J. Greenlee

Fixation First

Beginning a histology blog with a post about fixation is really the only fitting way to start.  Fixation, and most often formalin fixation, is a crucial step in histology that cannot be overlooked, yet as laboratories struggle with challenges ranging from heavy workloads to staffing shortages, unfortunately, it far too often is.

Formalin fixation is a process that involves immersing tissues in a solution of formaldehyde, which preserves the tissue by preventing decay and autolysis.

The importance of formalin fixation lies in the fact that it enables accurate and reliable examination of tissues. Without formalin fixation, tissues would rapidly degrade and the cellular architecture or morphology would be lost. This would make it difficult, if not impossible, to make a proper diagnosis.

Formalin fixation not only preserves the tissue structure but also ensures that the tissue remains in a state that is compatible with the various techniques used in histology. For example, formalin-fixed tissues can be processed and then embedded in paraffin wax, which allows for the cutting of thin sections with a microtome that are then prepared for staining and coverslipping and later examined under a microscope.

Proper formalin fixation is the key to a quality finished slide.  Formalin is an additive fixative.  Molecular components of additive fixatives like formalin are taken up and combine with the tissue.  In the case of formalin, crosslinking occurs.  Because of this characteristic, the volume and concentration of the formalin fixative used is very important for proper fixation.  Recommended ratios of tissue to formalin volume may range from 1:10 to even 1:20, and because the molecular components of the formalin fixative in solution are used up by the tissue, regularly exchanging the formalin at the grossing station or prior to processing is a must.

Time and tissue thickness are maybe the most important factors when it comes to formalin fixation.  The general rule of thumb is that formalin penetrates about 1 mm of tissue thickness per hour, but, of course, that depends much on the structures and features of the tissue itself.  For example, penetration of dense tissue is slower than that of tissues that are less dense or have vascular pathways that can act almost like highways to bring the formalin into the tissue.  The standard grossing thickness for formalin fixation is approximately 2 - 3 mm.  For tissues thicker than standard, the change in the surface area to volume ratio begins to hinder the ability of formalin to penetrate, which can lead to under-fixed and under-processed tissue.  It is important to remember that penetration rate is also NOT equal to fixation rate when it comes to formalin fixation.  The actual fixation of the tissue with formalin that is done by the cross-linking of proteins takes considerably longer than its penetration time.  

Another important yet often forgotten benefit of formalin fixation is that it also helps to reduce the risk of infection from the handling and processing of tissue. By inactivating potential infectious agents in the tissue, formalin fixation provides a safer working environment for histology professionals.

In summary, formalin fixation is a vital step in histology that cannot be understated. It is essential for preserving tissue structure, enabling accurate diagnosis, and ensuring a safe working environment. Histology professionals should pay close attention to this critical step in the histology process to ensure the best possible outcomes for their patients and other projects.
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