Ethanol Artifact

Processing tissues with inadequate fixation can have serious and uncorrectable consequences. This is especially the case when a simple formalin fixative is used. Formalin requires overnight for even minimal fixation, or several hours at elevated temperatures, to give protection from subsequent fluids, particularly ethanol. In most cases the dehydrant is ethanol but ethanol is also a fixative as well as a dehydrant, and quite a poor one at that. The effect of inadequate fixation with formalin is that the unfixed or partially fixed tissues will have fixation completed by the ethanol during the dehydration step.

If this is done with a modern processor in which the reagent temperatures may be increased, the negative effects of ethanol fixation will be exaggerated, and the results may well be unacceptable. This artifact is often erroneously referred to as overfixation and the time in formalin is then reduced even more, which merely serves to make the problem worse as even less protection is afforded the tissue. It is also termed the parched earth effect, but this appearance is simply due to unfixed tissue being fixed and dehydrated with (warm) ethanol and has been described in the early literature when ethanol fixation was first used.

Unfortunately, as the appearance is largely due to lack of protection for the tissue due to inadequate formalin fixation, the effects are permanent and reprocessing usually does not correct the appearance. The brittleness of the tissues may be overcome to some degree by using a very sharp knife, setting the section thickness to the thinnest possible and sectioning very slowly and evenly. Soaking the block face in ice cold water may also help.

The resolution to this artefact is to adequately fix the tissue initially before ethanol is used. One of the purposes of fixation is to protect tissue from reagents used subsequently, so if adequate fixation is given the artefact is not displayed. If there is insufficient time for adequate formalin fixation, even with heat, then another more aggressive fixative should be used.



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