Chapter 5:
Automation, Infiltration, and Embedding
Learning Objectives:
1. To be able to understand the histotechnique; in terms of preparation,
sequence, and activities.
2. To be able to discuss the effects of vacuum and heat in automated tissue
processing.
3. To be able to contrast manual and automated tissue processing.
4. To be able to name the two types of automated tissue processors, and discuss
each principles.
5. To be able to know the procedure of changing solutions.
6. To be able to describe and discuss the importance of infiltration or
impregnation.
7. To be able to know the selection of infiltration embedding medium to be used.
8. To be able to know the types of impregnation and embedding media.
Learning Objectives:
9.
To be able to know the factors qualifying paraffin wax as
the most common and best medium.
10.
To be able to know the properties of paraffin wax.
11.
To be able to know the melting point of paraffin wax
12.
To be able to discuss the problems associated with
infiltration process.
13.
To be able to know the ideal properties of infiltration and
embedding media.
14.
To be able to describe, discuss the importance, and know
the other names of embedding.
15.
To be able to discuss and contrast the process of
embedding frozen sections.
16.
To be able to know the importance and procedure for
tissue orientation.
Automated Tissue Processor
In modern laboratories - dehydration, clearing and infiltration are all carried
out by automated tissue processor.
It can also perform in accelerated mode (taking a shorter time period) by
subjecting it to vacuum and heat (40 degrees Celcius).
Smaller laboratories in developing countries however, still perform manually.
In the hands of a good technician, the manual procedure is in no
inferior to automated tissue processing but the former is tedious and requires
constant attention. Interruptions in the electricity supply and lack of service
support may cause problems in automated tissue processor.
Two broad types of automation processors;
a) tissue transfer
b) fluid transfer.
Tissue transfer
automatic processor
Tissue transfer – A series of stationary vats (containers), arranged in a
circle with a timing device. These vats hold reagents and paraffin
wax. The basket (receptacle) holds the cassettes and automatically
changes position and takes a bath in different reagents kept in vats at
a certain time, in order to accomplish dehydration, clearing and
infiltration.
Fluid transfer – fluids are pumped to and from a retort
in which the tissues remain stationary.
Fluid transfer automatic processor
Changing of Solutions
The fluids used in complete dehydration and
clearing tend to become contaminated with fluid
carried over from previous vat by the tissue.
After using them for 2 – 3 days, the last
solution in the series (e.g. xylene 2) is replaced by
fresh solution, and the previously used one is moved
forward while the first one (e.g. xylene 1) is discarded.
Infiltration (Impregnation)
Infiltration or impregnation is done after dehydration
and clearing.
Tissue must be infiltrated with the supporting
(embedding) medium, in order to hold the cells and intercellular
structures in their proper relationship while sections are being
cut.
The medium used to infiltrate the tissue is usually the
same medium utilized for impregnation.
There are generally four types of tissue impregnation and embedding
media, namely;
1.
Paraffin wax
2.
Celloidin
3.
Gelatin
4.
Plastic
(L to R) Paraffin pellets with paraffin block,
paraffin pellets
Paraffin wax remains the most common and the best medium because;
1.
large numbers of tissue blocks may be process in a short time
2.
serial sections are easily obtained, and
3.
routine and most special staining can be done easily
Paraffin wax properties:
1.
Is used for routine embedding
2.
Melting point at 56 – 58 degrees Celcius
3. It is a polycrystalline mixture of solid hydrocarbons
produced during the refining of coal and mineral oils.
4.
It is about two thirds the density and slightly more elastic
than dried protein.
Inadequate impregnation leads to drying and shrinking of
embedded tissues. In addition, during cutting, cracks and crumbles
develop if tissues are inadequately supported by wax.
On the other hand, excessive exposure to high temperature
(higher than 5 degrees melting point) will over harden the tissues
and will not yield good sections.
Ideally the infiltrating and embedding media should be:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Soluble in processing fluids
Suitable for sectioning and ribboning
Molten between 30 - 60 degrees Celcius
Translucent or transparent; colorless
Stable
Homogenous
Capable of flattening after ribboning
Non-toxic
Odorless
Easy to handle
Inexpensive
Embedding (Casting or Blocking)
It is a method of placing the infiltrated tissue in
warm liquid paraffin (embedding medium) that
solidifies into a firm block when it cools down to
room temperature.
This process provides support to the tissue to be
cut on a microtome.
During this process the tissues are placed into a
mold along with embedding medium which is
then hardened by cooling.
Embedding
Embedded Frozen Sections
The frozen sections
In frozen, un-fixed sections, the embedding medium is not
needed.
The water in the tissue is frozen, and as ice produces a firm
block of tissue, the ice acts as the embedding medium.
Tissue orientation in mold
(L to R) Metal mold, metal mold
with cassette, paraffin block
During embedding, it is important to orient the tissue in a way
that will provide the best information to the pathologist.
Ideally done at the time the pathologist is taking out the “bit or
block” from gross specimen. The pathologist may notch with a
scalpel or mark with india ink the side opposite that is to be cut.