Fixation is the first step in routine histology processes. It is the step which stops autolysis (enzymatic breakdown of tissue) and putrefaction (bacterial breakdown of tissue) which start immediately after blood and nutrients are no longer reaching the cells. Immediate fixation is important to preserve the tissue in a lifelike state.
Fixation makes the tissue more receptive to dyes by setting up amino acids and charges so staining works properly. Most stains are designed for formalin fixed paraffin embedded (FFPE) tissue. But beyond that, fixation also serves the purpose of bringing out differences in the refractive index, which is necessary for viewing under the microscope.
Types of Fixation
There are two main methods of fixation, chemical and physical. Chemical fixation is when we use reagents to remove water or denature proteins through chemical reactions. Physical also removes water and denatures proteins, but it utilizes heat in this process as opposed to chemical reactions.
Within chemical fixation, there are different categorizations, one being additive and non-additive. As you may have guessed, additive means something is physically added to the tissues, binding to the proteins to render them insoluble. This will alter the tertiary structure of the tissue and alter the charge, which becomes important during staining. Instead of physically becoming part of the tissue, non-additive fixation uses alcohol or acetone to remove water which denatures the proteins.
Another categorization of chemical fixation is coagulating vs non-coagulating. When you use a coagulating fixation, it creates a network inside the tissues that allows solutions to readily penetrate the interior of the tissue and it hardens them as it goes, which can be physically observed. Non-coagulating on the other hand, will create a gel between the proteins and fixative and will make fixation more difficult. Formaldehyde and potassium dichromate are examples of non-coagulating fixatives.
Factors Affecting Fixation
There are several factors that will impact the fixation process. For example, temperature. Most fixation is performed at room temperature, however, increasing temperature will increase speed of fixation whereas lowering temperatures will slow the speed. You will see increased temperatures used in automated processors and microwave processing. Another factor is tissue size. When you gross the tissue, you want to leave adequate space in the cassette. The penetration rate of the fixative can be limited by size. This is why you will see hollow organs bisected open and large specimens bread loafed so that the fixative can more easily penetrate these areas. You want to aim for a 20:1 ratio of fixative to tissue. This will allow for enough fixative to adequately bind to the tissue You must also be mindful of the time that the tissue is in the fixative. You want to allow enough time, particularly for the slower non-coagulating fixatives, but it is not recommended to leave in fixatives long-term as they can degrade or overharden.
pH can also be a factor influencing fixation, particularly in relation to electron microscopy. If the pH is not physiologic, ultrastructure and organelle changes will be seen. An indication of this is swelling or shrinking in the mitochondria, which are the first to react to changes in pH.
To learn more about fixation, check out NSH’s HT Prep Course, a webinar series that teaches core histology topics from the HT exam.