Fixation - Histology

What is Fixation?

Fixation is a critical process in histology that preserves biological tissues from the moment of removal from the organism until the tissue is processed for microscopic examination. It stabilizes the tissue to prevent autolysis and decomposition, thereby maintaining the structure and composition of the sample as closely as possible to its natural state.

Why is Fixation Important?

Fixation is essential for maintaining tissue integrity, preventing decay, and preserving cellular and subcellular structures. This preservation is crucial for accurate diagnostic and research purposes, enabling pathologists and researchers to study morphological features and molecular markers effectively.

What are the Types of Fixatives?

Fixatives can be broadly classified into two categories: chemical and physical.

Chemical Fixatives: These include formaldehyde, glutaraldehyde, ethanol, and acetic acid. Formaldehyde, often used as formalin (a 10% solution of formaldehyde in water), is one of the most commonly used fixatives.
Physical Fixatives: Methods such as cryopreservation (freezing) are used to preserve tissues without chemical alteration.

How Does Fixation Work?

Fixation works by cross-linking proteins and other macromolecules, which stabilizes tissue structure. Chemical fixatives typically work through cross-linking or precipitating proteins, which immobilizes them and prevents the enzymatic breakdown of tissues.

Formaldehyde: Ideal for preserving the overall structure of cells and tissues and is compatible with a wide range of staining techniques.
Glutaraldehyde: Provides excellent preservation of fine structure, making it suitable for electron microscopy.
Ethanol and Methanol: Used for preserving nucleic acids and are commonly used in molecular biology.
Acetic Acid: Often combined with other fixatives to preserve nucleic acids and nuclear structures.

Specimen Collection: Tissues are collected and immediately placed in the fixative to prevent autolysis.
Fixation Time: The tissue is left in the fixative for a specified period, which can range from a few hours to several days, depending on the tissue type and fixative used.
Post-Fixation Washing: Tissues are often washed to remove excess fixative before further processing.

What Factors Influence Fixation?

Several factors affect the efficacy of fixation:

pH and Osmolarity: The pH and osmolarity of the fixative should be close to physiological levels to prevent cellular damage.
Temperature: Higher temperatures can accelerate fixation but may also cause tissue distortion.
Fixative Concentration: The concentration of the fixative must be optimized to ensure adequate penetration and fixation without causing excessive hardening or shrinkage.
Fixation Time: Adequate time is necessary for complete fixation, but over-fixation can lead to tissue brittleness.
Tissue Size: Smaller tissue samples fix more quickly and thoroughly than larger ones.

What Are the Limitations of Fixation?

Despite its importance, fixation has several limitations:

Artifact Formation: Chemical reactions during fixation can introduce artifacts that obscure true cellular structures.
Incomplete Fixation: As fixatives penetrate tissues slowly, larger samples may not be completely fixed, leading to autolysis in the central parts.
Loss of Antigenicity: Some fixatives can mask or destroy antigenic sites, complicating immunohistochemical studies.

Conclusion

Fixation is a cornerstone of histological techniques, providing the means to preserve, study, and diagnose tissue samples accurately. Understanding the principles, types, and limitations of fixation is essential for achieving reliable and reproducible results in both diagnostic and research settings.