What is Fixation?
Fixation is the critical first step in the preparation of tissue samples for
histological examination. It involves preserving the tissue in as close to a life-like state as possible. This process prevents autolysis and putrefaction, maintaining the structural integrity and cellular details essential for accurate diagnosis and research.
Why is Fixation Quality Important?
The quality of fixation directly impacts the subsequent steps in histological processing, including embedding, sectioning, and staining. Poor fixation can result in
artifacts, tissue shrinkage, and loss of cellular details, thereby compromising the accuracy of microscopic evaluation. High-quality fixation ensures that the tissue morphology and molecular composition are preserved, allowing for reliable and reproducible results.
Types of Fixatives
There are several types of fixatives used in histology, each with its own advantages and disadvantages. Commonly used fixatives include: Formaldehyde: Widely used due to its ability to preserve a wide range of tissue types and its compatibility with various staining techniques.
Glutaraldehyde: Primarily used for electron microscopy due to its excellent fixation of cellular ultrastructure.
Alcohol-based fixatives: Rapidly penetrate tissues and are often used for cytological preparations.
Acetic acid: Often combined with other agents to enhance nuclear preservation.
Factors Influencing Fixation Quality
Several factors can influence the quality of fixation, including the choice of fixative, fixation time, temperature, and the size of the tissue sample. Choice of Fixative: The selection of an appropriate fixative is crucial. For example, formaldehyde is suitable for most routine histology, while glutaraldehyde is preferred for electron microscopy.
Fixation Time: Under-fixation can lead to incomplete preservation, while over-fixation can cause excessive cross-linking, making tissues hard and brittle.
Temperature: Fixation is typically carried out at room temperature, but some protocols may require refrigeration to improve fixation quality.
Sample Size: Smaller tissue samples fix more quickly and thoroughly than larger ones. It is essential to cut tissues to an appropriate size to facilitate efficient fixation.
Signs of Good and Poor Fixation
Good fixation is characterized by well-preserved cellular and extracellular structures, minimal artifacts, and uniform staining. Key signs of good fixation include: Intact cellular membranes
Well-preserved nuclear details
Minimal tissue shrinkage and distortion
Consistent and reproducible staining patterns
In contrast, poor fixation can result in:
Loss of cellular details
Presence of artifacts such as ice-crystal damage or formalin pigment
Uneven or weak staining
Tissue shrinkage and distortion
Improving Fixation Quality
To improve fixation quality, consider the following tips: Select the appropriate fixative for the tissue type and intended downstream applications.
Ensure adequate fixation time, avoiding both under- and over-fixation.
Maintain consistent fixation conditions, including temperature and pH.
Cut tissue samples to an appropriate size for efficient penetration of the fixative.
Use agitation or perfusion techniques to enhance fixative penetration in larger or more complex tissues.
Conclusion
Fixation quality is fundamental to the success of histological procedures. By understanding the various factors that influence fixation and implementing best practices, researchers and pathologists can ensure the accurate preservation of tissue samples, leading to reliable and meaningful
histological analysis.
