Basic and Advanced Laboratory Techniques in Histopathology and Cytology (Video)

Vocabulary-style flashcards covering key concepts, terminology, and techniques from the lecture notes on histopathology and cytology laboratory methods.

Fixation

The process of preserving tissue/cells by chemical or physical means to prevent autolysis and distortion, maintaining as close to the living state as possible.

10% Neutral Buffered Formalin

A common fixative (formaldehyde in phosphate buffer) used in routine histology; penetrates about 1 mm per hour; preserves morphology but can affect some biomolecules.

Glutaraldehyde

A fixative ideal for electron microscopy due to excellent protein cross-linking; poor tissue penetration and no lipid fixation.

Osmium Tetroxide

Fixative for lipids, used in EM; fixes unsaturated lipids and preserves membranes but has poor penetration and is toxic.

Bouin’s Fixative

Fixative containing picric acid; rapid penetration and good for glycogen, but produces yellow tissue and is not DNA-friendly.

Mercury Salt-Containing Fixatives

Fixatives such as Zenker’s/Helly’s/B5 containing mercuric salts; very rapid acting but highly toxic and can cause artefacts.

Formaldehyde (Formalin) - Components and Actions

Formaldehyde, typically as a 37\% aqueous solution (formalin), is diluted to 10\% neutral buffered formalin (4\% formaldehyde). It forms methylene bridges between proteins, cross-linking them but can cause protein denaturation.

Picric Acid - Properties in Fixation

A component in Bouin's fixative; a strong coagulant that enhances nuclear staining but leaves tissue yellow and can shrink connective tissue.

Mercuric Chloride - Properties in Fixation

A highly corrosive component of Zenker's, Helly's, and B5 fixatives; rapidly penetrates and yields excellent nuclear detail but is toxic and causes black mercuric pigment artefacts.

Fixative Effects on Tissue Characteristics

1. Hardening: Most fixatives cause tissue to become firmer. 2. Shrinkage/Swelling: Varies by fixative; Bouin's can cause shrinkage, some solutions cause swelling. 3. Color Changes: Picric acid (yellow), formalin (brown pigment). 4. Enzyme Inhibition: Prevents autolysis and bacterial degradation by inactivating enzymes.

Fixation Artefact

Unwanted morphological changes due to fixation, including formalin pigment, mercury pigments, or tissue distortion.

Formalin Pigment

A dark brown/black, crystalline artefact formed in acidic formalin solutions (acid hematin) due to the reaction of formaldehyde with hemoglobin.

Removal of Formalin Pigment

Can be removed by treating sections with saturated alcoholic picric acid or 1\% alcoholic sodium hydroxide before staining.

Mercury Pigment

Black, granular, and refractile deposits in tissue fixed with mercury-containing fixatives (e.g., Zenker's, B5); must be removed to avoid obscuring cellular detail.

Removal of Mercury Pigment

Removed by treating sections with iodine (e.g., Lugol's iodine) to convert the pigment to mercuric iodide, followed by sodium thiosulfate to decolorize the iodine.

Removal of Picric Acid Yellowing

The yellow discoloration caused by picric acid can often be removed by treating sections with alcoholic lithium carbonate or 70-80\% ethanol before staining.

Mechanism of Fixation

Cross-linking of proteins and potential dehydration; lipid fixation by osmium; fixation stabilizes tissues for subsequent processing.

Essential Precautions for Fixation

Use thin tissue slices (3–5 mm), adequate fixative volume (roughly 20x tissue volume), avoid blood, and maintain proper containers.

Factors Affecting Fixation

pH, temperature, duration, osmolarity, concentration, and agitation influence fixative performance.

Types of Fixation

Immersion, coating, vapour, perfusion, microwave, and freeze-drying fixation methods.

pH Characteristics of Common Fixatives

pH Characteristics of Common Fixatives

1. 10% Neutral Buffered Formalin: pH 6.8-7.2 (neutral) for optimal preservation and to prevent formalin pigment formation.
2. Bouin's Fixative: Acidic (due to picric acid and acetic acid) which enhances nuclear staining but can cause tissue shrinkage.
3. Mercury Salt-Containing Fixatives (Zenker's, Helly's): Typically acidic, which contributes to their rapid action and good nuclear detail.
4. Glutaraldehyde & Osmium Tetroxide: Usually buffered to a neutral or slightly alkaline pH (7.2-7.4) to preserve ultrastructure for electron microscopy.

Artefacts Associated with Specific Fixatives

Artefacts Associated with Specific Fixatives

1. Formalin: Formalin pigment (acid hematin) when acidic, protein denaturation, and slight tissue hardening.
2. Glutaraldehyde: Poor penetration leading to uneven fixation, and no lipid fixation.
3. Osmium Tetroxide: Poor penetration, toxicity, and unsuitable for routine light microscopy due to strong staining properties.
4. Bouin's Fixative: Distinct yellow discoloration of tissue, shrinkage of connective tissue, and poor preservation of DNA.
5. Mercury Salt-Containing Fixatives: Black mercuric pigment which obscures cellular detail, tissue hardening, and high toxicity.

End Point of Decalcification

When calcium has been sufficiently removed without excessive tissue damage, determined radiographically, chemically, or physically.

Clinical Significance of Decalcification

Essential for preparing bone, teeth, and calcified soft tissues for routine histological sectioning without damaging the microtome blade.

Acid Decalcification

Uses acids (e.g., HCl, nitric acid, formic acid, TCA) to remove calcium; rapid but can damage tissue, especially nuclei.

Chelating Decalcification (EDTA)

Use of EDTA to chelate calcium; slow but preserves morphology and nucleic acids well.

Factors Affecting Decalcification Rate

1. Concentration/Type of Acid: Stronger acids act faster but cause more tissue damage. 2. Temperature: Higher temperatures increase rate but also damage. 3. Agitation: Increases solution contact with tissue, speeding up removal. 4. Tissue Size: Smaller, thinner specimens decalcify faster.

End Point Detection in Decalcification

Radiography, chemical tests (calcium testing), or physical pliability tests indicate completion.

Surface Decalcification

Brief application of a decalcifying agent to the surface of a paraffin block containing small calcifications to allow sectioning.

Processing (Tissue Processing)

Removes water and replaces it with embedding medium (e.g., paraffin) via dehydration, clearing, and infiltration.

Infiltration and Embedding

Impregnating tissue with embedding medium (paraffin or resins) to provide support for sectioning.

Purpose of Embedding

To impregnate tissue with a solidifying medium (e.g., paraffin wax, resin) that provides rigid support, enabling thin and uniform sections to be cut with a microtome.

Paraffin Wax

Most common embedding medium; MP around 56–62°C; inexpensive and allows long-term storage but can cause shrinkage with over-embedding.

Alternative Embedding Media

1. Celloidin: Used for very delicate tissues (e.g., brain), but processing is lengthy. 2. Resins (e.g., acrylic, epoxy): Used for very thin or hard sections, especially for electron microscopy or specialized light microscopy. 3. Agar/Gelatin: Used as pre-embedding matrices for friable or multiple small samples.

Tissue-Tek System

Automated embedding systems for streamlined processing and block creation.

Tissue Orientation

Placing tissue to optimize cutting planes (e.g., Swiss roll for endometrium, transverse/longitudinal orientation).

Tissue Marking

Ink or marker methods (India ink, silver nitrate, Rose Bengal) to identify margins and planes of resection.

Sectioning the Paraffin Block

Cutting tissue into very thin ribbons with a microtome; knife angle and water bath alignment are critical.

Rotary Microtome

The most common type of microtome, used for cutting consecutive thin sections (3-5 \mu m) from paraffin-embedded tissue blocks; operates with a rotating flywheel and precise advance mechanism.

Sliding Microtome

Used for cutting large or hard paraffin blocks, or celloidin-embedded tissues; the blade slides across a stationary block, often producing thicker sections than a rotary microtome.

Ultramicrotome

Specialized microtome used for cutting extremely thin sections (<1 \mu m, typically 50-100 nm) from resin-embedded tissue for electron microscopy; uses glass or diamond knives.

Cryostat / Frozen Section

Instrument for rapid freezing and cutting of tissue for quick intraoperative diagnosis; stained with H&E or rapid stains.

Microtome Knife/Blade and its Importance

The sharp cutting edge (steel, glass, diamond) of a microtome is crucial for producing high-quality, continuous, and wrinkle-free sections; proper angle and condition prevent tearing or compression artefacts.