Histopathological and Cytologic Techniques - Tissue Processing

Flashcards covering key histotechnology, tissue processing, fixation, decalcification, dehydration, clearing, embedding, microtomy, and staining terminology from Lesson 7.

Histology

The microscopic study of normal tissues.

Histopathology

The microscopic study of diseased or abnormal tissues.

Autopsy

A detailed examination of a deceased patient, considered the gold standard for determining the cause of death.

Fixation

The first and most critical step in tissue processing, which preserves cells and tissue constituents by cross-linking proteins to prevent degeneration, autolysis, putrefaction, decomposition, and distortion.

Primary Aim of Fixation

To preserve the morphological and chemical integrity of the cell in as life-like a manner as possible.

Secondary Aim of Fixation

To harden and protect the tissue from the physical trauma of further handling.

Additive Fixation

A mechanism of fixation in which the fixative chemical combines with and becomes part of the tissue by forming cross-links or complexes (e.g., Formalin, Mercury, Osmium tetroxide).

Non-Additive Fixation

A mechanism of fixation where the fixative is not incorporated into the tissue, but stabilizes it by removing bound water (e.g., Alcoholic fixatives).

Microanatomical Fixative

A classification of fixative that permits general microscopic study of tissue structures without altering structural patterns or normal intercellular relationships (e.g., 10% NBF, Bouin's solution).

Cytological Fixative

A classification of fixative that preserves specific parts and microscopic elements of the cell itself, categorized into nuclear fixatives and cytoplasmic fixatives.

Nuclear Fixative

A cytological fixative containing glacial acetic acid that preserves and enhances nuclear constituents such as chromosomes and chromatin (e.g., Bouin's fluid, Flemming's fluid, Carnoy's fluid).

Cytoplasmic Fixative

A cytological fixative lacking glacial acetic acid, used to preserve cytoplasmic organelles and structures without dissolving them (e.g., Helly's fluid, Orth's fluid, Regaud's fluid).

Histochemical Fixative

A fixative used specifically to preserve chemical constituents present in tissue samples (e.g., 10% Formol Saline, Absolute Ethyl Alcohol, Acetone).

Paraformaldehyde

White crystalline precipitates formed in formaldehyde upon prolonged standing, which can be removed by filtration or by adding $10\text{\%}$ methanol.

Acid Formaldehyde Hematin

Brown to black granular pigment formed when unbuffered formalin reacts with hemoglobin in blood-rich tissues, removable using saturated picric acid or alcoholic potassium hydroxide.

10% Neutral Buffered Formalin (NBF)

The best overall general tissue fixative, buffered with double phosphate to maintain a neutral pH and prevent acid formalin pigment formation; ideal for preserving iron granules.

Dezenkerization

The process of removing black mercuric chloride pigments from tissues fixed in mercurial fixatives by washing with iodine solution followed by sodium thiosulfate.

Brasil's Alcoholic Picroformol Fixative

An excellent picric acid-based fixative recommended specifically for the preservation of glycogen.

Carnoy's Fluid

The most rapid alcohol fixative, requiring $1\text{-}3\text{ hours}$ of fixation time; used for fixing chromosomes, lymph glands, and urgent biopsies.

Secondary Fixation

The process of placing an already fixed tissue specimen into a second, different fixative to achieve further hardening or special preservation effects.

Post-Chromatization

A form of secondary fixation where a primarily fixed tissue is placed in an aqueous solution of $2.5\text{\%}\text{-}3\text{\%}$ potassium dichromate.

Washing Out

The process of removing excess fixative solution, pigments, or chemical artifacts from fixed tissue blocks before further processing.

Decalcification

The process of removing calcium or lime salts from bones and calcified tissues, typically using acid reagents or chelating agents at a recommended fluid-to-tissue ratio of $20:1$.

Perenyi's Fluid

A nitric acid-based decalcifying agent that simultaneously acts as a rapid decalcifier and a tissue softener.

Versene

The powdered preparation of ethylenediaminetetraacetic acid (EDTA), a chelating agent that removes calcium by sequestration.

Dehydration

The tissue processing step aimed at removing water and fixative from tissue using increasing concentrations of alcohol in preparation for impregnation.

Anhydrous Copper Sulfate

A chemical additive added to the final dehydrating alcohol bath that turns from white to blue in the presence of residual water, acting as an indicator for complete dehydration.

Clearing (Dealcoholization)

The step in tissue processing that removes the dehydrating agent using a reagent miscible with both alcohol and paraffin wax, making the tissue translucent.

Xylene

The most common routine clearing agent; fast-acting ($30\text{-}60\text{ minutes}$) but makes tissues excessively hard and brittle if exposed for longer than 3 hours.

Impregnation (Infiltration)

The processing step where clearing agents are removed and replaced by a supporting medium, such as melted paraffin wax, to fill tissue cavities and internal spaces.

Automatic Tissue Processor

An automated machine (such as an Autotechnicon) that sequentially transfers tissue cassettes through fixing, dehydrating, clearing, and infiltrating reagents under continuous agitation.

Vacuum Embedding

The process of wax impregnation under negative atmospheric pressure ($400\text{-}500\text{ mmHg}$) in a specialized vacuum chamber to accelerate penetration and remove trapped air bubbles.

Paraplast

A paraffin wax substitute composed of highly purified paraffin and synthetic plastic polymers, having a melting point of $56\text{-}57\text{ }^\text{o}\text{C}$.

Carbowax

A water-soluble polyethylene glycol embedding medium that does not require prior tissue dehydration or clearing.

Embedding (Casting/Blocking)

The process of placing an impregnated tissue specimen into a precise orientation within a mold filled with liquid embedding medium and allowing it to solidify.

Leuckhart's Embedding Molds

Adjustable embedding molds consisting of two L-shaped brass strips placed on a flat metal plate.

Compound Embedding Unit

An embedding system consisting of a series of interlocking metal plates forming multiple compartments to embed several tissue specimens simultaneously.

Plastic Embedding Rings

Embedding molds where a plastic ring is fitted over the specimen on a base plate, filled with wax, and used directly as the block holder during cutting.

Metal Base Molds

Reusable stainless steel embedding base molds fitted with plastic embedding cassettes.

Peel Away Molds

Disposable thin plastic embedding molds available in three sizes that are peeled off one by one after wax solidifies.

Paper Boats

Inexpensive, handmade paper molds used traditionally for embedding celloidin and paraffin blocks to match any custom tissue size.

Trimming

The process of removing excess wax from a solidified tissue block to shape it into a four-sided prism or truncated pyramid and expose the tissue face.

Microtomy

The process of cutting processed, embedded tissues into uniformly thin sections or ribbons using a microtome.

Honing

The first knife-sharpening stage that removes nicks and chips from the microtome blade using a honing stone in a heel-to-toe direction ($20\text{-}30\text{ double strokes}$).

Stropping

The second knife-sharpening stage that removes wire-edge burrs and polishes the microtome knife using a leather paddle strop in a toe-to-heel direction ($40\text{-}120\text{ double strokes}$).

Rotary Microtome

The most common microtome used in routine histopathology for cutting paraffin sections, invented by Minot in 1885–1886; operates via a handwheel connected to a pawl and ratchet feed mechanism.

Cryostat

A refrigerated cabinet containing a microtome used to cut unfixed, unembedded frozen tissue sections rapidly during intraoperative surgical consultations.

Floatation Water Bath

Laboratory equipment filled with water and maintained at approximately $10\text{ }^\text{o}\text{C}$ below the paraffin melting point ($\text{around } 46\text{ }^\text{o}\text{C}$), used to float, stretch, and flatten tissue sections.

Direct Staining

A staining method that uses simple aqueous or alcoholic dye solutions directly on tissue sections without a mordant or accentuator (e.g., Methylene blue, Eosin).

Indirect Staining

A staining method that requires an accentuator or mordant to intensify and facilitate the binding action of the dye to the tissue.

Progressive Staining

A staining method where dye is applied in controlled increments until the desired intensity is reached without applying a decolorizer.

Regressive Staining

A staining method where tissue is intentionally overstained first, and excess dye is subsequently differentiated/decolorized (e.g., Harris' hematoxylin).

Orthochromatic Staining

Staining in which the tissue component takes on the exact same color as the dye solution applied.

Metachromatic Staining

Staining in which tissue elements stain a distinct color different from the original color of the applied dye solution (e.g., Methyl violet, Bismarck brown).

Supravital Staining

Staining of living cells immediately after their removal from the living body (e.g., Neutral red, Janus green for reticulocyte counting).

Ripening

The process of oxidizing hematoxylin into its active staining substance, hematin, either naturally by exposure to air/sunlight or artificially using chemical oxidizers.

Poly-L-Lysine

A general-purpose glass slide adhesive widely used in immunohistochemistry that produces no background staining.

Coplin Jar

A glass staining jar equipped with internal vertical grooves designed to hold 5 to 9 slides during staining procedures.

Acid Alcohol

A differentiator solution used in H&E staining to remove excess hematoxylin and improve nuclear-cytoplasmic contrast.

Ammonia Water

A blueing agent used in H&E staining to shift the initial red-stained hematoxylin in nuclei to a permanent blue lake.

Canada Balsam

A natural resinous mounting medium extracted from Abies balsamea with a refractive index of 1.524 that gradually darkens and yellows over time.

Ringing

The procedure of sealing the outer edges of a coverslip using specialized ringing media (e.g., Kronig Cement) to prevent fluid evaporation and coverslip detachment.