Histotechnology: Fixation Fundamentals and Applications

Comprehensive practice questions covering tissue fixation definitions, mechanisms, specific reagents, safety protocols, and troubleshooting based on the provided text.

What is the literal meaning of the term autolysis?

Self-splitting; it refers to enzymes within a cell digesting the cell and destroying its structures.

How does denaturation affect the function of enzymes?

Denaturation changes the 3D structure of the protein; when the structure is changed, the enzymatic activities are lost.

What is the definition of refractive index in the context of tissue fixation?

The ratio of the velocity of light in air to the velocity of light in a liquid or solid medium.

What are the two primary physical methods of fixation mentioned in the text?

Heat fixation and desiccation (air drying).

Define the primary mechanism of nonadditive fixatives like acetone and alcohols.

They act on tissue by dissociating bound water molecules from tissue protein groups, causing the protein to lose solubility and precipitate.

According to Baker, why are noncoagulant fixatives considered inferior for paraffin infiltration?

They create a gel that makes penetration by subsequent processing solutions difficult.

What is the recommended volume ratio of fixative to tissue for adequate fixation?

The fixative volume should be at least $15-20\times$ greater than the tissue volume.

What mathematical law expresses the depth of fixative penetration?

Medawar's law of diffusion: d = k\text{\textsurd}t, where $d$ is the depth of penetration, $k$ is the coefficient of diffusibility, and $t$ is time.

What is the K value (coefficient of diffusibility) for 10\text{%} formalin?

$k = 0.78$ (often rounded to $1$).

In terms of penetration speed, order these ingredients from fastest to slowest: Formaldehyde, Picric acid, Acetic acid, Mercuric chloride.

Formaldehyde, Acetic acid, Mercuric chloride, Picric acid.

What is the difference between a hypertonic solution and a hypotonic solution regarding cell behavior?

In a hypertonic solution, water leaves the cell and the cell shrinks; in a hypotonic solution, the cell swells and may rupture.

Which fixative is preferred for the demonstration of urate crystals?

Nonaqueous fixatives such as absolute alcohol, because urates are water soluble.

What is the chemical composition of commercial stock solutions of formaldehyde?

37\text{%} to 40\text{%} formaldehyde gas by weight in water; this is incorrectly considered 100\text{%} formalin.

Why is methanol added to commercial 37\text{%}-40\text{%} formaldehyde solutions?

To help prevent the formation of paraformaldehyde, which is the long, insoluble polymeric form of formaldehyde.

What is the 'methylene bridge' in the context of formaldehyde fixation?

A cross-linkage formed when an additive aldehyde complex reacts with functional groups in macromolecules.

What causes the formation of formalin pigment (black acid hematin)?

A reaction between the heme part of hemoglobin and formic acid when the pH of the formalin solution drops below $5.5$.

How can formalin pigment be removed from tissue sections?

By treating sections with alcoholic picric acid or alkaline alcohol.

What are the three OSHA permissible exposure levels (PEL) for formaldehyde?

Time-weighted average (TWA): $0.75\text{ ppm}$; Short-term exposure limit (STEL): $2\text{ ppm}$; Action level: $0.5\text{ ppm}$.

Why can techniques using Schiff reagent (like PAS) produce false positives on glutaraldehyde-fixed tissue?

Glutaraldehyde is a dialdehyde; the extra aldehyde group remains free to react directly with Schiff reagent.

What is a major advantage of using Glyoxal as a fixative over formaldehye?

It is much less toxic than formaldehyde and is not a carcinogen.

How must mercury pigment be removed from a tissue section?

Treat the section with iodine to create soluble mercuric iodide, then remove the excess iodide with sodium thiosulfate.

What is the primary role of Osmium tetroxide in electron microscopy?

It is used as a postfixative to chemically combine with and insolubilize lipids, especially in cell membranes.

What is the main safety hazard associated with dry Picric acid?

It is an explosive compound and must contain at least 10\text{%} moisture to be safe.

How do Zinc salts improve immunohistochemical (IHC) results compared to NBF?

Zinc ions hold macromolecules in their native conformation via coordinate bonds, preventing the damaging cross-linkages created by formaldehyde and usually eliminating the need for antigen retrieval.

What are the components of Carnoy solution?

Absolute ethyl alcohol ($60\text{ mL}$), Chloroform ($30\text{ mL}$), and Glacial acetic acid ($10\text{ mL}$).

Which fixative is specifically recommended for the demonstration of chromaffin granules in pheochromocytomas?

Orth solution (a primary dichromate fixative).

What is B-5 fixative commonly used for?

Hematopoietic and lymphoreticular tissue, due to its ability to demonstrate beautiful nuclear detail.

Why does Zenker solution lyse erythrocytes while Helly solution preserves them?

Zenker solution contains glacial acetic acid, which lyses red blood cells, whereas Helly solution uses formaldehyde.

What is a major disadvantage of Bouin solution regarding storage?

Tissue cannot be held in it indefinitely; the maximum fixation time should be less than $24$ hours, or the acidic solution will hydrolyze DNA and extract RNA.

What are the hallmarks of good fixation visible under a microscope?

Well-defined blue nuclear membranes, crisp chromatin patterns, lack of cell shrinkage, and well-preserved cytoplasm staining with eosin.

How can autolysis be prevented in surgical specimens of the uterus or gastrointestinal tract?

Specimens should be opened (or pinned open) upon receipt so the fixative can immediately contact the endometrium or mucosal surface.

What causes the 'nuclear bubbling' artifact?

Incomplete fixation of specimens in formalin before dehydration is begun.

What is the purpose of Michel transport medium?

To transport unfixed tissue (like kidney biopsies for immunofluorescence) over long distances or for several days.

List two methods of removing formalin pigment.

1. Absolute alcohol saturated with picric acid. 2. 70\text{%} alcohol containing ammonium hydroxide.

What is the effect of acetic acid on collagen at a pH of $2.5$?

Collagen swells dramatically because links are broken and hydrophilic groups are exposed, absorbing water.