Specimen Fixation
Fixation is the most important step for both paraffin and resin embedded material. If the fixation is inadequate or unsuitable then the subsequent staining procedures will be impaired or unachievable. An ideal fixative should:
- Preserve the tissue and cells as life-like as possible, without any shrinking or swelling and without distorting or dissolving cellular constituents.
- Prevent putrefaction by bacteria and prevent autolysis by cathepsin-containing cells.
- Stabilize and protect tissues and cells against the detrimental effects of subsequent processing and staining procedures.
The choice of fixative will depend upon the type of specimen and the components to be demonstrated. The three most commonly employed fixatives for general use being neutral buffered formalin, glutaraldehyde and paraformaldehyde. Formalin, like other aldehyde fixatives, forms cross linking methylene bridges and Schiff bases between basic amino acid (lysine) residues of proteins. This cross linking stabilizes the proteins in situ, which is the basis of fixation. Formaldehyde produces mild cross linkages when compared to other aldehyde fixatives such as glutaraldehyde.
In addition to the choice of fixative, other important factors include fixation time, temperature and pH. Fixation time will depend upon the size of the specimen. In order to achieve adequate and consistent fixation it is essential that the specimens be sliced to a maximum thickness of 3 mm. Tissue such as lymph node (3 mm slices), skin and bone marrow trephines are routinely fixed for approximately 24 - 48 hours at room temperature. Dense tissue such as spleen may require extended fixation. Raising the ambient temperature can increase the rate of fixation. The pH of the formalin solution is generally between 5 and 7, which is governed by the pH of the local water supply.