LESSON 2 HISTOPATHOLOGY LABORATORY DECALCIFICATION DEHYDRATION CLEARING

Decalcification

The process of removing calcium/lime salts from tissue prior to dehydration.

Types of Decalcifying Agents

1. Acids 2. Chelating agents 3. Ion exchange resins 4. Electrophoresis.

Nitric Acid

Most common and fastest acid decalcifying agent, can impart a yellow color to tissue.

Formol–Nitric Acid

Rapidly acting decalcifying agent for urgent biopsies, must be used in a fume hood.

Perenyi’s Fluid

Decalcifies and softens tissue, providing good nuclear and cytoplasmic staining.

EDTA (Ethylene Diamine Tetraacetic Acid)

A chelating agent that binds calcium and magnesium; acts faster at pH 7 to 7.4.

Ion Exchange Resin

Ammonium form of polystyrene resin used to remove calcium from certain solutions.

Electrophoresis

Method where positively charged calcium ions migrate to the negative electrode for removal.

Calcium Oxalate Test

Tests for decalcification; precipitation indicates incomplete decalcification.

Dehydration

The process of removing intracellular and intercellular water from tissues prior to impregnation.

Ethanol

Best dehydrating agent, non-toxic and cost-effective for routine tissue dehydration.

Acetone

A cheap and rapid dehydrating agent, not recommended for routine use due to high volatility.

Dioxane (Diethylene Dioxide)

Excellent dehydrating and clearing agent but highly toxic due to vapor exposure.

Xylene (Xylol)

Commonly used clearing agent in histopathology, usually requires ½ to 1 hour clearing time.

Chloroform

Clearing agent that is slower than xylene and toxic to the liver upon prolonged inhalation.

Cedarwood Oil

Clears during embedding for both paraffin and celloidin sections; recommended for CNS studies.

Aniline Oil

Not routinely used for clearing; recommended for delicate specimens such as embryos and insects.