Carbs and Nucleic acids EX 2

Which sugars are lost during processing bc they are water soluble?

Monosaccharides (glucose, fructose)

Oligosaccharides (sucrose, lactose)

Carbs are __ charged.

Neutral or acidic (negatively)

NO BASIC

Mono carbs ex:

glucose, fructose

Oligo carb ex:

sucrose, lactose

Polysaccharide ex:

cellulose and glycogen

Polysaccharide groups:

1. Neutral polysaccharides/nonionic homoglycans

2. Glycosaminoglycans/acid mucopolysaccharides/anionic heteroglycans (think CT)→ carboxylated // sulfated and carboxylated

3. Glycoproteins/mucins/mucosubstances

4. Glycolipids (NT)

Neutral polysaccharides/nonionic homoglycans EX:

Glycogen (liver, skeletal, Type II muscle, hair follicles, ectocervix), starch, cellulose, chitin in fungal walls

Neutral polysaccharides/nonionic homoglycans has a Positive rxn with…

PAS

*N

Neutral polysaccharides/nonionic homoglycans have a Negative rxn with…

Colloidal iron, Alcian blue, Mucicarmine

*N

Neutral polysaccharides/nonionic homoglycans and pathology

Glycogen storage diseases, rhabdomyosarcoma, seminoma

Carboxylated Glycosaminoglycans/acid mucopolysaccharides/anionic heteroglycans are all ___ charged.

Acidic/negatively

Carboxylated Glycosaminoglycans/acid mucopolysaccharides/anionic heteroglycans is made of __ and is found…

Hyaluronic acid

ECM, ground substance of CT (submucosa, cartilage, umb cord, synovial fluid, virtuous humor)

Carboxylated Glycosaminoglycans/acid mucopolysaccharides/anionic heteroglycans differs from other GaGs bc…

it does NOT contain a protein core or sulfate group.

Sulfated Glycosaminoglycans/acid mucopolysaccharides/anionic heteroglycans…

are the most abundant.

Also, the Chondroitin sulfate and Keratan/Dermatan sulfates are found in CT.

What group is Heparin part of and where is it found?

Sulfated & carboxylated — Glycosaminoglycans/acid mucopolysaccharides/anionic heteroglycans.

Found in mast cell granules and are Metachromatic.

Chars of GaGs:

• All acidic/anionic

• Most abundant type of polysaccharide in the body

• All digestible with hyaluronidase

• Positive rxn with PAS, alcian blue, colloidal iron

• Negative rxn with mucicarmine

All GaGs have a positive rxn with…

PAS, Alcian blue, colloidal iron

*G

All GaGs have a negative rxn with

mucicarmine

GaGs pathology

Sarcomas, liposarcomas, histiocytomas, atherosclerosis, mast cell tumors

Glycoproteins/mucins/mucosubstances are also called …

Epithelial mucins

Glycoproteins/mucins/mucosubstances can be…

• Neutral

• Carboxylated (sialomucins)

• Sulfated and carboxylated (sulfomucins)

Neutral Glycoproteins/mucins/mucosubstances are found…

In gastric mucous cells, brunners glands, paneth cell granules, prostate epithelium, goblet cells (SI goblets have neutral and acidic), endocervical mucous cells.

Almost none in colon.

Carboxylated/Sialomucins Glycoproteins/mucins/mucosubstances are found…

Goblets in GI and respiratory tracts, mucous cells of salivary glands, endocervical mucous cells

They are also weakly acidic→ they have sialic acid but no sulfate

Sulfated and carboxylated/Sulfomucins Glycoproteins/mucins/mucosubstances are found…

Few SI goblets and ALL colon goblets.

Very Acidic → has sialic acid and sulfate

Glycoproteins/mucins/mucosubstances Chars:

• Different from GaGs bc of AA seq

• Range (neutral to acidic)

• Has a protein core and proteoglycans

• Resist hyaluronidase digestion

• + rxn with PAS

• Sialo and Sulfomucins only rx with mucicarmine, alcian blue, colloidal iron

Glycoproteins/mucins/mucosubstances have a positive rxn with…

PAS

*G

Sialo and sulfomucins only rx with…

mucicarmine, alcian blue, colloidal iron.

*S

Glycoproteins/mucins/mucosubstances Pathology

Carcinomas, Barret’s esophagus, adenocarcinomas and neoplastic processes

Glycolipids

Cerebrosides or phosphatides → Sphingomyelin

Positive rxn with PAS

Glycolipids pathology

MS, neurodegenerative disease, demyelination

Saccharide + lipid =

Sphingosine

PAS (Periodic Acid Schiff) Rxn purpose

Essentially all carbs demonstrated.

Basement membranes (renal disease) and brush borders

Detects mucin

Chitin and mucoid content in fungi

Also demonstrates amyloid, thyroid colloid, lipofuscin

Periodic acid oxidation

Hydroxyl groups (OH) of carbs are selectively ox (removes H) → forms 2 free aldehyde groups by periodic acid

→ Ox neutral carbs to make them negative

Schiff reagent contents:

Basic fuchsin, bisulfite/bisulfate and HCl

PAS main steps:

1. Periodic acid oxidation

2. Schiff reagent rxn

3. Aq rinse

4. Counterstain (usually hematoxylin)

Schiff rx

1. Bisulfite/bisulfate + HCl → sulfurous acid

2. Sulfurous acid + Basic fuchsin → clear solution = leucofuchsin or Schiff reagent

3. Sulfonic groups are added and rx with the aldehyde groups of carbs

Why do an aq rinse after Schiff reagent?

Removes the sulfonic group to restore the quinoid structure chromophore → attributes pink color

Acceptable fixatives for PAS

Alcohol is preferred for glycogen bc it avoids polarization but is not ideal for tissue in general

10% NBF, Bouins
Aq fixatives diffuse glycogen → artifact is called polarization

Fixatives to avoid for PAS

Glutaraldehyde: an aldehyde groups may rx with Schiff’s

Chromate fixatives (orth, helly, zenker): overoxidizes glycol group → weakens Schiffs

Results of PAS

Basement membranes, glycogen, fungal walls, neutral/acidic polysaccharides = Bright pink/magenta

Background = Counterstain (usually hematoxylin)

Testing quality of Schiff’s

Add it to 37-40% formaldehyde. Solution should immediately turn red/purple

Counterstains for demonstration of fungi for PAS:

Fast/light green

Doing a metabisulfite rinse after Schiff will…

remove unreacted leucofuchsin/Schiff’s reagent

Which type of hematoxylin is used for counterstaining Schiff’s?

Harris

Weak Schiff rxn is caused by…

• Not enough rinsing

• Used a chromate fixative

• Weak/overused Schiff reagent

• Low time in everything

• Low or over oxidation w/periodic acid

• Other ox agents used

Nonspecific Schiff rxn is caused by…

• Reagent carryover/contamination

• Deteriorated Schiff’s

• Did not rinse periodic acid or Schiff’s

• Glutaraldehyde fixative used

Overstaining with Schiff’s reagent is caused by…

Too much time in Schiffs.

If used a microwave, solution was overheated

PAS w/ and w/o digestion purpose:

Glycogen presentation

PAS w/ and w/o digestion diagnostic purpose:

Glycogen storage diseases, tumors with glycogen deposits, distinguishes glycogen from mucins and alpha-1 antitrypsin globules in hepatocytes

Diastase (beta and alpha) amylase functions to…

Catalyze hydrolysis of glycosidic bonds → depolymerize into smaller water soluble molecules (maltose and glucose) that are then rinsed away.

Basically, cleaves glycosidic bonds.

Fixatives to avoid with PAS w/ and w/o

Glutaraldehyde and Chromate.

ALSO avoid Picric acid fixatives like Bouins bc glycogen will become resistant to digestion

Slide without digestion results

Bright pink/magenta (glycogen)

Background = blue-purple

Slide with digestion results

No pink staining where glycogen was present

Background = blue-purple

Notes on PAS w/ and w/o:

• Other polysaccharides will stain positive on BOTH w/ and w/o slides.

• Liver w/large amounts of glycogen should not be used bc the rxn will be poor/weak, and depleted reagent will not be detected → False -

Disadvantages of diastase

• Alpha and Beta amylase makes tissue fall of slides

• Type II-A alpha amylase is preferred → BEST results

  • human saliva only has a-amylase

Digestion may fall because…

• Insufficient digestion time

• Overheated enzyme

• Tissue fixed w/picric acid

If digestion does not work → false negative

Best Carmine stain

Purpose: glycogen presentation

Old way of staining glycogen

Not as specific for glycogen as PAS and does not demonstrate as much glycogen.

Results: dark pink/magenta

Mucicarmine purpose

Most specific for acidic glycoproteins. Also mucoid capsule for fungus

No GaGs, no neutral sugars

Mucicarmine diagnostic purpose

Adenocarcinoma

*M

What hematoxylin cannot be used with Mucicarmine?

Gill (bc it stains mucin)

Mucicarmine solution reacts with…

acidic glycoproteins.

Mordants rxn with carmine dye → cationic dye rxn with acidic glycoproteins

Mucicarmine QC

Unautolyzed intestine (colon preferred), appendix

Mucicarmine Results

Acidic glycoproteins/fungal walls = dark pink

Nuclei = blue/black

Background = yellow/green

Weak/pale staining Mucicarmine

• If Weigert hematoxylin used, ferric ions can over ox dye lake = lost of staining

• Old/expired reagents

Nonspecific staining of Mucicarmine

• Overstained with mucicarmine

• insufficient rinsing after Mucicarmine

Mucins are obscured

• Over counterstaining

• Gill was used

Weak mucin staining in Mucicarmine

• Old/deteriorated mucicarmine solution

• Not enough time in mucicarmine

Absent mucin staining in Mucicarmine

• Autolyzed tissue

• Deteriorated mucicarmine

Alcian Blue pH 2.5 purpose

GaGs and acidic glygoproteins (sialomucins and sulfomucins)

Acidic will stain darker

Neural are not reactive

Also fungus

Alcian Blue pH 2.5 diagnostic

Adencocarcinomas

*AB2.5

Alcian Blue pH 2.5 steps

1. Acetic acid rinse - prevent water carryover

2. Alcian blue in acetic acid - copper based cationic dye; water soluble

3. Acetic acid rinse - remove excess stain and prevent nonspecific staining

4. Nuclear fast red = counterstain

Alcian Blue QC

Un autolyzed intestine, appendix

*AB

Alcian Blue pH 2.5 Results

Acidic glycoproteins = blue

GaGs = light blue

Background and nuclei = pink

Blue nuclear staining in Alcian Blue pH

• Prolonged alcian blue

• No rinse after alcian blue

Excessive background in Alcian Blue pH

• Too high pH

• Contaminated soln or too high [ ]

• Stained too long

• Section not rinsed w/acetic acid after alcian blue

Weak staining of acidic glycoproteins in Alcian Blue

• not hydrated enough

• dye soln weak or expired

• excessive acetic acid rinse

Cloudiness is caused by…

Not rinsing after using nuclear fast red

Alcian Blue pH 1.0 general purpose

Sulfated GaGs and sulfomucins

• NO sialomucins and hyaluronic acid, NO SI goblets or gastric mucins, No paneth, no mucous cells of salivary glands

Alcian Blue pH 1.0 diagnostic purpose

Colonic adenocarcinomas

Alcian Blue pH 1.0 differs from 2.5 bc…

the procedure uses HCl.

Still uses the same steps (acid rinse - AB w/acid - acid rinse - nuclear red fast counterstain)

Alcian Blue pH 1.0 results

Sulfated GaGs and Sulfomucins = pale blue

Background and nuclei = pink

Alcian Blue pH 2.5 w/Hyaluronidase purpose

Differentiate btwn acidic glycoproteins and GaGs, which are digested w/enzyme

Alcian Blue pH 2.5 w/Hyaluronidase diagnostic purpose

differentiate btwn carcinomas and sarcomas

Hyaluronidase reagent purpose

cleaves glycosidic link in anionic GaGs, digesting them

Alcian Blue pH 2.5 w/Hyaluronidase QC slides

2 slides of umb cord (w/ and w/o)

Intestine or appendix can be used as a 2nd control to demonstrate acidic glycoproteinsAlcian Blue pH 2.5 w/Hyaluronidase

Alcian Blue pH 2.5 w/Hyaluronidase results

Without digestion (GaGs and glycoproteins) = blue

W/ digestion = these will be absent and there is a loss of staining

Background = pink

Alcian Blue-PAS general purpose

differentiate btwn neutral and acidic glycoproteins

Alcian Blue-PAS diagnostic purpose

Barrett’s esophagus

Alcian Blue-PAS Steps

1. Acetic acid rinse

2. Alcian blue with acid

3. Periodic acid so OH on neutral mucins is ox

4. Schiff reagent to rx with aldehyde groups

5. Water rinse to restore chromophore

6. Optional Harris hematoxylin

Alcian Blue-PAS QC slide

Duodenum or cervix (must be endo and ecto)

• Bc they have neutral and acidic

Alcian Blue-PAS Results

GaGs and acidic glycoproteins = blue

Neutral mucosubstances and glycogen = dark pink/magenta

Mix of both = purple

Nuclei = blue/purple

Colloidal Iron (Muller-Mowry) purpose

Show acidic glycoproteins and GaGs

Also fungus.

***Not as specific as AB, but more sensitive for ALL acidic proteoglycans and will stain a wider variety. → Leads to more nonspecific staining.

Colloidal Iron diagnostic purpose

carcinomas, sarcomas (not differentiation), renal carcinomas

How Colloidal Iron works…

1. In an acidic environment, ferric ions are attracted to acidic carboxylated and sulfated mucosubstances.

2. Acetic acid rinse before and after colloidal iron prevents carryover

3. Ferric ions visualized by treatment with HCl K ferrocyanide → Forms Prussian blue pigment (ferric ferrocyanide).

4. Nuclear fast red (rinse after) or Van Gieson (no rinse after)

There is no “dye,’“ only iron is used

Fixatives to avoid for Colloidal Iron and High Iron Diamine

Chromate and metal fixatives bc they compete with ferric irons → masks mucosubstances

Colloidal Iron Results (normal, no PAS)

GaGs and acidic glycoproteins = blue

NFR = pink

Van Gieson
→ acid fuchsin makes collagen red
→ picric acid stains muscle/cytoplasm yellow

Colloidal Iron results if PAS is used as a counterstain

Neutral mucosubstances = bright pink/magenta

Acidic mucosubstances and proteoglycans (and fungus) = blue

Neutral and acidic mucosubstances = purple

Background or nonspecific staining in Colloidal Iron happens if…

• pH is greater than 2 (soln must be acidic)

• Old solution is used

High Iron Diamine purpose

Demonstrating sulfated proteoglycans (sulfomucins)

When combined with alcian blue, stain will differentiate btwn Sulfo and sialo mucins

High Iron Diamine diagnostic purpose

Differences in acidomucin subtypes in diseases like colorectal cancer and inflammatory bowel disease