L4. DECALCIFICATION

What is decalcification?

• A crucial preparatory process that ensures optimal tissue sectioning and microscopic examination of mineralized tissues, particularly bones and decalcified tissues.

Why is decalcification necessary in histologic specimens?

The presence of calcium deposits can impede proper tissue processing, embedding, and sectioning.

How does decalcification affect bone tissues?

• It removes calcium ions, making bones flexible and easier to cut.

What is the purpose of decalcification?

To soften hard, calcified tissues, facilitating sectioning and examination under a microscope

What are common specimens requiring decalcification?

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

What are the three main steps in decalcification?

Fixation, Decalcification, and Processing

What happens during the fixation step?

• Specimens are thoroughly fixed to preserve tissue structure.

What happens during the decalcification step?

Fixed tissues are immersed in a decalcifying agent until calcium is removed

What happens during the processing step?

Decalcified tissues are processed, embedded in paraffin, and sectioned for staining.

Why is decalcification a lengthy procedure?

Bone pieces must be left in the decalcifying agent for several days or weeks, depending on size

Why is it sometimes necessary to retain mineralized bone?

To differentiate mineralized bone from osteoid or perform morphometric measurements.

How can bone specimens be prepared without decalcification?

By infiltrating them with acrylic or epoxy resins and using specialized microtomy techniques

What are the two main types of decalcification agents?

Strong mineral acids and weak organic acids.

Give an example of a strong mineral acid used for decalcification

10% hydrogen chloride (HCl)

Give an example of a weak organic acid used for decalcification

5-10% formic acid (HCOOH).

What is the role of 14% ethylene diamine tetraacetic acid (EDTA) in decalcification?

It acts as a chelating agent, sequestering metallic ions, including calcium

What are the advantages of using hydrochloric acid (HCl) as a decalcifying agent?

Rapid decalcification.

What are the disadvantages of using hydrochloric acid (HCl) for decalcification?

It may damage tissue morphology and nucleic acids.

Why is nitric acid commonly used for decalcification?

It is the fastest decalcifying agent

What is a disadvantage of nitric acid?

It can cause yellow discoloration due to nitrous acid formation

What is Formol-Nitric Acid?

A decalcifying solution made of concentrated nitric acid, strong formaldehyde, and distilled water

Why is Formol-Nitric Acid used for urgent biopsies?

It is rapid-acting and provides relatively good nuclear staining.

What is a disadvantage of Formol-Nitric Acid?

It imparts a yellow color, which can impair staining

What is Perenyi’s Fluid?

A mixture of nitric acid and chromic acid used for decalcification

What is a disadvantage of Perenyi’s Fluid?

It is slow and not recommended for urgent biopsies.

What is Von Ebner’s Fluid?

A decalcifying solution using NaCl and hydrochloric acid.

What is a disadvantage of Von Ebner’s Fluid?

The extent of decalcification cannot be measured by a chemical test.

Why is formic acid commonly used for decalcification?

It preserves nucleic acids, making it compatible with molecular biology techniques

What is a disadvantage of formic acid?

It is relatively slow and not suitable for urgent specimens.

What is a chelating agent?

A substance that binds with calcium ions to facilitate calcium removal.

What is the most common chelating agent used in decalcification?

Ethylene diamine tetraacetic acid (EDTA).

What is the commercial name of EDTA?

Versene

Why is EDTA preferred for preserving nuclear DNA?

It allows histochemical methods for nucleic acids and enzyme activity studies.

Why is EDTA not used for urgent specimens?

It is slow and can take weeks instead of days

What are the advantages of chemical decalcification over physical methods?

It is more controlled and better preserves tissue morphology.

What is the disadvantage of chemical decalcification?

It can interfere with staining.

Why is electrophoresis used in decalcification?

It accelerates the removal of calcium ions from tissue

Why is microwave oven decalcification useful?

It speeds up the process.

What is a limitation of microwave oven decalcification?

It is only suitable for small bone fragments.

What factors influence the rate of decalcification?

Concentration, agitation, fluid access, and temperature

What are two main methods for determining the end-point of decalcification?

Physical test and chemical tests.

Why is proper end-point determination important?

To avoid overexposure, which can damage tissue morphology

What is the purpose of extensive washing after decalcification?

To remove acid residues.

What are two common chemicals used to neutralize acid after decalcification?

Saturated lithium carbonate solution and 5-10% aqueous sodium bicarbonate solution

What is surface decalcification?

A technique used to handle small calcium deposits in paraffin blocks.

What sensation indicates the presence of calcium during microtomy?

Resistance or a “grating” sensation

What solution is commonly used for surface decalcification?

10% hydrochloric acid.

Why must realignment of the tissue block be done after surface decalcification?

To ensure proper sectioning.

Why are tissue softeners used in histopathology?

To prevent microtome damage and improve sectioning of hard tissues.

What is Perenyi’s Fluid used for besides decalcification?

As a tissue softener.

What chemical is commonly used to soften tissues within 1-3 days?

4% aqueous phenol solution.

What are other examples of tissue softeners?

MOLIFLEX, 2% hydrochloric acid, 1% hydrochloric acid in 70% alcohol.