Comprehensive Guide to Conventional Tissue Processing and Fixation

Overview of Conventional Tissue Processing and Accessioning

Conventional tissue processing describes the series of steps required to take a tissue specimen from initial fixation to a state where it is completely infiltrated with a suitable histological wax. This process prepares the tissue for embedding and subsequent section cutting using a microtome. The standard sequence of steps in tissue processing includes Fixation, Decalcification, Dehydration, Clearing, Impregnation, Embedding, Trimming, Sectioning, Staining, Mounting, Ringing, and Labelling.

Every specimen requires proper identification and numbering. The accessioning process typically utilizes the code "S" "A" "C". The year of the specimen is usually indicated in two digits followed by the specific accession number assigned to that tissue block.

Fundamentals and Objectives of Fixation

A major objective in pathology is to maintain clear and consistent morphological features within a tissue sample. Fixation was classically defined as the killing, penetration, and hardening of tissues. Current definitions describe fixation as the alteration of tissues through protein stabilization, rendering the tissue resistant to further changes. The most critical reactions for routine histopathology are those that stabilize the proteins.

The primary aim of fixation is to preserve the morphologic and chemical integrity of the cell in a manner as life-like as possible. The secondary aim is to harden and protect the tissue from the trauma of further handling, which facilitates easier cutting during gross examination. Benefits of fixation include allowing thin sectioning by hardening the tissue, preventing autolysis, and inactivating infectious agents, with the notable exception of prion diseases. Fixation also improves the avidity of cells for special stains and reduces the risk of infection during handling. It makes cells resistant to damage and distortion, inhibits bacterial decomposition, and increases the optical differentiation of cells and tissue components. Furthermore, fixatives may act as mordants or accentuators, or they may inhibit specific dyes in favor of others.

Factors and Characteristics of Effective Fixatives

Many factors are involved in the fixation process, including the volume of the fixative, the pH (hydrogen ion concentration), the temperature, the thickness of the tissue section, the osmolality, the concentration of the fixative, the duration of fixation, and the time interval between removal and fixation.

A good fixative must possess several key characteristics: it should be cheap, stable, and safe to handle. It must kill cells quickly and inhibit bacterial decomposition and autolysis. It should produce minimum tissue shrinkage and permit rapid and even penetration. A good fixative must harden the tissue and, while it should ideally be isotonic, it must make cellular components insoluble to hypotonic solutions. Finally, it should permit the application of a wide variety of staining procedures.

Classification of Fixatives by Composition and Action

Fixation methods are divided into physical and chemical methods. Physical methods include heat, wave, and freeze-drying. Chemical fixatives are categorized into four major groups: Aldehydes, Oxidizing agents, Alcohol-based fixatives, and Metallic fixatives. According to composition, fixatives are either simple or compound. Simple fixatives include aldehydes like Formaldehyde and Glutaraldehyde; metallic fixatives such as Mercuric chloride (mnemonic BHZZ), Chromate fixatives (mnemonic CROP), and Lead fixatives; Picric acid (mnemonic BB); Alcohols (mnemonic MINCE); Osmium tetroxide; and Flemming’s Solution (with or without acetic acid).

According to action, fixatives are classified into three types. Microanatomical Fixatives permit general microscopic study of tissue structures without altering structural patterns or normal intercellular relationships. Examples include $10\%$ Formol saline, $10\%$ Neutral buffered formalin, Heidenhain’s Susa, Formol sublimate (formol corrosive), Zenker’s solution, Helly’s solution (Zenker-formol), Bouin’s solution, and Brasil’s solution. Cytological Fixatives preserve specific parts and elements of the cell. These are subdivided into Nuclear Fixatives (containing glacial acetic acid at $pH \leq 4.6$), such as Flemming’s fluid, Carnoy’s fluid, Bouin’s fluid, Newcomer’s fluid, and Heidenhain’s Susa; and Cytoplasmic Fixatives (never containing glacial acetic acid and at $pH > 4.6$), such as Flemming’s fluid without acetic acid, Kelly’s fluid, Formalin with post-chroming, Regaud’s fluid (Muller’s fluid), and Orth’s fluid. Histochemical Fixatives preserve the chemical constituents of cells and tissues, including Formol Saline $10\%$, Absolute Ethyl Alcohol, Acetone, and Newcomer’s Fluid.

Mechanisms and Secondary Processes of Fixation

Fixation involves two basic mechanisms. In one, the chemical constituents of the fixative are taken in and become part of the tissue. In the other, the fixing agent is not taken into the tissue but alters tissue composition to stabilize it and prevent bacterial decomposition. Fixatives are also grouped by their mechanism of action as Cross-linking fixatives, which create covalent chemical bonds between proteins and tissue to anchor soluble proteins to the cytoskeleton, and Non-cross-linking fixatives, which reduce the solubility of protein molecules and disrupt hydrophobic interactions.

Secondary fixation is the process of placing an already fixed tissue into a second fixative to improve the demonstration of a particular substance, enable special staining techniques, or ensure further hardening. This may be done before hydration or on deparaffinized sections before staining. Post-chromatization is a form of secondary fixation where tissues are placed in an aqueous solution of $2.5\%$ to $3\%$ potassium dichromate for $24 \text{ hours}$ to act as a mordant. Washing out is the process of removing excess fixative to improve staining and remove artifacts. Tap water is used to remove excess chromates (from Helly’s, Zenker’s, Flemming’s), excess formalin, or excess osmic acid. $50\%$ to $70\%$ alcohol is used to wash out excess picric acid from Bouin’s solution, and alcoholic iodine is used to remove excessive mercuric fixatives.

Specialized Fixation for Biological Components

Lipids are largely removed during routine preparation, so cryostat or frozen sections must be used for their demonstration. Fixatives with mercuric chloride and potassium dichromate are effective for lipids in cryostat sections, while aldehydes fix phospholipids and digitonin is used for cholesterol under electron microscopy. Carbohydrate fixation generally requires alcoholic fixatives like Rossman’s fluid or cold absolute alcohol for glycogen preservation. Alcoholic formaldehyde is superior to neutral buffered formaldehyde for human skin carbohydrates. Protein fixation typically employs neutral buffered formol saline or formaldehyde vapor for amino acid histochemistry.

For Electron Microscopy (EM), the primary fixatives are Osmium tetroxide, Glutaraldehyde, and Paraformaldehyde, with the procedure performed at $4^{\circ}C$. Karnovsky’s paraformaldehyde-glutaraldehyde is useful for electron histochemistry. Enzyme histochemistry tissues may be fixed in $4\%$ formaldehyde or formal saline overnight, while fresh frozen cryostat sections may use acetone. Immunofluorescence (IF) requires antigenicity preservation; sensitive cases use cryostat sections with brief fixation in absolute methanol or acetone. Immunohistochemistry (IHC) requires perfect fixation to immobilize antigens; while formalin is generally satisfactory, antigen retrieval may be necessary to uncover antigens and ensure antibody access.

Fixation Artifacts and Common Difficulties

Improper fixation leads to various difficulties. Failure to arrest early autolysis is caused by a failure to fix immediately or insufficient fixative. The removal of soluble substances results from the wrong choice of fixative. Artifact pigments, such as formalin pigments, occur due to incomplete washing. Formalin pigments are brown granules found in blood-rich tissues when using unbuffered formalin; they can be eliminated by using neutral buffered formalin or phenol-formalin. These pigments are removed using saturated alcoholic picric acid, ethyl alcohol with ammonia water, or acetone with hydrogen peroxide and ammonia water. Crush artifacts are often found in surgical specimens like liver biopsies, appearing as intense eosinophilic staining at the tissue center due to partial coagulation by ethanol or incomplete wax impregnation. Soft or feather-like tissue consistency results from the loss or inactivation of enzymes, while brittleness and hardness are often the results of over-fixation.

Detailed Review of Aldehyde and Metallic Fixatives

Formaldehyde (Formalin) is produced by the oxidation of methanol. A $10\%$ solution is used, as a pure $40\%$ stock is unsatisfactory. It is easy to prepare, compatible with most stains, and penetrates well, preserving fats, mucin, and glycogen. However, fumes are irritating, and unbuffered solutions reduce staining quality. Formalin waste is recycled by distillation, detoxified commercially, or disposed of by licensed haulers. Variations include $10\%$ Formol-Saline (recommended for CNS and post-mortem tissues, fixation for $24 \text{ to } 48 \text{ hours}$) and $10\%$ Neutral Buffered Formalin (best for tissues with iron pigments). Formal-Corrosive (Formol-Sublimate) contains mercuric chloride and is excellent for many stains but inhibits decalcification. Gendre’s (Alcoholic Formalin) fixes and dehydrates simultaneously, making it ideal for sputum and glycogen. Paraformaldehyde is a polymerized form of formaldehyde used for resin embedding and EM. Glutaraldehyde preserves structures better than formaldehyde and is recommended for EM and enzyme histochemistry at $4^{\circ}C$.

Metallic fixatives include mercuric chloride fixatives, which harden tissues rapidly and provide a greater affinity for acid dyes, making them the choice for tissue photography. Mercuric chloride penetrates poorly and can cause tissue shrinkage. Black mercury deposits are removed using $0.5\%$ iodine in $70\%$ ethanol for $5 \text{ to } 10 \text{ minutes}$ followed by $5\%$ sodium thiosulfate. Heidenhain’s Susa is recommended for tumor biopsies of the skin. Zenker’s Fluid (containing glacial acetic acid) is used for liver and spleen. Zenker-Formol (Helly’s Solution) is excellent for the pituitary gland and bone marrow. B-5 Fixative enhances nuclear detail in hematopoietic tissues with a rapid fixation time of $1.5 \text{ to } 2 \text{ hours}$.

Chromate, Lead, and Picric Acid Fixatives

Chromate fixatives include Chromic Acid ($1 \text{ to } 2\%$ solution), which precipitates proteins and preserves carbohydrates, and Potassium Dichromate ($3\%$ solution), which preserves lipids and mitochondria at $pH \text{ } 4.5 \text{ to } 5.2$. Regaud’s (Muller’s) Fluid is recommended for chromatin and mitochondria, though it deteriorates on standing. Orth’s Fluid is used to study necrosis and detect Rickettsiae. Lead fixatives ($4\%$ aqueous solution) are used for acid mucopolysaccharides; they can form insoluble lead carbonate, which is removed by filtration or acetic acid.

Picric acid fixatives, like Bouin’s Solution, are excellent for embryos, pituitary biopsies, and glycogen, though they are highly explosive when dry and not suitable for kidney structures. The yellow color of picrate fixatives must be removed using $70\%$ ethyl alcohol or lithium carbonate. Brasil’s Alcoholic Picroformol is less messy than Bouin’s and excellent for glycogen. Glacial Acetic Acid (GAA) solidifies at $17^{\circ}C$ and is essential in nuclear fixatives to counteract the shrinkage of other components.

Alcohols and Other Chemical Fixatives

Alcohol fixatives act by destroying hydrogen bonds. Ethyl Alcohol ($70 \text{ to } 100\%$) is used for blood, smears, and nucleic acids, providing usable DNA for PCR. Methyl Alcohol ($100\%$) is used for smears but penetrates slowly. Carnoy’s Fluid is the most rapid fixative and is recommended for chromosomes and rabies diagnosis (Nissl granules). Clarke’s Fixative is used for frozen sections. Newcomer’s Fluid is recommended for mucopolysaccharides. Osmium Tetroxide (Osmic Acid) fixes lipids permanently and is essential for EM, but it is expensive and volatile; it should be stored in dark bottles to prevent the formation of black osmic oxide precipitates. Flemming’s Solution is a chrome-osmium-acetic acid fixative used for nuclear preparations. Trichloroacetic acid is used for protein precipitation and acts as a weak decalcifying agent. Acetone, used at $-5 \text{ to } 4^{\circ}C$, fixes by dehydration and is recommended for water-diffusible enzymes and rabies diagnosis.

Physical Fixation and General Precautions

Physical methods include Heat fixation, used for bacteriologic stains and frozen sections, and Microwave fixation. Microwave-assisted fixation is carried out at $450 \text{ watts}$ and $55^{\circ}C$ for $1.5 \text{ to } 4 \text{ minutes}$. Freeze drying involves rapid freezing (quenching) followed by sublimation (desiccation) in a vacuum. Freeze substitution is similar but uses chemicals like Rossman’s formula or acetone for dehydration.

General principles for handling specimens include labeling all tissues and fixing surgical specimens immediately. If delayed, refrigerate but do not freeze to avoid ice crystal artifacts. Fresh tissue should be treated as potentially infectious. Tissues should usually be no more than $5 \text{ mm}$ thick. The volume of fixative should be $10 \text{ to } 20 \times$ the tissue volume ($5 \text{ to } 10 \times$ for osmium tetroxide, and up to $50 \times$ for museum preparations). Hollow organs should be packed with fixative-soaked cotton or opened. The human brain may be suspended by a cord under the Circle of Willis for a fixation period of $2 \text{ weeks}$. Eyes should not be dissected before fixation to prevent collapse. Muscle biopsies may be stretched or laid flat on filter paper to prevent rigor contraction. Hard tissues may be softened using $4\%$ aqueous phenol (Lendrum’s method). Fatty tissues require longer fixation in thin sections. Fixatives should be used only once to avoid contamination and should be stored in non-metal containers to prevent corrosion.