Chapter 7 - Carbohydrate & amyloid

What are carbohydrates and what do they include?

Carbohydrates (hydrated carbons) are organic compounds that include sugars, starch, cellulose, and polymers mostly linked to proteins.

How are carbohydrates chemically defined?

As ketone or aldehyde derivatives of polyhydroxy alcohols.

What are the three major classifications of carbohydrates?

1. Monosaccharides (1 sugar unit)

2. Oligosaccharides (few, 2–10 sugar units)

3. Polysaccharides (many sugar units)

What is the only monosaccharide found in the body in demonstrable quantity?

Glucose

Why is glucose not demonstrable in tissue sections?

Because it is extremely soluble in aqueous solutions and of small molecular size.

What is the general nature of carbohydrates found in tissue sections?

Most are conjugated to proteins or lipids, making histochemical analysis complex due to inconsistent terminology and classifications.

What are examples of glucose-containing neutral polysaccharides?

Glycogen, starch, and cellulose.

What is an example of a neutral polysaccharide containing N-acetyl-glucosamine?

Chitin

What is glycogen, and where is it stored in the human body?

A polymer of glucose used to store carbohydrates, primarily in the liver and skeletal muscles.

Why is prompt fixation important for glycogen demonstration?

Glycogen undergoes enzymatic breakdown after death, which can compromise its detection.

Is glycogen soluble or insoluble in aqueous solutions?

Relatively insoluble, allowing it to be demonstrated in tissue sections.

Which carbohydrate is the second most abundant in nature?

Starch

Why can humans not digest cellulose?

Humans lack the enzymes required for cellulose digestion.

Which histochemical stain does this group react positively and negatively with?

• Positive: Periodic Acid-Schiff (PAS)

• Negative: Alcian blue, colloid iron, mucicarmine

What are the three subtypes of acid mucopolysaccharides based on chemical groups?

1. Carboxylated (COOH): e.g., hyaluronic acid

2. Sulfated & Carboxylated (OSO₃H + COOH): chondroitin sulfates A, B (dermatan sulfate), C; heparin

3. Sulfated only (COOH-free): found in human aorta & bovine cornea

Where is hyaluronic acid found?

Connective tissue and umbilical cord.

Where are the following found?

• Chondroitin sulfate A & C:

• Chondroitin sulfate B (dermatan sulfate):

• Heparin:

• Chondroitin sulfate A & C: Cartilage, cornea, blood vessels, chondrosarcomas

• Chondroitin sulfate B (dermatan sulfate): Skin, connective tissue, aorta, lung

• Heparin: Mast cells, intima of arteries

What is the general charge and composition of Group II polysaccharides?

Acidic (anionic); believed to be protein-bound despite the absence of “protein” in their names.

What histochemical stains are these mucins positive and negative with?

• Positive: Alcian blue, colloid iron, mucicarmine

• Negative: PAS

What are the three main chemical subtypes of glycoproteins?

1. Neutral

2. Carboxylated (sialoglycoproteins without sulfate)

3. Sulfated & Carboxylated (sialoglycoproteins with sulfate)

What are examples of neutral glycoproteins?

Ovimucoid (egg white), stomach mucin, Paneth cell granules.

What are examples of carboxylated (non-sulfated) sialoglycoproteins?

• Submaxillary gland mucin

• Small intestine mucins

• Fetal mucins

• Colonic crypt (upper part)

• Human sublingual gland

• Serum glycoproteins

• Blood group substances

Where are sulfated + carboxylated sialoglycoproteins found?

Colonic mucins of sheep and humans.

Where do glycoproteins primarily occur?

Mostly in epithelial mucins, though some are found in connective tissue.

Are these glycoproteins PAS-positive?

They are potentially, but not necessarily PAS-positive.

What are the two main types of glycolipids and their characteristics?

1. Cerebrosides: Fatty residues bound to carbohydrate structures

2. Phosphatides: PAS-positive lipids that do not contain carbohydrates, such as lecithin, cephalin, and sphingomyelin

What does the term “acid mucosubstances” encompass?

Both acid mucopolysaccharides and acidic glycoproteins.

Do polysaccharides occur in pure or mixed forms in tissue?

They typically occur as mixtures.

Is it possible to histochemically differentiate long-chain vs. short-chain carbohydrate polymers based on core protein size?

No, but histologic localization may offer clues.

Which functional groups are primarily detected in histochemical staining of carbohydrates?

1. 1,2-glycol

2. Carboxyl (COOH)

3. Ester sulfate (OSO₃H)

What enzymatic digestion procedures can aid carbohydrate identification?

• Diastase

• Hyaluronidase

• Sialidase

What are blocking procedures used for?

To add specificity and aid in identification, although they are less commonly used in routine histopathology.

What is the purpose of the Periodic Acid–Schiff (PAS) reaction in histology?

To demonstrate polysaccharides, neutral mucosubstances, and basement membranes in tissue sections.

What is the fundamental chemical principle behind the PAS reaction?

The oxidation of certain tissue elements to aldehydes by periodic acid.

Which functional group is most commonly oxidized by periodic acid in PAS staining?

The 1,2-glycol group, though other groups may also be oxidized.

What is Schiff reagent made from?

Schiff reagent is made by treating basic fuchsin (pararosaniline) with sulfurous acid.

What happens chemically to basic fuchsin when treated with sulfurous acid?

The quinoid structure is reduced, and chromophores are masked, forming a colorless compound known as leucofuchsin.

How is the color restored after the Schiff reagent reacts with aldehydes?

Washing in running water removes the bound sulfurous acid group, allowing restoration of the quinoid structure, which produces the typical Schiff color.

What is the role of metabisulfite rinses in PAS staining?

1. To remove excess Schiff reagent

2. To prevent false colorization due to oxidation of any adsorbed reagent

What fixatives are recommended for the PAS reaction?

• 10% Neutral Buffered Formalin (NBF)

• Bouin solution

• For blood smears: fix in methyl alcohol for 10–15 minutes

At what thickness should paraffin sections be cut for PAS staining?

4–5 µm

At what thickness should kidney sections be cut for PAS staining?

1–2 µm

What is the most sensitive control tissue for the PAS reaction?

Kidney

What control tissue is used when the procedure is intended to demonstrate glycogen?

• Liver containing glycogen

• Cervix, including both endocervix and ectocervix

What are the reagents for PAS and how do you make them?

Periodic acid, 0.5% solution

• Periodic acid — 2.5 g

• Distilled water — 500 mL

1N hydrochloric acid

• Hydrochloric acid, concentrated (specific gravity, 1.19) — 83.5 mL

• Distilled water — 916.5 mL

• Add the acid to the water and mix well

Potassium metabisulfite, 0.55%

• Potassium metabisulfite — 2.75 g

• Distilled water — 500 mL

Schiff reagent

• Distilled water — 800 mL

• Basic fuchsin — 4 g

• Sodium metabisulfite — 4 g

• 1N hydrochloric acid — 80 mL

How do you test the quality of Schiff reagent?

• Place 10 mL of 37–40% formaldehyde in a beaker or flask.

• Add a few drops of Schiff reagent.

• Observe the reaction.

What indicates good quality Schiff reagent?

A rapid color change to reddish-purple.

What indicates Schiff reagent is deteriorating?

A delayed reaction and a deep-blue purple color.

What tissue elements stain positive (bright rose) with the PAS reaction?

• Glycogen

• Neutral mucosubstances

• Certain epithelial sulfomucins and sialomucins

• Colloid material of the thyroid and pars intermedia of the pituitary

• Basement membranes

• Fungal walls

What is the purpose of the PAS reaction with diastase digestion?

To demonstrate glycogen in tissue sections.

How does the PAS-diastase method work?

It is a sensitive method for glycogen detection. Diastase or α-amylase digests glycogen into smaller sugar units (maltose and glucose), which are then washed out of the section.

What happens after glycogen is removed from the tissue by digestion?

The PAS stain is applied, and absence of staining indicates glycogen removal, confirming its previous presence.

What enzyme activity is responsible for glycogen removal in PAS-diastase method?

Diastase and alpha-amylase enzymatic activity.

What fixatives are suitable for PAS with diastase digestion?

• 10% neutral-buffered formalin (NBF)

• Formalin alcohol

• Absolute alcohol

How should tissue sections be prepared for PAS with diastase digestion?

• Cut 2 paraffin sections at 4–5 µm thickness

• Label one section "with" (diastase treated) and the other "without" (untreated)

What tissues are recommended for use as control in PAS-diastase procedures?

• Two liver sections containing glycogen (one labeled "with", one "without")

• Cervix, including both endocervix and ectocervix, is also an excellent control

How is the quality of the Schiff reagent tested in this procedure?

• Mix formaldehyde with Schiff reagent

• A rapid reddish-purple color indicates good quality

• A delayed deep-blue purple color suggests reagent degradation

What are the reagents for PASD and how do you make them?

Periodic acid, 0.5% solution

• Periodic acid — 2.5 g

• Distilled water — 500 mL

1N hydrochloric acid

• Hydrochloric acid, concentrated (specific gravity, 1.19) — 83.5 mL

• Distilled water — 916.5 mL

• Add the acid to the water and mix well

Schiff reagent

• Distilled water — 800 mL

• Basic fuchsin — 4 g

• Sodium metabisulfite — 4 g

• 1N hydrochloric acid — 80 mL

Add the 80 mL of 1N HCl, cool completely, and add 4 g of sodium metabisulfite. Let the solution stand in the dark overnight; it should turn amber. Add 2 g of activated charcoal and shake for 1 minute. Filter and store in the refrigerator. Should be stable for 2-4 months.

Potassium metabisulfite, 0.55%

• Potassium metabisulfite — 2.75 g

• Distilled water — 500 mL

Malt diastase solution

• Diastase of malt — 0.1 g

• Phosphate buffer, pH 6 — 100 mL

Phosphate buffer, pH 6

• Sodium chloride — 8 g

• Sodium phosphate, monobasic — 1.97 g

• Anhydrous disodium phosphate (dibasic) — 0.28 g

• Distilled water — 1000 mL

• Adjust pH to 6 if necessary; store in refrigerator

How do results appear in PAS with diastase digestion?

• Section labeled "without" (no digestion): Glycogen stains bright rose red

• Section labeled "with" (digested): Glycogen is absent

What does the absence of staining in the "with" section confirm?

That the pink PAS-positive material seen in the untreated section was glycogen, digested and removed by diastase.

What is the primary purpose of the Mayer mucicarmine stain?

To stain epithelial mucin in tissue sections.

What type of mucin is Mayer mucicarmine particularly useful for identifying?

Epithelial mucin, especially in the context of adenocarcinomas.

Which types of mucins does Mayer mucicarmine stain?

Carboxylated and sulfonated mucins, but not neutral mucins.

What chemical interaction underlies Mayer mucicarmine staining?

• Aluminum forms a chelation complex with carmine

• This compound gains a net positive charge, allowing it to bind to acidic groups on mucins

What is the recommended fixative for Mayer mucicarmine staining?

10% Neutral Buffered Formalin (NBF)

What thickness should paraffin sections be cut for Mayer mucicarmine staining?

4–5 µm

What tissue types are recommended as quality control for Mayer mucicarmine staining?

• Unautolyzed colon

• Small intestine

• Appendix

How does mucin appear in Mayer mucicarmine staining?

Deep rose to red

How does the capsule of Cryptococcus spp. appear in Mayer mucicarmine staining?

Deep rose to red (same as mucin)

How do nuclei stain in Mayer mucicarmine staining?

Black

How do other tissue elements appear in Mayer mucicarmine staining?

Blue to yellow

What is the purpose of the Alcian Blue, pH 2.5 stain?

To demonstrate acid mucopolysaccharides in tissue sections.

What type of dye is Alcian Blue and what gives it its blue color?

Alcian Blue is a copper phthalocyanin basic dye, and its copper content gives it a blue color.

What solvent and pH are used with Alcian Blue for this staining technique?

A 3% acetic acid solution at pH 2.5.

What types of substances does Alcian Blue stain at pH 2.5?

• Sulfated and carboxylated acid mucopolysaccharides

• Sulfated and carboxylated sialomucins (glycoproteins)

What is the proposed mechanism by which Alcian Blue binds to tissue components?

It forms salt linkages with the acid groups of acid mucopolysaccharides.

What fixatives are appropriate for Alcian Blue, pH 2.5 staining?

• 10% neutral-buffered formalin (NBF)

• Bouin solution

What thickness should paraffin sections be cut for Alcian Blue staining?

4–5 µm

What are appropriate positive control tissues for Alcian Blue, pH 2.5 staining?

• Unautolyzed small intestine

• Appendix

• Colon

How do weakly acid sulfated mucosubstances, hyaluronic acid, and sialomucins appear on Alcian blue, pH 2.5?

Dark Blue

How does the background typically appear on Alcian blue, pH 2.5?

Pink to red

How are nuclei stained in Alcian Blue, pH 2.5?

Red

What are the Alcian Blue, pH 2.5, reagents and how do you make them?

Acetic Acid, 3% solution

• Glacial acetic acid — 15 mL

• Distilled water — 485 mL

Alcian Blue, 1 % solution

• Alcian Blue-8GX — 5 g

• Acetic acid, 3% solution — 500 mL

• Adjust pH to 2.5; filter and add few crystals of thymol

Nuclear-fast red (Kernechtrot) solution

• Nuclear-fast red — 0.5 g

• Aluminum sulfate — 25 g

• Distilled water

What is the purpose of Alcian Blue, pH 1.0 staining?

To demonstrate sulfated mucosubstances in tissue sections.

What solution and pH are used in Alcian Blue, pH 1.0 staining?

A 0.1N Hydrochloric acid solution at pH 1.0.

What substances are stained by Alcian Blue at pH 1.0?

• Sulfated acid mucopolysaccharides

• Sulfated sialomucins (glycoproteins)

Why aren’t carboxylated mucosubstances stained at pH 1.0?

Because they cannot ionize at this low pH, and thus cannot bind the dye.

What fixatives are appropriate for Alcian Blue, pH 1.0 staining?

• 10% Neutral Buffered Formalin (NBF)

• Bouin solution

What thickness should paraffin sections be cut for Alcian Blue, pH 1.0 staining?

4–5 µm

What are suitable control tissues for Alcian Blue, pH 1.0?

• Unautolyzed small intestine

• Appendix

• Colon

How do sulfated mucosubstances appear in Alcian Blue, pH 1.0 staining?

Pale blue

How does the background typically appear?

Pink to red

What color are the nuclei stained?

Red

What are the reagents for Alcian Blue, pH 1.0 and how do you make them?

0.1N Hydrochloric Acid solution

• Hydrochloric acid, concentrated — 8.2 mL

• Distilled water — 991.8 mL

1% Alcian Blue solution, pH 1.0

• Alcian Blue 8GX — 3 g

• Hydrochloric acid, 0.1N — 300 mL

Nuclear-fast red solution

• Nuclear-fast red — 0.5 g

• Aluminum sulfate — 25 g

• Distilled water — 500 mL

What is the purpose of the Alcian Blue with hyaluronidase digestion technique?

To differentiate epithelial mucins (glycoproteins) from connective tissue mucins (acid mucopolysaccharides).

What happens to Alcian blue staining after digestion with hyaluronidase?

• Staining disappears or is markedly reduced in tissues containing hyaluronic acid, chondroitin sulfate A, or chondroitin sulfate C (connective tissue mucins).

• Epithelial mucins (glycoproteins) are not affected.

What enzyme is used in this digestion method and what is its origin?

Testicular hyaluronidase

What types of mucins are affected by hyaluronidase digestion?

• Hyaluronic acid

• Chondroitin sulfate A

• Chondroitin sulfate C

Which types of mucins are unaffected by hyaluronidase digestion?

Glycoproteins (epithelial mucins)

What fixative is used for Alcian Blue with hyaluronidase digestion?

10% Neutral Buffered Formalin (NBF)