Tissue processing and paraffin embedding

Principle

• Water in fixed tissue is replaced by paraffin wax so the tissue becomes firm enough to cut thin sections.
• Steps: dehydration, clearing, infiltration, then embedding.

Specimen

• Well-fixed tissue in labeled cassettes, trimmed to about 3–4 mm thickness.

Reagents and equipment

• Graded alcohols (e.g. 70%, 95%, 100%), clearing agent (xylene or substitutes), paraffin wax (melting point about 56–58 °C).
• Automated tissue processor (with vacuum/pressure and heat), embedding center with hot and cold plates, metal molds.

Procedure

• Dehydration: pass through increasing alcohol concentrations to remove water gradually and limit distortion.
• Clearing: xylene removes alcohol; it is miscible with both alcohol and paraffin and makes tissue translucent.
• Infiltration: molten paraffin kept a few degrees above its melting point (typically about 58–60 °C) fills the tissue, usually with vacuum.
• A routine overnight schedule is typical; small biopsies can use shorter programs.
• Embedding: place tissue in a mold with molten wax, orient it correctly, attach the labeled cassette and cool quickly on a cold plate.
• Orientation examples: skin and mucosa on edge so all layers are cut; tubular structures in cross-section; cut surface face down; multiple small fragments on the same plane.

Quality control and pitfalls

• Incomplete fixation or dehydration leaves water, giving soft, mushy blocks that crumble or have holes.
• Overheated or prolonged paraffin makes tissue hard, brittle and shrunken.
• Rotate and replace reagents on schedule; contaminated alcohols and xylene degrade processing.
• Wrong orientation can make a diagnosis impossible (e.g. margins or depth of invasion).
• Check cassette labels against specimen records at each step to avoid mix-ups.
• Xylene is toxic and flammable; use ventilation and proper disposal.

Clinical use

• Produces formalin-fixed paraffin-embedded (FFPE) blocks for H&E, special stains, IHC and molecular tests; blocks can be stored long-term.
• Resin (plastic) embedding is used when very thin sections or hard tissue are needed.