Histology lab

Fixation Fixation

Stops autolytic and putrefactive processes in tissues to maintain basic morphology.

Physical Fixatives

Include heat and microwaves, often used in microbiology for bacterial cultures.

Chemical Fixatives

The most commonly used fixatives for histological investigations. They can be coagulants (contract relationships with denatured tissue proteins like acetic acid, ethyl/methyl alcohol) or non-coagulants. They can also be additives (contract chemical relationships with tissues) or non-additives.

Fixation by Perfusion

An ideal technique that fixes tissues at the moment of death, carried out by circulating fixative liquid in the vascular district of a tissue, but is limited to experimental animals.

Fixation by Immersion

The most common method in routine histological practice, involving immersing the sample in fixative liquid as quickly as possible, maintaining an adequate volumetric relationship (20:1 - 10:1) between fixative and tissue.

Fixation by Vapors

Uses vapors from heated fixatives, typically for cytological smears due to low penetration power.

Factors Influencing Fixation

Include pH (satisfactory between 6-8, cells undergoing anoxia lower pH), temperature (increases speed, but high T° risk accelerating autolytic processes), penetration power (varies per fixative), osmolarity (should be hypertonic, 400-450 mosm), fixative concentration, and timing.

Formaldehyde (Formalin)

The most used fixative, a non-coagulative, additive fixative that forms cross-links with tissue proteins. It has no effect on lipids (which can be lost if fixation is prolonged) and dissolves glucides. Neutral buffered formalin is the most common type. It is low cost, manageable, and hardens tissues, but is irritating and a carcinogen, and excessive fixation can damage nuclei and nucleic acids. Correct fixation time is 12-24 hours, depending on sample size.

Glutaraldehyde

An oily liquid, a non-coagulant additive fixative that preserves lipids and is most used in electron microscopy, preserving cellular structures like nuclei, mitochondria, and ribosomes. Drawbacks include low penetration, need for low temperatures, and high cost.

Osmium Tetroxide

A volatile, water-soluble fixative primarily used for electron microscopy and as an excellent cytological fixative due to its oxidizing action which impregnates tissue. It reacts with unsaturated lipids, causing blackening. Drawbacks include low penetration, loss of protein, high cost, and harmful vapors.

Ethyl Alcohol

A non-additive coagulative fixative that subtracts the mantle of hydration from proteins, causing coagulation, and is a solvent of lipids. It preserves some enzymatic activities and glycogen but extracts lipids and can cause nuclear detail distortion.

Picric Acid

A yellowish, toxic crystal, an additive coagulant protein fixative. It is widely used in histological techniques as a fixative, mordant, differentiating agent, and stain agent. Drawbacks include coarctation and hardening of tissue, making inclusion friable.

Mercury Chloride (HgCl2)

A white crystalline additive coagulant that provides good conservation of cellular structures. Drawbacks include tissue coarctation and precipitate formation.

Processing and Embedding

The phase of tissue preparation for light microscopy that follows sampling, involving dehydration, clarification, and impregnation.

Embedding

The terminal phase of processing, where fragments are definitively "included" in a cooled medium (like paraffin) to give them consistency for sectioning and permanent storage.

Purpose of Processing & Embedding

To include specimens in a hard, compact medium that makes them sectionable into 4-5 µm thick slices.

Dehydration

Removes fixative and water from tissues, facilitating cutting, typically using increasing concentrations of ethyl alcohol. Excessive dehydration can harden tissues too much.

Clarification (Diaphanization)

The transition phase where dehydrating liquid (alcohol) is replaced with a liquid miscible with the inclusion medium (e.g., paraffin), making the bioptic fragment translucent. Common clarifying agents are hydrocarbons like xylol.

Impregnation

The phase where the clarifying liquid is replaced by the embedding medium (e.g., paraffin), saturating tissue and cellular cavities to provide structural support.

Paraffin

The most routinely used embedding medium for light microscopy, a mixture of saturated hydrocarbons. It is insoluble in water but soluble in xylene, toluene, or chloroform. It is economical and easy to handle. Resin

Sampling

The process carried out by the pathologist (after macroscopic analysis) to select representative parts of a specimen.

Inclusion Inclusion

The same as embedding, where fragments are solidified within a medium like paraffin for sectioning and long-term storage.

Sectioning Sectioning

The process of obtaining thin slices of tissue (e.g., 4-5 µm thick for paraffin blocks) for microscopic examination.

Microtomes

Precision instruments used to obtain thin sections for microscopic viewing. Sledge Microtome

Minot Rotary Microtome

Has a fixed blade, with the piece holder moved vertically towards the cutting edge by a flywheel, allowing precise thickness selection.

Cryomicrotome

A rotary microtome placed in a refrigerated chamber (-20/-40°C) that allows dissection of unfixed, unprocessed samples by freezing the tissue's water. Used for rapid diagnosis (intraoperative exams), histochemistry (e.g., lipid stains), and immunohistochemistry.

Staining

A process to classify and highlight tissue structures for microscopic analysis.

Acid Stains

Neutral Stains

Result from the union of an acidic dye with a basic one.

Natural Stains

Derived from animals (carmine) or plants (hematoxylin).

Artificial Stains

Mordant

Used before or with the stain to make the bond with the substrate more stable, often powerful oxidants or alums.

Hematoxylin Eosin (H&E)

The most used routine staining. Hematoxylin stains nuclei blue/black, while eosin stains cytoplasm and connective tissue fibers pink/orange/red.

Hematoxylin

Derived from camping wood, its oxidation product Hematein has coloring capacity. Hematein needs a mordant (e.g., aluminum, iron) to stain negatively charged nuclei.

Aluminum Hematoxylins

The most used type in H&E, causing nuclei to stain "purple" initially, then blue/black after toning. Examples include Ehrlich's, Mayer's, and Harris's. Eosin

Trichrome Stains

Best highlight collagen fibers, using a nuclear dye (iron hematoxylin) and two acidic dyes with different penetration speeds. Examples include Masson's trichrome and Van Gieson trichrome.

Silver Stains

Used for reticular and nervous fibers (which appear black), exploiting their argyrophilia (ability to bind and reduce silver salts).

Elastic Fiber Stains

PAS Staining

Used for carbohydrates like glycogen and mucins.

Alcian Blue

Mucicarmine

Method for detecting acid mucopolysaccharides of epithelial nature (mucins), staining them from dark pink to red.

Reporting Pathologist's Aims

Achieving a correct diagnosis, achieving the correct prognosis, and establishing the correct therapy of diseases.

Diagnostic Report

Acts as a bridge between basic analysis and the clinical part, enabling the clinician to perform follow-up and therapy.

Immunohistochemistry (IHC)

A set of techniques that identifies substances in normal and pathological tissues through an antigen-antibody reaction, highlighted by a visible marker.

IHC Applications

Diagnostic: Identifying nature/origin of lesions (epithelial, muscular, nervous), typing lymphomas/leukemias, defining glomerulonephritis pathogenesis, typing malignant neoplasms; Prognostic: Assessing proliferative activity (Ki-67/Mib-1), identifying genetic mutations (p53, c-erbB-2), evaluating hormone receptors (ER, PgR), and microvessel count;

Therapeutic: Guiding therapies based on hormone receptor status (tamoxifen), C-erbB-2 status (trastuzumab), CD-117 (Glivec), EGFR (cetuximab, erlotinib), VEGF (Bevacizumab), chemoresistance in lung cancer (p53), and immunotherapy (PD-L1).

Main IHC Techniques

Immunoenzymatic (classical IHC), Immunofluorescence, and Immunogold. Immunoenzymatic Techniques

Immunoperoxidase

Uses peroxidase as the enzyme, often with DAB (Diaminobenzidine tetrachloride) as a chromogen to produce a brown color. Advantages of Immunoperoxidase

Direct Method (IHC)

Applies an antibody conjugated with peroxidase directly to the substrate; not very sensitive.

Indirect Method (IHC)

Uses two antibodies: a primary (specific to antigen) and a secondary (conjugated with peroxidase, directed against the primary), leading to a more intense image.

PAP Method

A more sensitive and specific method than the indirect method, using a primary antibody, an excess of secondary antibody, and an immune complex of peroxidases-antiperoxidases (PAP).

Avidin Biotin System

Exploits the high affinity between avidin (or streptavidin) and biotin, allowing for sensitive and specific signal amplification. Antigen Retrieval

Controls (IHC)

Negative controls (omission or substitution of primary antibody) guarantee specificity, while positive controls (known positive section, internal/external) confirm antigen presence and method sensitivity.

Polyclonal Antibodies

Produced by immunizing an animal, leading to numerous plasma cell clones that produce antibodies with slight and different specificity, potentially cross-reacting with other tissue molecules. Monoclonal Antibodies