2027 L2 Tissue Prep

Tissue Preparation

The process of collecting, fixing, processing, and embedding tissue samples for microscopic examination.

Tissue collection

Invasive vs non-invasive techniques, depends on intended tests.

Fine Needle Aspiration (FNA)

A minimally invasive technique for collecting cells using a fine needle, often guided by imaging. Primaraly used for enlarged lymph nodes and breast nodules.

Core Biopsy

A procedure that collects a cylindrical tissue sample for more comprehensive examination than a needle biopsy.

Formalin

An aqueous solution of formaldehyde stabilised with methanol used to fix tissues by cross-linking proteins.

Fixation

The process of preserving tissue structure by halting enzymatic activity and preventing decomposition (inhibits autolytic enzymes and kills bacteria).

Poorly fixed specimins may not yeild the appropriate information and can lead to diagnostic errors.

Fixation process

Adequate fixation takes around 12 - 24 hours, full fixation takes longer.

Over-fixation can hinder antigen detection and lead to brittleness.

Safety Concerns: Toxic, potential carcinogen - must be used with proper ventilation.

Dissection/ Grossing

The method of preparing tissue specimens by transforming a large sample into smaller, manageable pieces.

Cryostat

A specialized microtome housed in a freezer used for cutting thin sections of frozen tissue. Between -15 to -30 degrees. Tissue handled in here is unfixed, may contain high-risk organisms such as TB or HIV.

Sections fixed with alcohol & acetic acid solition before staining if required.

Inking margins

The process of applying ink to the edges of a surgical specimen to delineate margins for pathological examination, ensuring accurate assessment of tumor involvement.

Decalcification

The process of removing minerals from bone samples (Hydroxyapatite) to allow for easier sectioning for microscopic examination.

Acid decalcifiers

Nitric Acid - Fast acting, most damage to tissue if used for more than few hours. only syitable for urgent cases and hard bone (e.g. skull).

Hydrochloric Acid - Can be used alone, usually a part of solutions. Fast acting, less tissue damage than Nitric acid.

Formic Acid - Most popular, concentration between 5-30%. significantly slower acting, less tissue damage.

Embedding Medium

A supportive material, often wax or resin, used to encase tissue specimens for cutting thin sections.

Xylene

A clearing agent commonly used in tissue processing, which makes tissues optically clear.

Chelating Agents

“Calcium sponges”, mop up free ionic calcium that is always present around the bone mineral. Substances that bind metal ions, used in decalcification to slowly remove calcium from tissues. Can occur at natural pH.

Decalcification monitoring methods

• X-rays (expensive)

• Manual testing (not recommended)

• Chemical testing:

  • Decalcifying fluid sample taken and neutralised with hydroxide solution. Adding ammonium oxalate to this neutralised solution will show whether calcium is present by producing a precipitate.

Autolysis

The self-digestion or self-destruction of cells through the action of their own enzymes post-removal.

Stitches

Another term for sutures, used to mark surgical margins and orient specimens for analysis.

Investigating undecalcified sections

Only needed when actual process of bone mineralisation is being investigated. Samples need to be imbedded into a hard medium (e.g. resin)

• Osteoporosis and Rickets: Bone density and quality

• Osteoarthritis: Cartilage and bone interaction

• Bone tumours: Osteosarcoma

• Dental pathologies: Caries and periodontitis

Processing Machine

An automated device that prepares multiple tissue samples simultaneously for microscopic analysis.

Dehydration (Ethanol 70-100%) → Clearing (Xylene) → Impregnation (paraffin wax or resin)

Microtome

An instrument used to cut thin slices of tissue for microscope slides. Thickness from 0.1 to 50µm (typically 4 µm).

Thinnest sections required for electron microscopy and the thickest sections required for neuropathology techniques.

Paraffin Wax

The most commonly used embedding medium in histology for supporting tissue sections during cutting.

Isopentane

A less volatile liquid used to cool and freeze delicate tissues for rapid sectioning.

Formic Acid

A decalcifying agent used for gentle removal of calcium from tissues, particularly suitable for histopathology.

Embedding/ Blocking out

The process of enclosing a tissue sample in a solid medium to provide support for sectioning. Correct orientation essential. Wax takes 30 mins to solidify.

Cryotomy

Rapid tissue processing method. Provides immediate pathological assesment (e.g. during surgery). No need for paraffin embedding, or chemical fixatives.

Limitations: Inferior section quality, limited for special stains.

Immunohistochemistry

A technique used to identify specific antigens in tissue sections through labeled antibodies.

Rapid freezing methods

Liquid Nitrogen (-196 degrees)

• Boils when contacting warmer tissue, creating insulation that slows freezing

• Boiling can damage delicate tissues (e.g., kidney biopsies, skin

Freezing & tissue damage

Freezing too slow can lead to extensive damage. Ice crystals may occur intracellularly and rupture cell membranes (ice-crystal artefact).

Artefacts can lead to misinterpretations (misdiagnoses).