Carbohydrates CH 7 Flashcards
Single most abundant class of organic molecules in nature.
The metabolic precursors
of virtually all other biomolecules
Formula: (CH₂O)n
Including: Sugars, starch, cellulose…
Carbohydrates
Major saccharides of concern
Glucose
Glycogen (storage form of glucose)
Additional types of concern: Homoglycans, heteroglycans, mucins, glycolipids
1.) Type of saccharide is Glucose
2.) Type of saccharide is Glycogen (storage form of glucose)
1.) Monosaccharide
2.) Polysaccharide
Carbohydrate Pathological Conditions
1.) Name the carbohydrate metabolism enzyme deficiencies.
2.) Glycogen may have diagnostic significance in
several types of tumors including what?
1.)
Von Gierke’s disease
Pompe’s disease
2.)
Carcinoma
Mesothelioma
Rhabdomyosarcoma
Carbohydrate Pathological Conditions
◦ Mucins function as lubricants or assist in cell adhesion or host defense
◦ Can be from epithelial or connective tissue origin
What are mucins produced by?
By many tumors including
- Carcinoma
- Liposarcoma
- Mesothelioma
Enzyme deficiencies can cause abnormal systemic production of mucins
- Hurler syndrome
- Hunter disease
For histopathology concerns, there are four main groups of carbohydrates for demonstration. They are:
- Neutral Polysaccharides (Nonionic homoglycans)
- Acid Mucopolysaccharides (Anionic heteroglycans also known as glycosaminoglycans)
- Glycoproteins (Mucins, Mucoid, Mucoproteins,
mucosubstances) - Glycolipids
Neutral Polysaccharides (Nonionic homoglycans) group
- Either glucose containing
- Like glycogen, starch, cellulose
- Or N-acetyl-glucosamine containing
- Like chitin
- Group is VERY positive PAS
- Negative with other common carbohydrate stains
Acid Mucopolysaccharides (Anionic heteroglycans) group
- Either carboxylated: like hyaluronic acid
- Or chondroitin sulfated: Connective tissue compounds
- Or heparin (mast cells); sulfated and carboxylated;
- Sulfated only: Aorta
- Group is considered proteoglycans (attached to protein)
- PAS negative. Positive with other common carb stains
Glycoproteins (mucins, mucoid, mucoprotein, mucosubstances) group
- Either neutral: Stomach mucins, Paneth cell granules
- Or carboxylate, sialomucins: goblet cells, serum, blood or sulfated and carboxylated groups
- Group is mostly epithelial mucins
- Potentially but not always PAS positive. Demonstrated
with other stains or techniques
Glycolipids group
- Either cerebrosides: fatty residue bound to a carbohydrate structure
- Phosphatides: PAS positive noncarbohydrate containing lipids
- Not commonly demonstrated in clinical laboratory
Carbohydrate Special Stains
1.) PAS (Periodic acid, Schiff): PAS with Diastase
2.) Best Carmine
3.) Mayer Mucicarmine
4.) Alcian Blue, pH 2.5
- Alcian Blue, pH 1.0
- Alcian Blue with Hyaluronidase
- Alcian Blue-PAS-Hematoxylin
5.) Colloidal Iron
PAS (Periodic Acid Schiff) Reaction
- Demonstrates polysaccharides, neutral mucosubstances, basement membranes
(measure invasiveness of skin tumors) and fungus (fungal wall) - Sections cut at 4 to 5 microns (1 to 2 microns for kidney)
- Control tissue: kidney or small or large intestine (liver to demonstrate glycogen only)
- Fixative: 10% NBF or Bouin solution
PAS (Periodic Acid Schiff) Reaction
- Reaction is based on oxidation of certain tissue elements to aldehydes by periodic acid.
- Schiff’s is compound of basic fuchsin and sulfurous acid (forming a colorless compound)
- Schiffs reaction with aldehydes (attachment of dye structure), followed by washing in running water, causes the attached dye color to become visual by breaking the sulfurous group away from basic fuchsin
- Results: PAS positive for neutral mucosubstances, glycogen, basement membranes, fungal walls – BRIGHT ROSE/MAGENTA
PAS (Periodic Acid Schiff) Reaction Typical Stain Protocol
- Periodic acid solution
- Rinse in running water
- Schiff reagent
- Rinse in metabisulfite**
- Running tap water for 5 to 10 minutes
- Counterstain with hematoxylin
- Metabisulfite rinses may be used to remove excess Schiff reagent, preventing false staining of oxidized absorbed reagents (i.e. highly chlorinated water). Must follow with running water after sulfite rinses.
PAS Reaction Troubleshooting
- Glutaraldehyde can NOT be used (di-aldehyde)
- Open aldehyde stains
- PAS false positive
- Chromate containing fixatives may “over- oxidize” tissue
- Schiff reagent is typically stored between 2ºC to 12ºC.
PAS with Diastase Digestion
- Demonstration of glycogen
- 2 slides are prepared:
1.) First slide WITH enzyme diastase (or α-amylase) digests glycogen from tissue section
2.) Second slide of same tissue WITHOUT diastase treatment
- Diastase digestion shows “where glycogen was” when compared to slide WITHOUT diastase (otherwise, it was not glycogen).
- Fixative: 10% NBF, formalin alcohol, absolute alcohol
- Avoid picric acid fixatives; glycogen is more resistant to digestion
PAS with Diastase Digestion
- Typical Staining Protocol
- Section WITH Diastase, 1 hr at 37ºC
- Running water 5 minutes
- Both sections in Periodic acid
- Continue as with standard PAS protocol
Best Carmine
- Demonstration of glycogen
- Sections cut at 4 to 5 microns
- Control tissue: liver
- Results: Glycogen-Pink to Red; Nuclei-Blue
- Fixative: Absolute alcohol, Carnoy solution, Bouin solution
Best Carmine
- Typical Staining Protocol
- Harris hematoxylin
- Running water 15 minutes
- Working Carmine solution 30 minutes
- Differentiate 30 seconds
- Rinse quickly 80% alcohol
- Run Down
- Not as specific as PAS with Diastase.
- Ammonium hydroxide component toxic, corrosive
Mucicarmine
- Demonstration of epithelial mucins
- Sections cut at 4 to 5 microns
- Control tissue: Small or large intestine, appendix
- Results: Mucin-Deep Rose to Red; Nuclei- Black; Cryptococcus neoformans-Deep Rose to Red
- Fixative: 10% NBF
Mucicarmine
- Typical Staining Protocol
- Weigert hematoxylin
- Running water
- Working Mucicarmine solution (alum Carmine)
- Rinse and remove water quickly
- Mentanil yellow
- Run down
Alcian Blue pH 2.5
- Demonstrates acid mucopolysaccharies
- Section at 4 to 5 microns
- Control Tissue: small or large intestine, appendix
- Results: acid mucosubstances-DARK BLUE, background-PINK TO RED,
- Fixative: 10% NBF or Bouin solution
Alcian Blue pH 2.5
- Typical Staining Protocol
- 3% acetic acid solution
- Alcian blue solution for 15 minutes at 37ºC
- Rinse in 3% acetic acid
- Running water for 10 minutes
- Counterstain with nuclear-fast red for 5 minutes
- Copper based dye that is colored blue due to copper content
Alcian Blue pH 1.0
- Demonstrates sulfated mucosubtances
- Section at 4 to 5 microns
- Control Tissue: small or large intestine, appendix
- Results: sulfated mucosubstances-PALE
BLUE, background-PINK TO RED, - Fixative: 10% NBF or Bouin solution
