Histopathologic Techniques

primary purpose of histopathologic techniques

To prepare tissue samples for microscopic examination in order to diagnose diseases, while preventing autolysis and preserving tissue morphology

Autolysis

self-digestion of tissues post-mortem

temperature, enzyme content, and tissue type (e.g., liver vs. connective tissue)

what factors influence autolysis?

Fine-Needle Aspiration (FNA)

A biopsy technique using a thin needle to extract cells; it collects individual or small clusters of cells without preserving tissue architecture.

core needle biopsy

It uses a larger needle to obtain a cylindrical tissue sample, preserving tissue architecture and allowing for better diagnostic evaluation.

incisional biopsy

removes part of a lesion

excisional biopsy

removes the entire lesion with surrounding tissue.

squash technique

A tissue sample is compressed with a coverslip to spread cells; often used with supravital staining for cytological evaluation.

xylene

It is miscible with both alcohol and paraffin wax, making it ideal for replacing alcohol and preparing tissue for embedding.

Rapid penetration, long shelf-life, compatibility with stains, preservation of tissue architecture, inactivation of enzymes and pathogens, and support for macromolecule recovery.

What are key properties of an ideal fixative?

embedding

To orient and support tissues in a solid medium (e.g., paraffin) for sectioning and microscopic analysis.

curved ribbons (trim block), thick/thin sections (adjust clearance angle), chatter (soften tissue), and incomplete sections (reprocess or re-embed)

What are common problems during paraffin sectioning and their solutions?

3-aminopropyltriethoxysilane (APES)

What adhesive is best for cytology or protein-rich specimens?

3-aminopropyltriethoxysilane (APES)

forms covalent bonds and ensures permanent tissue adherence.

stains nuclear material blue/purple when oxidized to hematein and used with a mordant (e.g., aluminum or iron salts).

How does hematoxylin stain tissues?

metachromasia

When a dye (like toluidine blue) stains tissue components a different color than its own due to molecular interactions (e.g., cartilage stains reddish-purple instead of blue).

Fixation (10% buffered formalin), dehydration (gradual alcohol series), clearing (xylene/toluene), impregnation (paraffin), and embedding (paraffin for 3 hours)

Describe the standard manual processing protocol for tissues.

Custom programs, faster processing, reduced fume exposure, improved reagent handling, and start-time delays.

What are the advantages of automated tissue processors?

teasing or dissociation

It involves separating cells in NSS/LRS with needles and examining them live on a wet mount.

Live cell evaluation (e.g., mitotic figures)

Pros of teasing or dissociation

Not permanent; cannot be preserved

Cons of teasing or dissociation

touch/impression smear?

A tissue is gently pressed onto a slide, transferring surface cells for analysis without disrupting intercellular architecture.

For thick secretions (e.g., sputum, blood, serous fluids); allows cytologic evaluation either fresh or fixed.

In what situations is the smear method particularly useful?

streaking

Involves direct zigzag motion on a slide to spread material without prior teasing—used when sample is already loose.

spreading

uses a needle to create a moderately thick film, better preserving cellular relationships; used for mucoid secretions.

microtome

It sections paraffin-embedded tissue blocks into thin slices for mounting on slides.

Rotary (Minot)

Block moves, used for routine tissue.

Base Sledge

Knife moves, good for large blocks and hard tissue (e.g., brain, eye).

flotation

Tissue ribbons are floated in a water bath (10°C below wax melting point) to flatten before mounting.

Chatter: Soften tissue, fix angle
Sections rolling: Use sharper blade, adjust angle
Sections not sticking: Improve adhesive or mounting

What are common issues in microtomy and their fixes?

section adhesives

Substances like albumin or APES used to help tissue sections adhere to slides, especially during staining.

Nuclei (blue/purple).

what does hematoxylin stain

Cytoplasm and connective tissue (pink/red).

what does eosin stain

mordants

Metal salts (e.g., aluminum, iron) that bind hematein to tissues, allowing nuclear staining.

Binding site availability
Dye properties
Reaction rate and duration
Reagent loss
Metachromasia (e.g., toluidine blue turns purple on mast cells)

What factors influence stain uptake?

Feulgen reaction

A specific DNA stain; involves acid hydrolysis to expose aldehyde groups, stained magenta by Schiff’s reagent.

special stains

To highlight specific tissue components (e.g., lipids, fibers, microorganisms) not easily visualized with H&E.