TOPIC IV A: PARAFFIN SECTION

Paraffin Section

A method of tissue processing in which tissues are thinly cut or sliced and embedded in paraffin wax.

Dehydration

The process of removing water from fixed tissue by replacing it with alcohol.

Clearing

The process of dissolving alcohol from dehydrated tissue using a clearing agent, such as xylene, to prepare it for embedding.

Infiltration

The process of impregnating cleared tissue with paraffin wax to provide support, preserve tissue structure, and enable precise sectioning.

Embedding

The final step in tissue processing, in which the infiltrated tissue is placed in a mold with the embedding medium (paraffin wax) and allowed to solidify.

Paraffin Wax

The most common embedding medium used in routine tissue processing, which has advantages such as preserving tissue structure and enabling precise sectioning.

Paraffin Wax Substitute

Alternative embedding mediums, such as Paraplast, Embeddol, Ester Wax, and Water Soluble Waxes, used in special cases where paraffin wax may not be suitable.

Gelatin

A substance used as an embedding medium in double embedding techniques.

Agar

Another substance used as an embedding medium in double embedding techniques.

Double Embedding

A technique in which tissues are embedded in two different mediums, such as paraffin wax and gelatin or agar, to achieve specific objectives.

Tissue Marking Inks

Inks used to mark tissues for identification purposes during tissue processing.

Calcified Tissues

Tissues that contain calcium deposits, which require special decalcification techniques before processing.

Decalcification

The process of removing calcium deposits from calcified tissues using decalcifying agents.

Decalcifying Agents

Substances, such as strong mineral acids, weaker organic acids, and chelating agents, used to dissolve calcium deposits in calcified tissues.

Surface Decalcification

A technique used to selectively remove calcium deposits from the surface of calcified tissues.

Factors Affecting Decalcification

Various factors, such as tissue density, type of decalcifying agent, and duration of decalcification, that can influence the effectiveness of the decalcification process.1. Paraffin:A substance that becomes solid at room temperature and is used as an embedding reagent in histology to provide consistency in supporting tissue during cutting.

Vacuum and pressure

Techniques used to remove air from reagents and allow faster penetration rate during the dehydration process in histology.

Dehydration

The process of removing water from tissue samples after fixation, in preparation for impregnation with wax. It involves the gradual substitution of water with organic solvents, such as alcohol.

Wax

A hydrophobic substance used in histology that hates water, thus the need to remove water first during the dehydration process.

Bound water

Water molecules that are still present and bound to cellular molecules in tissue samples during the dehydration process.

Drying

The complete exhaustion of moisture, including the removal of bound water, resulting in tissue samples being completely dried out.

Alcohol

Considered the best dehydrating agent in histology, commonly used to gradually remove free water from tissue samples.

Absolute ethanol

The final concentration of alcohol used in the dehydration process to achieve complete dehydration of tissue samples.

Acetone

A rapid-acting dehydrating agent used for urgent biopsies, but not recommended for routine purposes due to poor penetration and brittleness it can cause.

Dioxane

An excellent dehydrating and clearing agent that is miscible with xylene, water, alcohol, and paraffin wax. It causes less shrinkage compared to alcohol but can produce poor tissue ribbons.

Cellosolve (Ethylene glycol monoethyl ether)

A dehydrating agent that is rapid and allows tissues to be stored for months without distortions, but it is combustible, toxic, and expensive.

Triethylphosphate

A dehydrating agent used to dehydrate smears and sections following certain stains, causing very little distortion and hardening of tissue.

Tetrahydrofuran

A dehydrating agent that is miscible with paraffin wax but is toxic when inhaled or ingested, causing nausea and headache.1. Paraffin:A substance used in histology to dehydrate tissues without causing shrinkage or distortion.

Clearing agent

A substance used to remove alcohol from tissues during the histological process, should be miscible with paraffin and the preceding alcohol.

Xylene

A commonly used clearing agent in histology that is miscible with alcohol and rapidly removes dehydrating agents, but is toxic and can cause various health disorders.

Toluene

A clearing agent that is more tolerant of small amounts of water than xylene, but is more toxic and expensive.

Benzene

A clearing agent that penetrates and clears tissues rapidly, but is carcinogenic and not commonly used nowadays.

Chloroform

A clearing agent that is slower in action than xylene and causes less brittleness, but is not a good cleaning agent and does not make tissues translucent.

Cedarwood oil

A clearing agent used for both paraffin and colloidin sections, recommended for CNS tissue and cytological studies, but requires two changes to clear solutions and produces crystals with acetic-alcohol fixed tissues.

Aniline oil

A clearing agent used for clearing embryos and delicate specimens.

Clove oil

A clearing agent that is slow and causes brittleness, but removes anilin dyes.

Carbon tetrachloride

A clearing agent similar to chloroform, but toxic.

Tetrahydrofuran

A non-toxic clearing agent with an offensive odor.

Dioxane

A clearing agent that can be transferred directly after fixation, but is toxic to the liver.

Limonene

A clearing agent found in citrus pills.

Coconut oil

A clearing agent that solidifies at lower temperatures.

Infiltration

The process of replacing the clearing agent with a medium to fill tissue cavities and provide firm consistency to the specimen, allowing for thin sections to be cut without damage or distortion to the tissue and its cellular components.1. Manual Infiltration:The process of infiltrating tissue using an infiltration oven, commonly used in histology labs.

Automatic Tissue Processor

An automated machine used for tissue processing, which includes a vacuum feature for efficient infiltration.

Vacuum Processor

A machine used for tissue processing that utilizes vacuum to aid in tissue infiltration.

Embedding

The process of placing infiltrated tissue into a mold and positioning it with a medium, such as paraffin wax.

Tissue Block

The final product of embedding, where the infiltrated tissue is solidified within the embedding medium.

Paraffin Wax

The most common and widely used medium for routine tissue embedding, composed of a mixture of solid hydrocarbons.

Gelatin

An embedding medium often used in conjunction with other mediums, not typically used as a standalone embedding medium.

Agar

An embedding medium often used in conjunction with other mediums, not typically used as a standalone embedding medium.

Plastic

An embedding medium often used in conjunction with other mediums, not typically used as a standalone embedding medium.

Paraplast

A paraffin wax substitute composed of highly purified paraffin and synthetic plastic polymers.

Embeddol

A paraffin wax substitute used for tissue embedding.

Ester Wax

A paraffin wax substitute used for tissue embedding.

Water Soluble Waxes

Paraffin wax substitutes that are soluble in water.

Advantages of Paraffin Wax

Simplest and most common medium for routine embedding, solid at room temperature, can be purchased with different melting temperatures, and allows for good tissue-wax adhesion.

Disadvantages of Paraffin Wax

Can cause damage to tissue if left in the oven for too long, hardness and shrinkage of tissue can occur with prolonged infiltration, and not recommended for fatty tissues due to the removal of fat during processing.1. Paraffin Section:A method of tissue preparation where the tissue is impregnated with paraffin wax to make it easier to cut thin sections for microscopic examination.

EMBEDDOL

A synthetic wax similar to paraplast, used in paraffin sectioning. It has a melting point of 56-58 °C and is less brittle and compressible than paraplast.

Ester Wax

A type of wax with a lower melting point (46-48 °C) and harder consistency than paraffin. It is suitable for heavy-duty microtome use and can be performed without prior clearing of the tissue.

Water Soluble Waxes

Plastic polymers, mostly polyethylene glycols, incorporated in paraffin to improve adhesion, hardness, and plasticity. The most commonly used is Carbowax, which is soluble in and miscible with water and does not require dehydration and clearing of tissue.

Gelatin

A rarely used embedding medium with a low melting point (35-40 °C). It is suitable for histochemical and enzyme studies, miscible with water, and used for friable tissues. It does not require dehydration and clearing.

Agar Gel

An embedding medium used for friable and fragmented tissue, such as cytology and endometrial curettings. It does not provide good support for section cutting but is suitable for double embedding.

Double Embedding

A technique where tissue is infiltrated first with a supporting medium to facilitate cutting of large blocks of tissues. It is now considered obsolete and not recommended.

Embedding Arranged in the Mold with the Medium

The orientation of different types of tissues in the embedding mold, such as tubular structures, skin, nails, epithelial tissue, and multiple layers.

Mold Embedding System

Different types of molds used for embedding, including peel-a-way plastic molds, plastic ice trays, and paper boats. Each has its advantages and is suitable for different purposes.

Leuckart or Dimmock Embedding Irons

Metal tools consisting of adjustable L-shaped strips used for routine processing in embedding.

Embedding Rings or Cassettes

Special steel base molds fitted with plastic embedding rings that serve as block holders during cutting. They are commonly used in modern laboratories.

Compound Embedding Unit

A device used for embedding tissues, typically consisting of a heated plate and a mold holder. It provides controlled temperature and pressure for embedding.1. Embedding Agent:A substance used to embed tissues for sectioning that has specific characteristics such as solubility, stability, melting point, homogeneity, non-toxicity, translucency, and suitability for sectioning.

Tissue Marking Inks

Inks used to mark and identify tissues, typically India ink or silver nitrate, that remain on the surface and do not penetrate too far, do not react with stains, and are visible both macroscopically and microscopically.

Calcified Tissues

Bones or hard deposits in soft tissues that require decalcification before sectioning.

Decalcification

The process of removing calcium ions from calcified tissues to make them soft enough for sectioning.

Decalcifying Agents

Substances used to remove calcium ions from calcified tissues, including strong mineral acids, weaker organic acids, and chelating agents.

Nitric Acid

A strong mineral acid commonly used as a decalcifying agent, but can impair staining and affect subsequent staining processes.

Perenyi's Fluid

A decalcifying agent composed of nitric acid, chlorine acid, and absolute alcohol, which is slower than aqueous nitric acid but impairs staining.

Phloroglucin-Nitric Acid

A decalcifying agent composed of concentrated nitric acid and phloroglucin, used for urgent cases but may result in poor nuclear staining.1. Paraffin section:A technique used in histology to prepare tissue samples for microscopic examination.

Weak organic acids

Gentler acids that are slower in action compared to strong mineral acids, and are less likely to interfere with nuclear staining.

Decalcification

The process of removing calcium salts from tissues, commonly used in histology to prepare bone samples for microscopic examination.

Formic acid

A weak acid commonly used for decalcification, particularly suited for bone marrow samples.

Organic acids

Acids such as acetic and formic acid that are better suited for decalcification of bone marrow, but act more slowly on dense cortical bone.

Trichloroacetic acid (TCA)

An acid that has been used for decalcification.

Chelating agents

Substances that capture calcium salts from tissues, facilitating the removal of calcium salt deposits.

Sonication with EDTA

A technique that uses sound waves along with chelating agents to expedite the decalcification process.

Microwave treatment

A technique that uses resins to facilitate further ion exchange during decalcification.

Ion-exchange resins

Resins that allow the migration of ions during decalcification.

Electrophoresis

A technique that can be used for decalcification.

Factors affecting decalcification

Concentration, temperature, agitation, fluid access, size, and consistency of the tissue sample can all affect the decalcification process.

Determining end-point of decalcification

A process that involves removing the sample from the decalcifier, rinsing it, and immersing it back in formalin to determine if decalcification is complete.

Physical test

A test that involves bending, probing, or trimming the sample to determine if decalcification is complete.

Chemical test

A test that involves using ammonium oxalate to detect the formation of calcium oxalate crystals, indicating the presence of calcium.

Surface decalcification

A technique used to remove unexpected calcium deposits from the surface of a tissue sample.

Formol-Nitric acid

A decalcifier formula that consists of concentrated nitric acid, formaldehyde, and distilled water.

Hydrochloric acid

A decalcifier formula that consists of hydrochloric acid and distilled water.

Von Ebner's solution

A decalcifier formula that consists of sodium chloride solution, distilled water, and hydrochloric acid.

Formic acid decalcifier formulas

Various decalcifier formulas that include formic acid, sodium citrate, sodium formate, or formaldehyde.

Trichloroacetic acid decalcifier

A decalcifier formula that consists of trichloroacetic acid and formal saline.

Flaming's fluid

A decalcifier formula that consists of chromic acid, osmium tetroxide, and glacial acetic acid.