Histotechnology: Fixation Fundamentals and Applications

Fundamental Definitions and Purposes of Fixation

• Fixation: The process of transforming soluble cellular components into insoluble substances to prevent their loss during subsequent tissue processing. This is achieved primarily through protein denaturation.

• Purposes of Fixation:

  • To prevent autolysis and putrefaction.

  • To maintain proper relationships between cells and extracellular substances (collagen, reticulin, elastin).

  • To bring out differences in refractive indexes and increase contrast between tissue elements.

  • To render cell constituents insoluble, specifically stabilizing tissue proteins.

  • To make tissue firmer for easier gross dissection and collection of thin sections.

• Autolysis: Literally "self-splitting"; a process where cellular enzymes digest the cell, destroying the structures of nuclei, cytoplasm, organelles, and membranes. This occurs rapidly in enzyme-rich tissues like the liver, pancreas, and brain.

• Putrefaction: Tissue breakdown caused by bacterial attack.

• Denaturation: The change in the $3\text{-dimensional}$ ($3D$) structure of macromolecules, usually proteins or nucleic acids. When the shape (conformation) of a protein enzyme is changed, its biological activity is lost and its solubility changes.

• Classical Definition: Collectively, fixatives are said to kill, penetrate, and harden tissue. Hardening provides the rigidity needed for the tissue to withstand processing chemicals.

Fixative Actions and Physical Methods

• Mordant Action: Fixatives make tissue more receptive to dyes and often act as mordants to link the dye to the tissue.

• Heat Fixation: A physical method where added energy breaks internal bonds, causing molecules to unwind and form new stabilized bonds (analogous to cooking an egg).

• Microwave Fixation: Uses nonionizing radiation. Dipolar (charged) molecules like water or polar protein side chains oscillate at a rate of $2.5 \times 10^{9}\,\text{Hz}$ ($2.5\,\text{billion times per second}$), creating molecular friction and instantaneous heat.

  • Temperature Ranges: Critical ranges are $50^{\circ}\text{C}\text{--}68^{\circ}\text{C}$ or $45^{\circ}\text{C}\text{--}55^{\circ}\text{C}$. Exceeding $68^{\circ}\text{C}$ causes pyknotic, overstained nuclei, loss of enzyme activity/antigenicity, and red cell lysis.

• Desiccation: A physical method of fixing protein through air drying, most frequently used for touch preparations (e.g., Wright staining).

Chemical Classification of Fixatives

• Additive Fixatives: These reagents chemically link or add themselves to tissue macromolecules, potentially changing the electrical charge at the attachment site.

  • Examples: Mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, and zinc salts.

• Nonadditive Fixatives: Predominantly organic compounds that act without chemically combining with the tissue. Their primary mechanism is dissociating bound water molecules from tissue protein groups, causing them to precipitate.

  • Examples: Acetone and alcohols.

• Coagulant Fixatives: These establish a network in the tissue that allows solutions to readily penetrate into the interior.

  • Examples: Zinc salts, mercuric chloride, cupric sulfate, ethyl alcohol, methyl alcohol, acetone, and picric acid.

• Noncoagulant Fixatives: These act by creating a transparent gel that makes subsequent penetration by processing reagents difficult.

  • Examples: Formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, potassium dichromate, and acetic acid.

Factors Affecting Fixation Quality

• Temperature: Increasing temperature increases the rate of fixation and the rate of autolysis. Room temperature ($25^{\circ}\text{C}$) is often preferred for ultrastructural preservation over traditional refrigerator temperatures ($0^{\circ}\text{C}\text{--}4^{\circ}\text{C}$). Fixative temperatures up to $45^{\circ}\text{C}$ have little negative effect on morphology.

• Size: Reagent penetration depends on tissue thickness. For routine processing, sections should be $\leq 3\,\text{mm}$ thick. Large specimens (colon, solid organs) must be opened or "bread-loafed" to allow fixative access to the interior.

• Volume Ratio: The fixative volume should be at least $15\text{--}20 \times$ greater than the tissue volume. Inadequate volume compromised the fixative composition through the displacement of water and serum proteins.

• Time:

  • Ischemic Time: The interval between loss of blood supply and placement in fixative. Should be minimized to prevent autolysis.

  • Duration: Formalin requires at least $6\text{--}8\,\text{hours}$ of action. HER2 guidelines for invasive breast cancer require fixation in $10\%\,\text{NBF}$ for a minimum of $6\,\text{hours}$ and a maximum of $72\,\text{hours}$.

• Penetration Rate: Described by the Medawar law of diffusion: $d = k\sqrt{t}$, where $d$ is depth, $t$ is time in hours, and $k$ is the coefficient of diffusibility (Medawar constant).

  • $K$ values: $10\%$ formalin has a $K \approx 0.78$.

  • Order of speed (fastest to slowest): Formaldehyde, acetic acid, mercuric chloride, methyl alcohol, osmium tetroxide, picric acid.

• pH: Influences reactivity and ultrastructure preservation.

• Osmolality:

  • Body fluids measure $\sim 340\,\text{mOsm}$ ($0.3\,\text{Osm}$).

  • Hypertonic: Cell shrinks as water leaves (solution has more particles than cytosol).

  • Hypotonic: Cell swells/ruptures as water enters (solution has fewer particles than cytosol).

  • Isotonic: Equal concentrations; normal saline ($0.9\%\,NaCl$) is often used as a holding solution.

Reactions of the Cell with Fixatives

• Nucleus:

  • Contain DNA, RNA, and histones.

  • Formaldehyde does not react with DNA/RNA at room temperature; reactions start at $45^{\circ}\text{C}$ for RNA and $65^{\circ}\text{C}$ for DNA.

  • Acetic alcohol and Carnoy solution are preferred for nucleic acids.

• Proteins:

  • Primary (sequence), Secondary (hydrogen bonds, alpha helix, beta sheet), Tertiary ($3D$ shape/R group interactions), and Quaternary (multiple chains).

  • Fixatives stop enzyme degradation by changing protein structure.

• Lipids: Only osmium tetroxide and chromic acid insolubilize lipids for paraffin processing. Osmium reacts with double bonds of unsaturated lipids.

• Carbohydrates: Mostly lost in aqueous fixatives unless trapped within crosslinked protein networks.

Common Fixative Reagents

Acetic Acid ($CH_{3}COOH$)

• Properties: Concentrated form is "glacial acetic acid" (freezes at $16.6^{\circ}\text{C}$).

• Actions: Does not fix lipids or carbohydrates; penetrates very rapidly; leaves tissue soft.

• Specific Uses: Precipitates nucleoproteins and DNA. Added to compound fixatives to counteract the shrinkage of other ingredients.

• Disadvantages: Increases protein swelling ($pH < 4.0$), specifically collagen at $pH\,2.5$. Lyses red blood cells (RBCs).

• Safety: PEL of $10\,\text{ppm}$. Causes severe burns; add acid to water.

Formaldehyde ($CH_{2}O$)

• Properties: Colorless gas, commercial stock is $37\%\text{--}40\%\,\text{formaldehyde}$ (often incorrectly called $100\%\,\text{formalin}$).

• Calculation: $10\%\,\text{formalin}$ is a $1:10$ dilution of stock ($3.7\%\text{--}4.0\%\,\text{formaldehyde}$).

• Chemistry: Forms methylene hydrate ($HO\text{--}CH_{2}\text{--}OH$). Paraformaldehyde is the insoluble polymeric form.

• Actions: Noncoagulant and additive. Two-stage reaction: (1) Addition of formaldehyde to functional groups (amines, purines), (2) Formation of methylene bridges (cross-linking).

• Pigment: Formalin pigment (black acid hematin) forms at $pH < 5.5$ in blood-rich tissues. Prevented by buffering to neutral $pH$. Removed by alcoholic picric acid or alkaline alcohol.

• Safety: Carcinogen. OSHA Pel TWA: $0.75\,\text{ppm}$; STEL: $2\,\text{ppm}$; Action Level: $0.5\,\text{ppm}$.

Glutaraldehyde ($C_{5}H_{8}O_{2}$)

• Properties: Dialdehyde with two reactive groups.

• Actions: Penetrates slowly/poorly but fixes as it penetrates. Preserves ultrastructure best among aldehydes.

• Disadvantages: Leaves a free aldehyde group that reacts with Schiff reagent, causing false-positive PAS (periodic acid-Schiff) stains. Overhardens tissue.

• Safety: ACGIH threshold limit value of $0.05\,\text{ppm}$. Sensitizer and irritant.

Glyoxal ($C_{2}H_{2}O_{2}$)

• Properties: Smallest dialdehyde, supplied as $40\%\,\text{aqueous solution}$.

• Actions: Rapid ($4\text{--}6\,\text{hours}$). Does not give off vapors (no odor). Not a carcinogen.

• Disadvantages: unsatisfactory silver staining for Helicobacter pylori; may leach iron or lyse RBCs in some formulations.

Mercuric Chloride ($HgCl_{2}$)

• Actions: Powerful protein coagulant; leaves tissue highly receptive to dyes.

• Pigment: Mercury pigment (crystalline/amorphous brown precipitate). Removed by iodine followed by sodium thiosulfate.

• Safety: Extreme toxicity; affects central nervous system; hazardous waste; corrosive to metals.

Osmium Tetroxide ($OsO_{4}$)

• Actions: Used primarily for electron microscopy (postfixation) to preserve lipids. Makes membranes electron-dense.

• Safety: Vapor is dangerous and fixes the conjunctiva and cornea. OSHA TWA: $0.002\,\text{ppm}$.

Picric Acid ($C_{6}H_{3}N_{3}O_{7}$)

• Actions: Strong coagulant of nucleoprotein; leaves DNA soluble (do not use for nucleic acid stains). Decalcifies small calcium deposits.

• Handling: Must be washed out (usually with $50\%\text{--}70\%\,\text{alcohol}$) to prevent staining deterioration.

• Explosive Hazard: Becomes explosive when dry ($< 10\%\,\text{moisture}$). Keep cap tight and reagent damp.

Zinc Salts ($ZnSO_{4}$)

• Actions: Substitute for mercury; preserves antigenicity. Zinc ions hold macromolecules in native conformation via coordinate bonds.

• Maintenance: Can precipitate in processors; remove with dilute acetic acid ($5\%\text{--}20\%\,$).

Compound and Combined Fixatives

• B-5 Fixative: Mercuric chloride, sodium acetate, and formaldehyde. Preferred for hematopoietic/lymphoreticular tissue (bone marrow/lymph nodes) for nuclear detail.

• Bouin Solution: Saturated aqueous picric acid ($750\,mL$), formaldehyde ($250\,mL$), glacial acetic acid ($50\,mL$). Best for trichrome stains and GI biopsies. Lyses RBCs.

• Davidson Solution: Ethanol, NBF, glacial acetic acid, water. Used for eyes and testes. Lacks picric acid safety hazards.

• Gendre Solution: Alcoholic Bouin; excellent for carbohydrates (glycogen).

• Hollande Solution: Modification of Bouin with copper acetate. Stabilizes RBCs and granules. Decalcifies bone.

• Orth Solution: Potassium dichromate, sodium sulfate, water, and formaldehyde ($added just before use$). Preferred for chromaffin granules (pheochromocytomas).

• Zamboni (PAF): Paraformaldehyde and buffered picric acid. Stable general-purpose fixative; good for electron microscopy.

• Zenker and Helly:

  • Stock: Mercuric chloride and potassium dichromate.

  • Zenker: Stock + Glacial acetic acid (lyses RBCs, best for muscle striations).

  • Helly: Stock + Formaldehyde (preserves RBCs, unstable solution).

Nonaqueous and Transport Solutions

• Acetone: Nonadditive coagulant. Rapid ($cold, 4^{\circ}C$). Used for enzymes (acid/alkaline phosphatase), rabies diagnosis (brain), and IHC frozen sections. Causes extreme shrinkage.

• Ethyl Alcohol: Coagulant. Preserves water-soluble components (urate crystals in gout, glycogen). Dissolves fat.

• Carnoy Solution: Absolute ethanol ($60\,mL$), chloroform ($30\,mL$), glacial acetic acid ($10\,mL$). Lyses RBCs. Used in cytology. Chloroform is a suspected carcinogen.

• Michel Transport Medium: For transporting unfixed tissue over long distances (e.g., kidney biopsies). Maintain $pH\,7.0\text{--}7.2$. Not for muscle biopsies. Wash in PBS-sucrose before freezing.

Troubleshooting and Hallmarks of Fixation

• Hallmarks of Good Fixation:

  • Crisp chromatin patterns and distinct nuclear membranes.

  • Absence of bubbling, smudginess, or fading.

  • No cell shrinkage or artifactual spaces between cells.

  • Cytoplasm that stains well with eosin.

• Problem: Autolysis: Caused by delayed fixation. Results in loss of nuclear chromatin and epithelial desquamation.

• Problem: Incomplete Fixation:

  • Results in "nuclear bubbling" or smudgy nuclei.

  • Tissue components may separate on the flotation bath.

  • Correction: Increase fixation time, use thinner sections, use agitation, or ensure fixative isn't depleted.

• Ultrastructural Quality (Electron Microscopy):

  • Uniform, undilated space between nuclear membranes.

  • No mitochondria swelling (most sensitive indicator).

  • Regular width of endoplasmic reticulum channels.