Types of Fixative in Histopathology Explained

In histopathology, the process of tissue preservation is as vital as the analysis itself. Fixatives are at the core of this preservation, halting decay and stabilising cellular structures for accurate examination. So, we need to explore the science behind fixation, such as the principles, properties, and various types of fixative in histopathology.

From routine solutions like formalin to specialised options for complex studies, the role and selection of fixatives is undeniable. With increasing focus on safer alternatives and evolving research needs, we shall also address practical considerations for choosing the most effective fixative for a purpose.

What is Fixation in Histopathology

Fixation in histopathology is a chemical process designed to preserve cells and tissues for analysis by stabilising their structure and morphology. It prevents autolysis, which begins immediately after tissue removal due to disrupted cell balance and enzymatic activity, creating conditions for microbial growth that can destroy tissue architecture.

All types of fixative in histopathology work by cross-linking proteins, converting the tissue’s semifluid state into a semisolid form and preserving its in vivo relationships. This stabilisation halts degeneration, facilitates tissue handling, and maintains structural details for accurate examination.

Rapid immersion in an appropriate volume of fixative is essential to preserve tissue integrity and prevent post-removal changes. Fixation also enhances optical differentiation, ensuring the tissue is adequately prepared for subsequent analysis.

Principal of Fixation in Histopathology

The principle of fixation in histopathology involves transforming the semifluid state of tissues into a semisolid form by cross-linking proteins, leading to their denaturation or coagulation. This preserves the in vivo relationships of cellular and extracellular components, enabling easy manipulation and analysis. The types of fixatives used in histopathology stabilise proteins and other cellular elements by forming cross-linked gels, while some tissue components remain unaffected but are trapped by fixed structures.

Types of Fixatives Used in Histopathology

Classification of fixation in histopathology is based on chemical composition, the number of structures fixed, and tissue types targeted. Chemical classifications include aldehydes, oxidizing agents, and coagulants. Fixatives are also divided into simple (e.g., formaldehyde) or compound (e.g., Bouin’s fluid), while histochemical, microanatomical, and cytological categories focus on tissue-specific preservation needs.

Type of Fixative	Examples
Aldehydes	Formaldehyde, Glutaraldehyde, Acrolein
Coagulants	Ethanol, Methanol, Picric Acid, Acetic Acid
Oxidizing Agents	Osmium Tetroxide, Potassium Dichromate
Miscellaneous	Mercuric Chloride, Glyoxal, Genipin
Compound Fixatives	Bouin's Fluid, Zenker's Fluid, Formol Saline, Heidenhain's Susa, Helly's Fluid, Rossman's Fluid
Cytologic Fixatives	Champy's Fluid, Glacial Acetic Acid, Alcohol, Formol Saline, Carnoy's Fluid, Clarke's Fluid, Newcomer's Fluid, Flemming's Fluid

Properties of Fixatives

Coagulation and Precipitation: Fixatives induce coagulation and precipitation of proteins, stabilising tissue structures.

Volume Changes: Fixatives influence cell volume due to altered membrane permeability and respiratory inhibition.

Ideal Characteristics: The types of fixatives used in histopathology should be affordable, non-toxic, non-flammable, and capable of preserving tissues over prolonged periods.

Common Fixatives Used in Histopathology

Formaldehyde
Discovered in 1859, formaldehyde is the most common type of fixative in histopathology. It stabilises proteins and nucleic acids, forming a nucleic acid-protein complex. Typically used as 10% formalin, it minimises tissue shrinkage while conserving lipids. White deposits (paraformaldehyde) in stored solutions can be reduced by low temperatures or methanol. Its acidic nature can be neutralised with magnesium carbonate.

Glutaraldehyde
Developed in 1963, glutaraldehyde is a potent protein cross-linker with slow tissue penetration. It preserves ultrastructure, making it ideal for electron microscopy but less suitable for light microscopy due to overhardening. It is highly reactive, forming oligomers and glutaric acid; but stable at pH 5 and 4°C. Prolonged exposure may irritate the skin, respiratory, and digestive systems.

Osmium Tetroxide
Water-soluble osmium tetroxide cross-links proteins and stabilises lipids. It is a secondary type of fixative. It is used in electron microscopy to improve tissue contrast. Though effective, it removes carbohydrates and proteins during fixation and may cause tissue swelling, reducible with sodium or calcium chloride. Its vapours can harm the eyes, requiring careful handling in sealed glass ampules.

Mercuric Chloride
Mercuric chloride is classified under the types of fixative in histopathology. It reacts with cysteine, amines, and sulfhydryl groups, effectively hardening tissues and enhancing nucleic acid fixation. Common in Helly’s and B-5 fixatives, it offers strong staining affinity but is toxic and unsuitable for metals. With slow penetration, it is mainly reserved for hematopoietic tissues and requires thin specimens for effective use.

Glyoxal
Introduced in 1943, glyoxal serves as a formalin alternative with fewer safety concerns. A bifunctional aldehyde, it preserves cellular details but can cause erythrocyte lysis. Often used in microwave fixation, glyoxal is part of the types of fixative in histopathology and is stable and non-volatile at room temperature. It is commercially available as an aqueous solution, it includes trimers, dimers, and ring structures.

Picric Acid
Picric acid coagulates proteins and is preferred for glycogen preservation. While not suitable for DNA/RNA fixation, it brightens tissue staining. It dissolves calcium deposits effectively. Alcohol washing and lithium carbonate neutralisation prevent cellular distortion. Its acidic nature requires careful handling to maintain tissue integrity during processing.

Ethanol and Methanol
Ethanol and methanol are coagulants that denature proteins, disrupting hydrogen bonds. Classified under the types of fixative in histopathology, ethanol preserves glycogen but distorts cellular details, while methanol is favoured for cytological smears and blood films. Effective at high concentrations, both solvents are widely used despite their dehydrating and denaturing properties.

Acetone
Acetone is categorised within the types of fixative in histopathology. It is essentially a lipid solvent that fixes tissues while causing brittleness. Its volatility and flammability limit its application in automated processors. Commonly used in dehydration, acetone's rapid penetration makes it valuable for specific histopathological needs, though it is not ideal for routine processing.

Acetic Acid
A non-coagulant, acetic acid stabilises nucleic acids and coagulates nuclear proteins. This fixative is faster penetration and allows quick fixation, though solo use causes cell swelling. When combined with ethanol, it becomes a robust cytological fixative, suitable for conserving nucleic acids during processing.

Potassium Dichromate
Potassium dichromate is one of the non-coagulant types of fixative in histopathology. Unless combined with acids, it acts as non-coagulant. It conserves mitochondria and enhances eosin staining by reacting with hydroxyl and carboxyl groups. Washing tissues thoroughly after use prevents insoluble chromate suboxide formation, which could hinder subsequent processing.

Bouin’s Fixative
Invented in 1897, Bouin’s fixative blends picric acid, acetic acid, and formaldehyde. Bouin’s Fixative excels in preserving delicate tissues but compromises hybridisation studies. This picrate-based fixative requires careful handling to prevent adverse chemical interactions during processing.

Acrolein
Acrolein, a monoaldehyde, provides precise structural preservation and retains viral antigenicity. Its reactivity enables rapid tissue penetration, making it suitable for enzyme histochemistry among the types of fixative in histopathology. However, its instability at alkaline pH and polymerisation risks limit its widespread use.

Genipin
Derived from Geniposide, Genipin cross-links amino acids, forming stable collagen structures. Its non-toxicity and efficacy in biomedical applications like cartilage engineering highlight its versatility. Soluble in alcohols, genipin’s mild reaction conditions and stable cross-links make it promising fixative in histopathology.

Simple and Compound Fixatives in Histopathology

Fixation and fixatives in histopathology are categorised as simple or compound. Discussing them helps select the appropriate fixative for specific diagnostic or research purposes.

Simple fixatives consist of a single active ingredient, such as formaldehyde, glutaraldehyde, or picric acid. They are used to preserve specific tissue components effectively. In contrast, compound fixatives are mixtures of multiple agents designed to combine the properties of different fixatives for broader applications. Examples include Bouin’s fluid, Zenker’s fluid, and formol saline.

While simple fixatives target specific reactions, compound fixatives are versatile. They provide high stabilisation for diverse cellular and extracellular components, making them ideal for specialized studies.

Choosing the Right Fixative

Choosing the right fixative from the many types of fixative in histopathology is not straightforward. It depends on the laboratory’s needs and practices. Many established labs rely on neutral buffered formalin for its flexibility and compatibility with various specimen types.
It supports special stains, immunohistochemistry (with antigen retrieval), and molecular techniques like ISH. Formalin-fixed tissues can also be stored long-term without significant damage, making it a reliable choice.
However, with growing concerns about formalin’s toxicity, alternatives are being explored. When selecting or replacing a fixative, consider:

• Toxicity and safety measures are required.
• Volatility and exposure risks.
• Flammability.
• Effects of prolonged fixation.
• Storage requirements.
• Compatibility with tissue processors.
• Practical and legal disposal needs.

Careful evaluation is essential before transitioning to a new fixative.

Conclusion

When exploring the types of fixative in histopathology, let’s consider advancements in eco-friendly and biodegradable fixatives. These emerging options aim to reduce environmental and health risks without compromising tissue preservation quality.
As histopathology evolves, adopting sustainable practices could redefine standard protocols, offering safer alternatives for labs worldwide. Staying informed about these developments ensures laboratories remain innovative while maintaining precision in tissue analysis.