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Alternatives To Fixation

The most common approach to making microscope slides is to use fixation followed by paraffin processing, sectioning and staining. This sequence is not the only means of making such preparations, neither is chemical fixation the only initial step that may be taken.

Frozen sections

Unfixed frozen sections are now commonplace in histotechnology, used for many purposes from rapid diagnosis to immunoenzyme histochemistry. Some years ago there was a move to replace routine paraffin processing with cryostat sectioning and diagnosis. In many cases it was satisfactory, but whenever frozen section diagnoses are given, there are a number of cases where the diagnosis is not clear enough that the pathologist defers a full report until paraffin sections are available. This emphasizes the major problem with such a system, that paraffin sections must still be made, both for accuracy of diagnosis and for storage for future reference. This effectively doubles the workload, or more, since high quality, thin frozen sections are often more difficult to obtain.

A secondary concern is with small biopsies. It may be that there is insufficient tissue left after cryostat sectioning to permit an adequate examination from paraffin sections, and what tissue there is may have ice crystal artefact present from the slow freezing obtained from a cryostat heat sink that is in constant use. It should also be noted that many special stains do not stain completely as expected on frozen sections. In practice, this system of replacing paraffin processing with cryostat sectioning never became commonplace.


Before the introduction of the cryostat, frozen sections were made with a clinical microtome after the tissue was frozen with carbon dioxide or on a freeze platform which used the Peltier effect (thermoelectric cooling). Prior to freezing it was a common practice to “fix” a 1-2 mm thin slice of tissue, usually from a breast biopsy, by dropping it into boiling formalin or water for one minute immediately after removing the heat. This rapidly fixed the tissue proteins by heat denaturation and preserved the structure well enough for frozen section diagnosis. The practice was rarely, if ever, used for paraffin processing, although the remaining tissue was transferred to formalin for fixation and processed so that the diagnosis given could be compared with the frozen section diagnosis. However, there is no reason that it could not be used with paraffin processing providing that the quality of the results is satisfactory for the purpose.

This means of tissue “fixation” is rarely used any more, but increasing the temperature of formalin to speed up fixation of tissues is quite common. Its former use does emphasize that increasing the temperature too much may have some untoward effects, as the heat itself can become a factor in denaturing the proteins directly rather than just increasing the speed of the chemical reactions which cross link the proteins.

Freeze drying

This procedure is a means of infiltrating unfixed tissue with support media, often paraffin wax. A small piece of tissue is first instantaneously frozen in isopentane that has been cooled with liquid nitrogen until it turns to slush. While still frozen the tissue is transferred to a cold chamber which is then evacuated of air, causing a vacuum. A very cold probe, often cooled by liquid nitrogen, is then brought close to the tissue. The temperature of the tissue is increased so that it becomes warmer than the probe, although it is still kept frozen. As a consequence, ice in the tissue sublimes and condenses on the probe. After a while the tissue will have become dehydrated without the ice having melted. At that point the tissue may be transferred to paraffin wax, or some other embedding medium, and slowly warmed up. As it does so, the medium melts and infiltrates the tissue. The block may then be hardened and sections cut.

It must be remembered that the tissue is unfixed and all subsequent handling must be done with that in mind, including storage of the blocks. Due to the inherently time consuming nature of the procedure, it has not gained much popularity in diagnostic pathology, although it remains a valuable research technique.

Freeze substitution

As with freeze drying, freeze substitution begins with freezing of a small piece of tissue in isopentane cooled with liquid nitrogen. Following freezing, the temperature of the tissue is allowed to rise to a point where it is still frozen, but at which ethanol or acetone, or perhaps some other dehydrant, is still liquid. The tissue is placed into the liquid at this cold temperature and the ice in the tissue is replaced (substituted) by the liquid. Because this replacement is done at such cold temperatures, there is almost no fixation effect from the liquids. When the ice has been replaced, the tissue may be placed into an alcohol miscible embedding medium and the tissue infiltrated. The block is then hardened and sections cut.

It is also possible to transfer the tissue to a clearing agent at subzero temperatures, and from there to paraffin wax.

It must be remembered that the tissue is largely unfixed, or only very minimally fixed, and care must be taken at all subsequent steps because of this, including storage of the blocks. This procedure has not become popular in diagnostic pathology due to its time consuming nature, but it remains a valid research technique.