Clearing

Most clearants (clearing agents) are hydrocarbons of one kind or another. Many clearants are inflammable and this is a serious concern. The saying that "familiarity breeds contempt" is valid, and many technologists treat clearing agents as if they are as safe as water. They are not. Always keep in mind that using such fluids on an electric machine with the possibility of sparking is inherently unsafe, especially when the fluids are heated to assist with their action. Fire blankets and appropriate fire extinguishers must be available in a place where they can be easily accessed should a fire begin. Do not let this give a false sense of security though, and if the fire is beyond simple measures to extinguish, leave and call the fire department. Do not put your life in jeopardy.

As with dehydration there is some shrinkage during clearing, but it is not excessive and varies according to the particular fluid used.

The derivation of the term "clearing" is explained in two different ways. One view is that the term refers to the dehydrant being "cleared" (i.e. removed) from the tissue. The other view is that it refers to making the tissue "clear" (i.e. translucent). Another term is occasionally encountered because of the latter view, "wax antemedium", which refers to any fluid used immediately prior to infiltration. Most technologists do not differentiate between the two synonyms.

Many clearing agents may be recycled through distillation. Keep in mind that the commonly available stills are set up to redistill xylene or toluene. Before you use them for another clearant you must check with the manufacturer or vendor to ensure the equipment can redistill that fluid. Do not just assume it can and try to redistill, as an example, light naphtha in a still set up for xylene. That could be disastrous. Put your own safety before anything else.

As with dehydration, clearing is a matter of diluting out the dehydrant from the tissue until the amount remaining is well below the tolerence level of the embedding medium. Usually fewer changes of clearant are required because paraffin wax tolerates the presence of minute quantities of ethanol and other dehydrants better than it tolerates the same amounts of water. It will usually be observed that if dehydration is complete that clearing is quite rapid. For that reason, it is more advantageous, overall, to assign a greater proportion of the available time to dehydration so that clearing may proceed more rapidly.

Minimalist processing
There are two concerns with regards to clearing during minimal processing. One is that inadequate dehydration may inhibit thorough clearing due to residual moisture. The other is that when a barely adequate time in a clearing agent is allowed, small amounts of dehydrant may remain to resist infiltration with the wax. Fortunately, paraffin waxes can tolerate a minute amount of dehydrant better than they can the same amount of moisture, but small amounts of either will still affect the quality of the block. This may show up by the tissue shrinking back some time after sectioning. The resolution is to extend processing times, but this may not always be possible in a diagnostic setting.

Starting point
A few technologists begin with a mixture of dehydrant and clearant, but it is not really necessary. Placing the dehydrated tissue into a sufficiently large quantity of clearant is all that is required.

Number of changes
Since the tolerance level of paraffin wax for clearant is greater than it is for moisture, fewer changes are required than for dehydration. An absolute minimum of two changes should be given, but three or four will be found advantageous.

Agitation
As with dehydration, gentle agitation can improve clearing and reduce the time necessary for it to be done. Emphasis must be on the "gentle", as too vigorous an agitation can cause fragments of tissue to detach and they may contaminate other specimens.

Heat
Gentle heat may also be applied during clearing. Warming a fluid usually reduces viscosity, and this helps penetration into the tissue. It may also increase the fat solvent effect, but can increase hardening and brittleness.

Vacuum
Although often used, it is not clear whether applying vacuum increases the speed or thoroughness of clearing. Even if it does not, it causes no harm and is often applied on that basis.

Time
This varies depending on the particular clearing agent used. Generally, the less dense the clearant, the faster it will remove the ethanol. However, most clearants are reasonably comparable to xylene. The exceptions are that chloroform and limonene are reported to take a little longer than xylene, and cedarwood oil takes a lot longer. Carbon disulphide is faster.

Shrinkage
Little shrinkage occurs during clearing. Xylene causes a little, but it is not significant.

 


 

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