Decalcification

decalcification

Os the removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut for the microscopic examination.

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

Purpose

Bones, teeth, calcified tumors, and calcified heart valves

Common specimens

Metallic bone disease

Is being investigated and it is necessary to differentiate mineralized bone from osteoid, or if morphologic measurements are required, it may be necessary to retain and demonstrate the mineral content by producing sections of undecalcified bone

Mineralized bone

Is a hard material, there is a limited range of techniques abailable to produce sections from it

Principle of decalcification

Based on the removal of calcium ions by using chemical agents that create soluble salts.

10% hydrogen chloride

Strong mineral acids

5-10 % formic acid

Weak organic acids

HCOOH

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

14% ethylene diamine tetraacetic acid (EDTA)

An ideal chelating agent that sequesters metallic ions, including calcium, in aqueous solutions

• Ideal reagent for decalcification of mineralized tissues such as bone

can result in poor tissue integrity, inadequate staining, and difficulties in microtome sectioning

Common errors

• Tissue become fragile and difficult to handle

• Loss of cellular details leading to poor staining integrity

Consequences of poor fixation

• Acids

• Chelating agents

• Strong minerals

• Nitric acid

• Formal-nitric acid

• Perenyi’s fluid

• Hydrochloric acid

• Von ebner’s fluid

Decalcifying agents

Strong acids

Rapidly dissolves calcium salts making them efficient but may damage tissue morphology and nucleic acids.

Hydrochloric acid, nitric acid, sulfuric acid

Examples of Strong acids

Weak acids

Slower but better at preserving tissue structure

Formic acid, citric acid, acetic acid

Example of Weak acids

Chelating agents

Are substances which combine with calcium ions and other salts to form weakly dissociated complexed and facilitate removal of calcium salt

• specimen size

• Density

• Choice of decalcifying agent

• Agitation

• Monitoring decalcification

Factors influencing decalcification

Ethylene Diamine Tetra Acetic Acid

The most common chelating Agent in the market. Recommended only for detailed microscopic studies, as an anticoagulant, tissue preservation, and water softener

Versene

Commercial name of EDTA

Binds calcium and magnesium ions

EDTA Mechanism of Action

Preferred when nuclear DNA preservation is important

EDTA Advantages over acids

Nitric acid

Is the most common and the fastest decalcifying agent used so far.

Rapid in action

Formol-Nitric acid

Rapid acting, recommended for urgent biopsies

Nuclear staining is relatively good

Perenyi’s fluid

Recommended for routine purposes

Slow decalcifying agent for dense bones; not recommended for urgent biopsies

Hydrochloric acid

Is inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue produced on the decalcified section.

Von Ebner’s Fluid

It permits relatively good cytologic staining

Iy is a moderately rapid decalcifying agent

Weak acids

Preferred when a more controlled and less agressive decalcification process is required

Formic acid

Ia a moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion, and is safer to handle than nitric acid or Hydrochloric acid

Ion exchange resin

Hastens decalcification by removing calcium ions from formic-acid containing decalcifying solutions

Electropherosis

Suitable for small bone fragments only

Can process only limited number of specimens at a time m.

• Concentration

• Fluid access

• Agitation

• Temperature

Factors influencing the rate of decalcification

• Physical tests

• Chemical tests

Determining the End-point of decalcification

Tissue softeners

Unduly hard tissues can damage microtome knifes and may require softening before sectioning

• Perenyi’s fluid

• 4% aqueous phenol solution

• Molliflex

• 2% Hydrochloric acid

• 1% hydrochloric acid in 70% alcohol

Tissue softeners