Decalcification (Gregorios)

Flashcards about the decalcification process.

What is decalcification?

The removal of calcium ions from bone or calcified tissues through a histological process.

What types of specimens often require decalcification?

Bones, teeth, calcified tumors, and calcified heart valves.

Why is decalcification necessary?

To enable the histotechnologist to cut soft sections of bone using a microtome.

What tools and techniques are commonly used for grossing and cutting bone specimens?

High-speed saws and long periods in decalcifying solution.

What is the ideal thickness to reduce undecalcified surgical specimens of hard tissue to?

2-3 mm.

When might it be necessary to retain and demonstrate the mineral content of bone?

To differentiate mineralized bone from osteoid and if morphometric measurements are required.

How can mineralized bone be processed into thin sections?

By sawing it into thin wafers and grinding it using abrasive surfaces.

What types of acids are used in decalcification?

Strong mineral acids or weak organic acids.

How do acids facilitate decalcification?

They form soluble calcium salts in an ion exchange that moves calcium into the decalcifying solution.

What is an example of a chelating agent used in decalcification?

Ethylene diamino tetracetic acid (EDTA).

How does EDTA work as a decalcifying agent?

It sequesters metallic ions, including calcium, in aqueous solutions.

Besides acids, what other materials can bones be infiltrated with to avoid decalcification?

Acrylic or epoxy resins.

What is generally a satisfactory fixative for bone?

Buffered formalin.

What alternatives to buffered formalin are used when bone marrow preservation is important?

Zinc formalin mixtures, B-5, formol-acetic alcohol, or Bouin’s solution.

Why is thorough fixation crucial prior to decalcification?

To protect cellular and fibrous elements of bone from damage caused by acids.

What specimens contain calcium that can interfere with histologic section evaluation?

Bones, teeth, and other calcified tissues like tuberculous lungs.

What happens to poorly-fixed specimens during decalcification?

They become macerated and stain poorly.

Why should large specimens be cut into thin slices as soon as possible?

To enhance fixative penetration.

Why should high-quality fine tooth saws be used for preparing bone slices?

To avoid mechanical damage and forcing bone fragments into soft tissues.

What are toenails composed of that make them unsuitable for decalcification?

Insoluble keratin filaments.

What are the three main types of decalcifying agents?

Strong mineral acids, weaker organic acids, and chelating agents.

What cellular components can be extracted by acidic decalcifying agents if specimens are exposed for too long?

RNA and purine/pyrimidine bases from DNA.

Provide examples of strong mineral and weak organic acids used for decalcification

10% hydrogen chloride (HCl) or 5-10% formic acid (HCOOH).

Why are tissue pieces suspended in liberal amounts of decalcifying solution?

To ensure complete decalcification and protect the tissue from precipitates.

Why should the thread be dipped in melted paraffin wax, and why should metal cap containers be avoided?

To avoid corrosion from the acid.

What can result from using strong acids for longer than necessary?

Loss of nuclear staining and tissue maceration.

What is the most common strong acid used in proprietary rapid decalcifiers?

Hydrochloric acid.

How do rapid decalcifying agents affect cell nuclei staining?

Failure of nuclear chromatin to take up hematoxylin and other basic dyes.

How can the effects of decalcifying agents on H&E staining be reduced?

Post-decalcification and removal, and by adjusting the staining procedure.

What is the most common and fastest decalcifying agent?

Nitric acid.

What are the disadvantages of using nitric acid?

Inhibiting nuclear stains and destroying tissues, especially in concentrated solutions.

Why must the endpoint of decalcification with nitric acid be carefully monitored?

To prevent tissue damage and impaired staining.

How is the yellow color imparted by nitrous acid formation prevented/neutralized?

5% sodium sulfate and washing in running tap water for at least 12 hours.

What are the advantages of Perenyi’s Fluid?

It decalcifies and softens tissues at the same time and avoids maceration.

How is a precipitate in Perenyi's fluid dissolved to determine complete decalcification?

By adding glacial acetic acid drop by drop and then adding ammonium oxalate.

What is the advantage of Phloroglucin-Nitric Acid?

It is the most rapid decalcifying agent so far, recommended for urgent cases.

Why is Hydrochloric acid (HCl) inferior to nitric acid as a decalcifying agent?

Slower action and greater distortion of tissue.

What do slow proprietary decalcifying mixtures usually contain?

Buffered formic acid or formalin/formic acid.

For what specimens is Von Ebner's Fluid recommended?

Teeth and small pieces of bone.

Which type of acids are suited to bone marrow?

Organic acids such as acetic and formic acid.

Which weak acid used extensively as a primary decalcifying agent?

Formic acid.

What are the advantages of gentle formic acid?

It's gentler in action and less likely to interfere with nuclear staining.

When should formic acid be used?

Routine decalcification of postmortem research tissues.

How can formic acid be applied?

10% aqueous solution or combined with formalin or buffer.

For what specimens is Tricholoracetic acid suitable?

Small bone spicules.

For what cases is Sulfurous acid suitable?

Minute pieces of bone.

What is Chromic acid combined with in Flemming's Fluid?

Osmium tetroxide.

What happens when tissue is dehydrated with alcohol after being decalcified with Chromic acid?

Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol.

What are the main components of Citric Acid-Citrate Buffer Soultion?

Citric acid (monohydrate) aqueous solution and Ammonium citrate (anhydrous.

Advantage of Citric Acid-Citrate Buffer Soultion?

It does not produce cell or tissue distortion.