Processing Without Clearing

There are a few fluids which permit wax infiltration without the use of clearing agents. These are sometimes referred to as wax antemedia. Strictly speaking, the term refers to the final fluid used immediately prior to the paraffin wax bath. Since "ante-" has the meaning of "before", it would include all clearing agents. Usually, however, those are referred to as simply "clearing agents", and the term "wax antemedia" is used for those fluids which are miscible with molten paraffin wax but do not fit the classic definition of a clearing agent. In particular, it is used for those few fluids which may be employed to dehydrate tissue, then permit the tissue to be infiltrated with the wax without using a clearing agent. Iso-propanol, acetone and dioxane are the most well known, with iso-propanol being used somewhat more commonly than the other two, although still not very much.

Why do this? The dehydration, clearing, wax sequence is so firmly entrenched and works so well, why step out of the mold? The infrequent use of this process emphasises the validity of that view, but there are occasions when the standard process is not as effective as it usually is. Often this is due to excess hardening brought about by xylene and similar clearing agents. Xylene has the unfortunate characteristic of rehardening decalcified bone, so that it may return almost to its pre-decalcified texture. Similarly, small uteri with dense cervixes are often very hard and occasionally one is encountered that simply cannot be sectioned following treatment with xylene. Other clearing agents may help, if available, but the fluids under discussion here can often be relied upon to produce a block soft enough to be sectioned.

Wax infiltration after these fluids usually takes longer than with xylene. The antemedia also tend to be poorer fat solvents than most clearing agents, and fatty tissues are often poorly infiltrated with a lot of residual fat. This affects the sectioning characteristics of the block, of course. For those reasons these fluids cannot be recommended as a replacement for the usual processing schedules. They are special case procedures.

iso-propanol This fluid is likely the one that is least difficult to use. It is a reasonably safe alcohol, easily available, and some laboratories use it as a routine dehydrant instead of ethanol. Following thorough dehydration the tissue is simply drained and placed into molten paraffin wax. Three changes are usually sufficient, but the times should be increased somewhat, particularly in the first wax as the miscibility of iso-propanol and paraffin wax is poorer than xylene and paraffin wax. An increase of about 50% in the first wax bath and 25% for subsequent wax baths should be satisfactory with suitable agitation.

It should be noted that there is no visual cue as to when infiltration is complete. The tissue remains opaque throughout and the refractive index is not raised at all. The tissue is not transparent, and remains so after the paraffin wax has hardened, so the tissue always looks as if it has not completely infiltrated even when it has. It is the appearance to be expected when using this fluid as a wax antemedium.

During sectioning tissues will be found to be less hard than with xylene, otherwise they are much the same with the exception that fatty tissues will not have infiltrated completely. I strongly recommend this fluid for those rare occasions when this kind of processing is necessary.

acetone Acetone is used in a similar manner to iso-propanol, and a comparison of the formulas indicates the close relationship between the two. The tissues are first dehydrated, often with the acetone, then the excess drained and the tissues immersed in molten paraffin wax. Acetone is a little more efficient than iso-propanol as a fat solvent, so tissues with a little higher fat content may be successfully infiltrated, but the difference is not great and it is also considerably inferior to xylene in that regard. It takes about the same time as with iso-propanol for infiltration to be successful and, as with iso-propanol, there are no visual cues that it is complete.

Acetone is more volatile than iso-propanol and is more inflammable, with a distinct odour. For those reasons I prefer iso-propanol, but both produce wax infiltrated tissues that are very similar.

If you are considering the use of dioxane, read this first. You should also read the Wikipedia article on dioxane. As it is a suspected carcinogen and can cause liver damage, it is likely more sensible to avoid this fluid. Fortunately, it is not popular. If it is used, do make sure to do so in a working fume chamber and to avoid inhaling any fumes.

Dioxane is often confused with dioxin, but they are not the same thing. Read this Wikipedia article on dioxin and follow the links describing the environmental pollutants.



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