMARY OF CARBOHYDRATE STAINING
- PAS is always negative in mesenchymal and connective tissue mucopolysaccharides
- Alcian blue is always negative in neutral mucins
- PAS+Alcian blue is key to distinguish ectopic gastric mucosa or eccentric gastroesophageal junction from Barret’s mucosa in the esophagus.
Comment your queries and suggestions!! Thank you!
Check this link for Key Points on Hematoxylin and eosin staining
