Posted in Histopathology, Staining

Confusing Carbs!! – Carbohydrate staining simplified

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

TABLE OF CONTENTS
1. Types of carbohydrates
2. PAS( Periodic acid Schiff) staining
3. Alcian blue staining
4. Combined PAS + Alcian blue staining
5. Combined Alcian blue + High iron diamine staining
6. Summary
TABLE OF CONTENTS

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
  • Surface epithelia of the stomach

  • Brunner glands of the duodenum

  • Prostatic epithelium
  • Bronchial submucous glands

  • Goblet cells

  • Salivary glands
  • Bronchial mucus glands
    Group 3

    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.

    carbohydrate staining- PAS stained gastric foveolar cells
    Carbohydrate staining – Periodic acid-Schiff (PAS) stain -Positive gastric foveolar cells6. SUMMARY OF CARBOHYDRATE STAINING

    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

    Carbohydrate staining- Periodic acid-Schiff (PAS) stain highlights the microvillous along the apical surface of the absorptive cells.
    Carbohydrate staining- Periodic acid-Schiff (PAS) stain highlights the microvillous along the apical surface of the absorptive cells.

    Diseases in which PAS staining can be used for diagnosis

    1. Glycogen storage disease
    2. Adenocarcinoma
    3. Paget’s disease of the breast
    4. Staining macrophages in Whipple’s disease
    5. Fungal infection
    6. Alveolar soft part sarcoma
    7. Erythroleukemia
    8. α1-antitrypsin deficiency
    9. Ewing sarcoma
    10. 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 staining of barrett mucosa
    Alcian blue staining of Barrett mucosa at a pH of 2.5- Goblet cells are stained blue and gastric mucous cells are clear.

    Alcian blue stain is frequently combined with PAS and High iron diamine for diagnostic purposes (discussed below).

    Alcian blue staining of cartilage at pH 0.5
    Alcian blue staining of cartilage at pH 0.5

    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.

    Batter esophagus showing goblet cells
    Barrets esophagus showing goblet cells
    PAS + Alcian blue stain showing purple colored goblet cells and magenta colored gastric foveolar cells
    PAS + Alcian blue stain showing purple colored goblet cells and magenta colored gastric foveolar cells at pH 2.5.

    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

    Carbohydrate staining- Sulfomucins stained brown with High iron diamine and few sialomucins stained blue.
    Sulfomucins stained brown with High iron diamine and few sialomucins stained blue.

    6. SUMMARY OF CARBOHYDRATE STAINING

    1. PAS is always negative in mesenchymal and connective tissue mucopolysaccharides
    2. Alcian blue is always negative in neutral mucins
    3. 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

    Posted in Histopathology, Staining

    10 Key points- Hematoxylin and Eosin staining in Histopathology

    Key points in Hematoxylin and Eosin staining

    1. Hematoxylin is extracted from the heartwood of the tree Hematoxylin campechianum.
    2. Hematoxylin is not a stain itself. Hematin, a major oxidation product of hematoxylin is responsible for the color.
    3. Hematin is produced in two ways (a) Natural oxidation OR Ripening (b) Chemical oxidation.
    4. Ripening is done by exposing the solution to light and air. This is a slow process and takes up to 3-4 months.
    5. Ehrlich’s hematoxylin and Delafield’s hematoxylin are obtained by natural oxidation.
    6. A mordant is a substance, usually a metal which helps bind the dye with the tissue strongly.
    7. Sodium Iodate is the mordant used in Meyer’s hematoxylin and Mercuric Oxide used in Harris hematoxylin.
    8. Aluminum is the most common mordant.
    9. Carrazi’s hematoxylin has a staining time of 1 minute- shortest time for any hematoxylin stain. Hence it is used to stain frozen section slides.
    10. Iron hematoxylins are preferred connective tissue stains. Due to the acidity of dye solutions in connective tissue staining ( picric acid in Van Gieson staining), standard alum hematoxylins are decolorized. Iron hematoxylins such as Wiegert’s hematoxylin are resistant to the acidic environment and should be used in these techniques

    Examples of ALUM HEMATOXYLIN are:

    1. Erhlich hematoxylin

    2. Delafield hematoxylin

    3. Meyer hematoxylin

    4. Harris hematoxylin

    5. Cole hematoxylin

    6. Carrazi’s hematoxylin.

    Check out this post on Carbohydrate staining.

    Examples of IRON HEMATOXYLIN are

    1. Weigert’s

    2. Heidenhain’s

    3. Loyez for myelin

    4. Verhöeff’s for elastin fibers.