Once tissues have been fixed and
processed, they are subjected to microscopic examinations, and in many
instances, to histochemical evaluation, particularly involving enzyme studies.
In the latter studies, the tissue should have been chemically active, that is,
the important chemical constituents should not have been removed, altered and
displaced. In most instances, frozen section is deemed to be the most ideal and
the preferred means of preserving tissues in order to avoid complete or partial
loss of enzymes consequent to chemical fixation. Difficulties, however, arise
in obtaining thin and serial sections of uniform thickness; cut sections tend
to disintegrate and cannot be easily handled. These disadvantages shall have to
be considered in determining the necessity and availability of such sections.
There are methods that may be restored
to, if chemical fixation of tissue blocks is to be avoided, namely:
1.
Freeze–drying
2.
Freeze substitution
3. Freeze frozen tissue sectioning
All have the common principle of
rapidly preserving the tissue block by freezing (quenching), to produce instant
cessation of cellular activity thereby preventing chemical alteration of tissue
constituents and displacement of cellular tissue components. Freezing must be
rapid, being accomplished within seconds to prevent the formation of ice
crystal artifacts in tissue blocks and produce optimum tissue preservation. The
freezing agent commonly employed is Liquid Nitrogen. The use of isopentane,
pentane and propane and most recently of dichlorodifluoromethane, which can be
cooled to very low temperature in order to retain the fluidity of the freezing
agents, have contributed much in giving higher conductivity to this liquefied
gas.
Freeze–drying
Freeze–drying is a special way of
preserving tissues by rapid freezing (quenching) and removing water
(dessication) by a physical process from the still frozen block without the use
of any chemical fixative.
A tissue around 2mm thick is plunged
into isopentane or propane – isopentane mixture which has been chilled to –160oC
to –180oC with Liquid Nitrogen. This will effectively solidify the
tissue in 2 – 3 seconds, thus preventing the formation of large ice crystals,
autolysis and putrefaction. The frozen tissue is then transferred into a high
vacuum drying apparatus maintained at a temperature of –30oC to –40oC
depending upon the size of the tissue. Water is sublimated and dehydrated from
the tissue, thereby completing dessication within 24 – 48 hours. Once drying is
completed, the tissue is removed and embedded, either in molten paraffin wax,
water soluble waxes or celloidin. Infiltration and impregnation are usually
performed in a vacuum embedding oven. The tissue is then sectioned in the usual
routine manner and specific staining is applied, depending upon individual
necessity.
This technique is generally time–consuming
and expensive. Furthermore, freeze–dried materials are generally more
difficult to section than ordinary paraffin blocks. The tissue is brittle and
inadequately supported due to the relative short period for wax impregnation;
hence, it is not advisable as a routine manner, and tissues are usually flattened
directly into an albuminous glass slide with the aid of the finger. Water must
be avoided and warm alcohol, acetone, mercury are preferred.
However, it has also many outstanding
good features. It produces minimum tissue shrinkages, and allows tissues to be
processed in a fresh state, thereby allowing minimal chemical change of the
cells, most especially on the protein components, and less displacement of
tissue and cell constituents. This is particularly important as far as enzyme
studies are concerned.
Freeze
substitution
Freeze substitution is similar to
freeze–drying in preparing and preserving tissue blocks for subsequent
sectioning because both involve the rapid freezing of tissues and the
subsequent infiltration and embedding of the frozen tissue block in paraffin or
celloidin. The only variation is that, the frozen tissue, instead of being
subjected to dehydration in an expensive vacuum drying apparatus, is fixed in
Rossman’s fluid or in Osmium tetroxide in 1% acetone for 1 – 6 days at a
temperature of –60oC to –70oC and dehydrated in absolute
alcohol at room temperature or in acetone at –70oC, respectively.
Infiltration and embedding is then carried out in the same way as in paraffin
sections.
This technique is relatively more economic
than freeze–drying and is more suitable for routine process.
Fresh
Frozen Tissue Sectioning
Fresh frozen tissue requires that the
tissue be maintained in the frozen solid state during cutting of section,
thereby supporting and protecting the tissue from damage and distortion by the
knife during the process of cutting.
The tissue must be sufficiently cold
and hard to prevent compression and displacement of cell and tissue structure
as the knife passes through it. Otherwise, the thin section would completely
melt and form a sticky distorted mass at the edge of the knife.
Fresh frozen tissue, unlike fixed
frozen tissues in the first two techniques, requires that the microtome knife
be chilled and maintained at low temperature to prevent complete melting of the
tissue, thereby forming a sticky, distorted mass along the knife edge. When the
tissue is too cold, on the other hand, resistance to cutting increased, the
tissue becomes brittle and is broken down into fragments upon cutting.
The success of fresh tissue
sectioning, therefore, depends to a large extent on the temperature, both of
the tissue and the knife.
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