Introduction To Fixation
One of the most fundamental principles in the preparation of high quality slides for microscopic examination is that fixation is the foundation on which all subsequent procedures build. If this step is not carried out effectively, then it is not possible to rectify it at a later date and what may be done with the fixed tissue will always be limited by what was done initially.
The purpose of fixation is to preserve the tissue in such a way that it appears the same in a microscopic preparation as it appeared while living and functioning. Of course, we must acknowledge that this is an impossible aim, as the very act of fixation stops all functions and distorts in some fashion the appearance. Nevertheless, the aim should be to approach this ideal as closely as possible and many combinations of chemicals have been recommended for it with varying degrees of success.
Fixatives not only preserve tissues, they chemically alter them in some fashion, usually due to denaturing their protiens. This can be by precipitation, as with ethanol, or by forming compounds with them, as formaldehyde. In both cases the proteins are preserved in situ with little distortion, thus keeping the tissue structure intact. There may be effects on carbohydrates and lipids as well, but it is the proteins which are the main target.
Actually fixing carbohydrates and lipids, as in chemically altering and rendering them inert, is usually considered to be a specialist procedure requiring separate treatment. An exception is when carbohydrates and lipids are in the form of complexes with proteins, glycoproteins and lipoproteins for instance, when fixation can occur via the protein component. In other cases carbohydrates are simply trapped within a meshwork of fixed protein. Lipids may be unaltered and simply dissolve out during processing.
When properly and thoroughly fixed, blocks of tissue should be able to resist the effects of other reagents used during the processing procedure. In particular, resistance to the effects of ethanol is important as it is capable of fixing proteins in its own right. If the tissue is not properly fixed before the ethanol for dehydration is applied, there is a distinct likelihood that it will complete the fixation while dehydrating. Unfortunately, when used alone as a fixative, ethanol is quite poor, causing shrinkage and making tissues hard and brittle. One side effect of the modern minimalist approach to fixation and processing, in which formalin is applied barely long enough to stabilise the proteins but certainly not long enough to cross link them, is that ethanol fixation during processing has become fairly commonplace. In fact, this is so common that it has been referred to as "parched earth" artifact, or "overfixation", a rather ironic term considering that it is caused by not fixing the tissue long enough in formalin. The resolution to this problem is to fix tissues thoroughly before processing. If there is insufficient time for this to be done with formalin, then another fixative that can do it should be used.
Any microorganisms present in the tissue are likely to be killed and preserved along with the tissue, although there may be a few isolated types of bacteria which can resist this treatment. Similarly, the enzymes that cause autolysis are usually rendered inactive, either through denaturation because they are themselves proteins or because their substrate, cellular components and contents, have been denatured. In either case, putrefaction and autolysis come to an end. Once the tissues have been removed from the fixative, however, microorganisms in the environment may colonise the tissue and putrefaction may begin again unless precautions are taken against it. These precautions include the completion of processing. It is generally accepted that no bacteria can resist the complete process from fixative to paraffin infiltration, and paraffin processed tissues are considered to be safe to handle and stable indefinitely. It is known that prions can resist this complete process, so precautions must be taken to ensure personal safety if the tissue is from this source.
One of the observed effects of fixation is that the tissue tends to harden somewhat, the degree being dependent on which specific chemicals are used to fix. In moderation this is of benefit as it tends to even out the differences in hardness of the individual components and layers, and this helps during sectioning. It can be overdone, of course, and excessive hardening is a detriment. Often, excessive hardening is due to the tissue being left too long in the fixative, particularly if harsh precipitant chemicals are used. Fortunately, the hardening from formalin is relatively mild.
Tissues may also become brittle, which can make sectioning very difficult as the tissues may shred and shatter as the sections are cut. Sectioning much thinner, cooling the block and soaking the tissue in ice cold water may all help when this is encountered. It is more prevalent with areas of proteinaceous exudates, colloids and similar such materials, but it can occur in many tissues with harsh fixatives.
Most fixatives cause a degree of shrinkage, or give inadequate protection to the tissue so that shrinkage occurs during dehydration and clearing. It can be easily seen that most shrinkage occurs during the infiltration step in hot wax. It can be quite striking to observe the first wax bath during the first 30 minutes of application as the tissue diminishes in size quite rapidly. Shrinkage not only occurs overall with the piece of tissue becoming smaller in dimensions. It also occurs at a cellular level, and cells and fibres may separate from each other introducing spaces where non existed during life. Simple formalin solutions permit significant shrinkage even when the tissue is thoroughly fixed. Harsher, protein precipitant solutions tend to resist it more.
It cannot be stressed enough that tissues should be thoroughly fixed before proceeding to the dehydration, clearing and infiltration steps. Unfortunately, in the quite understandable rush for quick diagnoses this basic principle has often been forgotten, with a consequent reduction in quality. Most often this shows with the use of simple formalin mixtures, probably the most commonly used fixative, which is often carried out for an inadequate length of time, allowing subsequent treatments with harsh reagents to modify the microscopic appearance. Formalin on its own requires at least overnight at room temperature, and preferably several days, but it is often applied for just a few hours or even less. Frequently, this is accompanied by subsequent complaints of poor quality sectioning, staining and appearance.
Under these circumstances it would make more sense to use a faster fixative so that fixation is complete within the available time. This may not be possible in practice as many of the fixatives which would be useful for this contain mercuric chloride, a chemical which may no longer be used in some jurisdictions or whose use is deprecated even if not under outright ban. As well, the modern use of immunological methods requires a fixative that permits various antigens to be identified in fixed tissue, and the characteristic of formalin that allows cross linking of proteins to be reversed by moist heat (HIER) is useful. When sufficient tissue is available one sample may be processed as quickly as possible while a second is fixed more thoroughly and then processed, but small biopsies often do not permit this. On occasion, increasing the temperature of the formalin to about 50°C or 60°C will usually speed up fixation significantly, but this is not a panacea as there is a reduction in the quality of preservation and there may be consequences from the heat itself, which can denature proteins by cooking.
The best resolution, from a technical perspective, is patience. Chemical reactions take some time to complete, and fixation is no different than any other chemical reaction. I strongly recommend that sufficient time be allowed for the fixative to fix, and that processing be delayed until that is so. It causes only a short delay in diagnosis, and for those specimens where a very rapid diagnosis is required frozen section diagnoses may be used.
A sometimes overlooked factor in fixation is the amount of fixative used. There must be enough to permit free access to all parts of the tissue. Cramming a large piece of tissue into a small container, then pouring a little fixative over it is just not good enough, but it is frequently encountered. The tissue must be able to float freely in the fixative and there should be not less than 10 times the volume of tissue, and preferably about 20 times the volume.
If the piece of tissue is large enough to require a long time for the fixative to penetrate to the centre, then it should be described and "breadloafed", or cut into slices no thicker than 1 cm. This will allow the fixative to penetrate more effectively. Intestines should be opened and any contents removed. It may then be replaced into a large volume of fixative, or if small enough, pinned out with plastic pins (to avoid corrosion) and floated in the fixative.
There are a limited number of chemicals used in fixatives.