Histology - Module 2, Unit 2

What is the chemical formula for formaldehyde?

HCHO.

What is the chemical formula for water?

H2O.

What is the chemical formula for methylene glycol?

HO-CH2-OH.

What type of fixative is formaldehyde?

Additive and non-coagulant.

How is formalin made?

By pumping formaldehyde gas into water until saturated.

What is the concentration of commercial formalin when purchased?

37% w/w or 40% w/v.

What is the formula to convert % w/w to % w/v?

% w/v = % w/w x density (SG).

What is the calculation to convert 37% w/w formalin to % w/v?

37 g x 1.08 g/mL = 0.3996 g/mL = 39.96 g/100 mL = 40% w/v.

What is the physical state of pure formaldehyde?

Colorless gas.

What is the physical state of formalin?

Colorless liquid.

What hazard label is required for formaldehyde?

DANGER: Irritant and potential cancer hazard, authorized personnel only.

What is formaldehyde formed from chemically?

Oxidation of methyl alcohol, becoming an aldehyde

An aldehyde is the oxidation product of what?

A primary alcohol.

What kind of chemical agent is formaldehyde?

Very strong reducing agent.

What are the main safety hazards of formaldehyde?

Strong irritant to eyes and respiratory epithelium, possible carcinogen.

What safety precautions are required for formaldehyde?

Use fume hoods, eyewear, and monitor exposure.

What device is used to monitor formaldehyde exposure?

Formaldehyde Multimode Monitor.

What type of reaction does the formaldehyde monitor use?

Colorimetric reaction to exposure.

What is the concentration of commercial formaldehyde?

40% w/v (100% formalin).

What is the concentration of routine working formalin?

4% formaldehyde (10% formalin).

What is a 1/10 dilution of commercial formalin called?

Routine working formalin (10% formalin).

Calculate: 5% formalin = ? % formaldehyde.

2% formaldehyde.

What is the formula to calculate formaldehyde concentration from formalin dilution?

(100% formalin / % formalin dilution) = (40% formaldehyde / X% formaldehyde).

What is paraformaldehyde?

Polymerized formaldehyde (formalin) created by dehydration.

What does paraformaldehyde look like?

Insoluble white precipitate.

How is paraformaldehyde formation prevented?

Addition of 10% methanol.

Why is methanol added to concentrated formaldehyde?

To stop polymerization into paraformaldehyde.

What are two properties of methanol in formalin?

It has some fixing properties and can cause a small amount of precipitate.

What type of fixing properties does methanol have?

Coagulant fixing properties.

What can methanol cause in formalin?

A small amount of precipitate.

First way paraformaldehyde can be removed?

Filter, but this also removes formaldehyde and fixation molecules.

Second way paraformaldehyde can be removed by hydrolysis?

Use NaOH and heat to add water and break the bonds.

Third way paraformaldehyde can be removed by filtering and drying?

Filter and dry precipitate, weigh it, and use it to make up a 4% solution in distilled water.

What does an aldehyde become when oxidized?

An acid.

What acid does formaldehyde slowly oxidize to in dilute solution?

Formic acid.

What happens to the pH of 10% formalin when formic acid forms?

It drops to approximately 3-4.

as pH decreases H+ increases

Why does formalin have a lower pH when oxidized?

Due to the formation of formic acid.

What happens to [H+] when pH drops in formalin?

[H+] increases.

What does a drop in pH in formalin result in?

Loss of fixing properties and damage to tissues.

How can the loss of fixing properties from oxidation be prevented?

Store concentrated, buffer it, or use diluted formalin (soon)

What buffer is added to 10% formalin to make 10% NBF?

Phosphate buffers.

What is the pH of 10% Neutral Buffered Formalin?

6.8.

Formalin cross-links between which groups on proteins?

Free amine groups (-NH2) - called Methylene bridges.

What type of gel does formalin form?

Non-coagulant or non-precipitant gel of inactivated protein.

What happens to methylene bridges during normal processing after adequate fixation?

They are washed out.

Why is protein reactivity reversible in formalin fixation?

Because CH2 can be washed out without proteins refolding or reactivating. (reversibility)

What is the time frame for adequate fixation?

4 to 8 hours - just enough to stop autolysis and putrefaction

Why is staining not hindered in adequate fixation?

Because normal processing washes out most methylene bridges, leaving amines free.

What is the time frame for complete fixation?

Up to 7 days.

In complete fixation, what happens to the methylene bridges?

All possible reactions have occurred, All possible bridges have formed.

Most or all amine groups are tied up. Normal processing is not enough to remove these bridges

Why is staining with colored anions poor in completely fixed tissue?

Because most or all amine groups are tied up by methylene bridges.

How can staining be improved in completely fixed tissue?

Thoroughly wash prior to processing - This will restore staining ability

What is the charge of tissue before fixation?

Overall net positive charge.

It attracts anions and repels cations.

What happens to the charge of tissue after complete fixation?

It has no charged amine groups.

Decreased attraction to anions, decreased repulsion of cations.

Is eosin an anionic or cationic dye?

Anionic.

in tissue = decrease +ve charge

Is hematoxylin an anionic or cationic dye?

Cationic.

in tissue = decrease staining by coloured anions (-ve)

What is the effect of complete fixation on eosin/hematoxylin staining?

Eosin staining is poor.

Hematoxylin staining is a bit overdone.

What happens to the staining when methylene bridges are left in tissue?

1. Decreased anionic staining

2. Increased cationic staining.

Why is a water station used in processors before the first dehydrating alcohol?

Because tissues often undergo complete fixation and have been fixed for a long time

What happens to the buffer during prolonged fixation (weeks/months)?

It weakens or gets used up.

therefore the ph drops due to the formation of formic acid.

During prolonged fixation, does washing remove methylene bridges?

Yes, therefore restores anionic staining

Why is cationic staining (pale nuclei) still poor even after washing in prolonged fixation?

Because there is poor restoration of COO- carboxyl groups.

If prolonged fixation is anticipated, what should be done before processing?

Complete fixation in formalin, then transfer to alcohol for better staining

At what pH does formic acid react with hemoglobin to produce formalin pigment?

What is another name for formalin pigment?

Acid formaldehyde hematin.

What complex is formalin pigment formed from?

Reaction of formic acid with hemoglobin to produce a porphyrin-formalin complex.

What does formalin pigment look like?

Dark, brown, black, refractile granules.

What optical property does formalin pigment have?

Birefringent (splits light) able to rotate polarized light

Where is formalin pigment found?

Extracellular, near bloody areas (spleen, liver, blood vessels).

What is the best way to prevent formalin pigment?

Use NBF at pH 6-6.8.

What buffer is used in NBF to stabilize pH?

Sodium phosphate buffer.

What is a less effective method to prevent formalin pigment?

Use 10% aqueous formalin with magnesium or calcium carbonate on the bottom of the container.

How does the less effective method of preventing formalin pigment work?

It neutralizes formic acid as it is formed, though pH still ends up at 6 or less.

What is the most common method to remove formalin pigment?

Deparaffinize/hydrate, place in absolute alcohol saturated with picric acid (10 min - 3 hrs), wash with water.

What is the second method to remove formalin pigment?

Deparaffinize/hydrate, 100 mL 70% alcohol + 2-3 mL ammonium hydroxide (30 min - 3 hrs), wash with water, rinse in 1% acetic acid, wash well again.

Does formalin cause tissue shrinkage?

No, the hot wax does.

How does formalin compare to other fixatives regarding shrinkage?

It allows less shrinkage in subsequent processing steps.

What happens to iron in tissue if unbuffered formalin has a low pH?

Some iron is lost (acid dissolves iron).

* Important in iron stains - therefore, if iron is to be demonstrated, watch the ph of the fixative

What is the ingredient and property of 10% aqueous formalin?

Formaldehyde (40%) 100ml + distilled water 900ml; very hypotonic, may produce formalin pigment.

What is the ingredient and property of 10% formal saline?

Formaldehyde (40%) 100ml + sodium chloride 9g + distilled water 900ml; isotonic, may produce formalin pigment.

What is the ingredient and property of alcoholic formalin?

Formaldehyde (40%) 100ml + ethyl alcohol 650ml + distilled water 250ml; compound fixative, tissue can be stored indefinitely.

What is the ingredient and property of 10% neutral formalin?

Formaldehyde (40%) 100ml + distilled water 900ml + calcium or magnesium carbonate to excess; becomes acidic after withdrawal.

What are the ingredients and properties of 10% Neutral Buffered Formalin?

Formaldehyde (40%) 100ml, distilled water 900ml, monobasic sodium phosphate 4g, dibasic sodium phosphate 6.5g; Most widely used, pH 6.8, hypotonic.

What is the ingredient and special use of Calcium formalin?

Formaldehyde (40%) 100ml + distilled water 900ml + calcium chloride 10g; recommended for preservation of phospholipids.

What is IBF tissue fixative?

A proprietary fixative formulation containing isopropanol/Methanol, BaCl2, and low percentage formaldehyde (< 3% by weight).

What are the properties of IBF?

Sold ready to use, penetrates quickly and uniformly, intolerant fixative (do not store long), forms acid hematin pigment.

What is IBF good for?

Biopsies (especially prostate, better nuclear detail than formalin), sometimes bone marrow/lymphatic tissue.

What do eosinophil fixatives contain?

all contain formaldehyde but use different buffers

Note: Different buffers with formaldehyde affect cytoplasm and granule appearance.

What is glycogen?

Polysaccharide (polymer of glucose), mostly in liver and muscle as a storage form of energy

How is glycogen fixed by formalin?

The portion bound to protein is fixed well, most of the rest is trapped, some free glycogen may dissolve out of tissues

Why must tissue be fixed promptly to demonstrate glycogen?

Glycogen breaks down into glucose very rapidly by enzymes after tissue death.

What is the best fixative for maximum glycogen preservation?

Alcoholic formalin.

Why is alcoholic formalin best for glycogen?

Glycogen is not soluble in alcohol, so it precipitates and is better preserved.

What is glycogen streaming artefact?

Glycogen appearing polarized to one side of the cell, caused as fixative moves through the cell.

Does formalin fixation fix neutral lipids?

No, it preserves but does not stabilize them.

Why are lipids lost during processing?

Due to solvents (such as xylol).

How can lipids be demonstrated in formalin-fixed tissue?

Cut as a frozen section.

What do calcium acetate and formalin (formal Ca) do for phospholipids?

Fixes phospholipids well enough for paraffin processing.

How does calcium link to lipids?

It links up with unsaturated bond areas.

Calculate: 20% formalin = ? % formaldehyde.

8% formaldehyde.