HISTOLOGY

AN INTRODUCTION TO TISSUE PROCESSING Methods of Microtechnique

❖ Microscopic analysis of cells and

Paraffin Section Alternatively, we can
tissues requires the preparation of infiltrate our tissue
very thin, high-quality sections specimen with a liquid
(slices) mounted on glass slides and agent that can
appropriately stained to demonstrate subsequently be
normal and abnormal structures. converted into a solid that
has appropriate physical
properties, which will allow
MICROTECHNIQUE thin sections to be cut
from it. Paraffin wax is
such an agent. This
❖ is a method of preparation of tissues produces so-called
for microscopic examination. “paraffin sections”. It uses
paraffin for the
impregnation of tissues at
MICROTOME room temperature.

❖ A microtome is a cutting tool used to Frozen section We can freeze the tissue
and keep it frozen while
produce extremely thin slices of
we cut the sections. These
material known as sections. sections are called “frozen
Important in science, microtomes sections”. It is a quick
are used in microscopy, allowing for technique in which the
the preparation of samples for tissues are hardened at
observation under transmitted light low temperatures by the
or electron radiation. A microtome is use of a cryostat of the
freezing microtome.
a specialised precision cutting
instrument, which accurately and Celloidin section uses celloidin as an
repeatedly slices sections from a embedding medium useful
block of embedded tissue. Different for cutting large objects
kinds of microtomes are used to (e.g. brain) and for hard
section paraffin and and brittle material (e.g.
cartilage).
plastic-embedded tissues.
❖ Most fresh tissue is very delicate
and easily distorted and damaged,
and it is thus impossible to prepare Tissue Processing
thin sections from it unless it is
chemically preserved or “fixed” and ❖ describes the steps required to take
supported in some way whilst it is an animal or human tissue from
being cut. Broadly, there are fixation to the state where it is
methods that can be employed to completely infiltrated with a suitable
provide this support: histological wax and can be
 embedded ready for section cutting whole of the specimen has been
on the microtome. Tissue processing processed (“all in”). There is no
can be performed manually (hand spare tissue. There is no diagnosis.
processing), but where multiple There is, however, a patient to whom
specimens must be dealt with, it is an explanation has to be provided.
more convenient and much more
efficient to use an automated tissue
Overview of Steps in Tissue Processing for
processing machine (a “tissue
Paraffin Sections
processor”). There are two main
types of processors: the
tissue-transfer (or “dip and dunk”) 1.Obtaining a fresh specimen
machines where specimens are ● Fresh tissue specimens will come
transferred from container to from various sources. It should be
container to be processed, and the noted that they can very easily be
fluid-transfer (or “enclosed”) types damaged during removal from the
where specimens are held in a patient or experimental animal. It is
single process chamber or retort and important that they are handled
fluids are pumped in and out as carefully and appropriately fixed as
required. Most modern fluid-transfer soon as possible after dissection.
processors employ raised Ideally, fixation should take place at
temperatures, and effective fluid the site of removal, perhaps in the
circulation and incorporate operating theatre, or, if this is not
vacuum/pressure cycles to enhance possible, immediately the following
processing and reduce processing transport to the laboratory.
times.
2. Fixation
● The specimen is placed in a liquid
The Importance of Tissue Processing
fixing agent (fixative) such as
formaldehyde solution (formalin).
● Most laboratory supervisors would This will slowly penetrate the tissue
emphasize to their staff the causing chemical and physical
importance of tissue processing. It is changes that will harden and
worthwhile to stress that the use of preserve the tissue and protect it
an inappropriate processing against subsequent processing
schedule or the making of a steps. There are a limited number of
fundamental mistake (perhaps in reagents that can be used for
replenishing or sequencing of fixation as they must possess
processing reagents) can result in particular properties that make them
the production of tissue specimens suitable for this purpose. For
that cannot be sectioned and example, tissue components must
therefore will not provide any useful retain some chemical reactivity so
microscopic information. This can be that specific staining techniques can
disastrous if you are dealing with be applied subsequently. Formalin,
human diagnostic tissue where the usually as a phosphate-buffered
 solution, is the most popular fixative ● Because melted paraffin wax is
for preserving tissues that will be hydrophobic (immiscible with water),
processed to prepare paraffin most of the water in a specimen
sections. Ideally, specimens should must be removed before it can be
remain in fixative for long enough for infiltrated with wax. This process is
the fixative to penetrate into every commonly carried out by immersing
part of the tissue and then for an specimens in a series of ethanol
additional period to allow the (alcohol) solutions of increasing
chemical reactions of fixation to concentration until pure, water-free
reach equilibrium (fixation time). alcohol is reached. Ethanol is
Generally, this will mean that the miscible with water in all proportions
specimen should fix for between 6 so that the water in the specimen is
and 24 hours. Most laboratories will progressively replaced by the
use a fixative step as the first station alcohol. A series of increasing
on their processor. concentrations is used to avoid
excessive distortion of the tissue.
● Following fixation, the specimens ● A typical dehydration sequence for
may require further dissection to specimens not more than 4mm thick
select appropriate areas for would be:
examination. Specimens that are to
be processed will be placed in
70 % Ethanol 15 minutes
suitably labelled cassettes (small
perforated baskets) to segregate 90 % Ethanol 15 minutes
them from other specimens. The
duration of the processing schedule 100 % Ethanol 15 minutes
used to process the specimens will 100 % Ethanol 15 minutes
depend on the type and dimensions
of the largest and smallest 100 % Ethanol 30 minutes
specimens, the particular processor
employed, the solvents are chosen, 100 % Ethanol 45 minutes
the solvent temperatures, and other
factors. At this point, all but a tiny residue of tightly
bound (molecular) water should have been
● Reagents: 10% neutral formalin, removed from the specimen.
10% formal saline, Bonin's solution,
Zenker's solution, Potassium Purpose: to remove water from the cell or
bichromate, Osmic acid tissue

Purpose: to preserve tissue morphology Dehydrating agent: graded concentration of

and chemical composition sets as a ethyl ROH (70 -100%)
disinfectant, hardening tissue and permitting
better staining reaction. 4. Clearing
● Unfortunately, although the tissue is
3. Dehydration now essentially water-free, we still
 cannot infiltrate it with wax because Although many different reagents
wax and ethanol are largely have been evaluated and used for
immiscible. Therefore, we have to this purpose over many years,
use an intermediate solvent that is paraffin wax-based histological
fully miscible with both ethanol and waxes are the most popular. A
paraffin wax. This solvent will typical wax is liquid at 60°C and can
displace the ethanol in the tissue, be infiltrated into tissue at this
then this, in turn, will be displaced by temperature and then allowed to
molten paraffin wax. This stage in cool to 20°C, where it solidifies to a
the process is called “clearing” and consistency that allows sections to
the reagent used is called a “clearing be consistently cut. These waxes
agent”. The term “clearing” was are mixtures of purified paraffin wax
chosen because many (but not all) and various additives that may
clearing agents impart optical clarity include resins such as styrene or
or transparency to the tissue due to polyethylene. It should be
their relatively high refractive index. appreciated that these wax
Another important role of the formulations have very particular
clearing agent is to remove a physical properties which allow
substantial amount of fat from the tissues infiltrated with the wax to be
tissue, which otherwise presents a sectioned at a thickness down to at
barrier to wax infiltration. A popular least 2 µm, to form ribbons as the
clearing agent is xylene, and sections are cut on the microtome,
multiple changes are required to and to retain sufficient elasticity to
completely displace ethanol. flatten fully during flotation on a
warm water bath.
A typical clearing sequence for specimens
not more than 4mm thick would be: A typical infiltration sequence for specimens
not more than 4mm thick would be:
● xylene 20 min
● xylene 20 min ● wax 30 min
● xylene 45 min ● wax 30 min
● Purpose: to impregnate tissue with a ● wax 45 min
paraffin solvent.
Purpose: paraffin penetrates all intercellular
Clearing agents: Xylol (xylene) – most spaces and even into the cells making the
commonly used, Ether, Cedarwood oil, tissue more resistant to sectioning.
Benzene, Carbon tetrachloride

5. Wax infiltration 6. Embedding or blocking out

● The tissue can now be infiltrated ● Now that the specimen is thoroughly
with a suitable histological wax. infiltrated with wax, it must be
 formed into a “block” which can be another helps in the identification of tissues
clamped into a microtome for section and its some special structure
cutting. This step is carried out using
an “embedding center” where a 9. Mounting
mould is filled with molten wax and ● Make use of a colour slip with a few
the specimen placed into it. The drops of Canada balsam as a
specimen is very carefully oriented mounting medium.
in the mold because its placement
will determine the “plane of the
Xylene Free Processing
section”, an important consideration
in both diagnostic and research ● Although xylene is used widely as a
histology. A cassette is placed on clearing agent for tissue processing,
top of the mold, topped up with more it is a toxic reagent. Some
wax, and the whole thing is placed laboratories prefer to use less-toxic
on a cold plate to solidify. When this alternatives such as isopropanol or
is completed, the block with its other xylene substitutes. For this
attached cassette can be removed method to be successful, higher wax
from the mold and is ready for temperatures are required so that
microtomy. It should be noted that, if isopropanol can be eliminated from
tissue processing is properly carried specimens during infiltration.
out, the wax blocks containing the
tissue specimens are very stable
and represent an important source Steps to Better Processing and Embedding
of archival material.
From patient to pathologist, preparing tissue
7. Cutting specimens for histological examination
● Sectioning of tissue by use of a requires care, skill, and sound procedures.
microtome about 3-5u in thickness. This guide provides practical advice on
best-practice techniques and simple ways to
8. Staining avoid common errors.
● It is a process of artificially colouring
the tissue with dye or reagents. 1. Use an Appropriate Schedule
Hematoxylin and eosin stain.
Hematoxylin, a basic dye, gives a - An appropriate schedule is
bluish or purple colour to the chosen for the tissue type
nucleus. The eosin, an acid dye, and size.
imparts a reddish or pinkish colour to - An inappropriate schedule is
the cytoplasm. chosen. For example, a very
long schedule for a small
Purpose of Staining: endoscopic biopsy or a very
short schedule for a large,
to observe and appreciate the appearance fatty breast specimen.
of the tissue to differentiate one tissue from
2. Provide Additional Fixation
 ensure high-quality blocks
- For optimal processing and that are easy to cut.
good morphology, tissue - Cheap, poor-quality wax from
should be well fixed before little-known sources is used
processing. Where for infiltration and
specimens are incompletely embedding. Poor quality wax
fixed, additional formalin produces blocks that are
fixation is provided in the difficult to cut.
processing schedule.
- Incompletely fixed specimens 5. Avoid Hazardous Reagents
go directly into
alcohol-producing zonal - Where possible, xylene-free
fixation formalin fixation for protocols are used (such as
the outside of the specimen, those available when using
alcohol fixation for deeper Leica Biosystems PELORIS).
areas). This provides a safer
laboratory environment
3. Maintain Reagent Quality without compromising
processing quality.
- Processing reagents are - No consideration is given to
replaced strictly according to the health effects of xylene
established guidelines use. The possibility of using
(ideally using our agent alternatives has not been
management system in an considered.
advanced tissue processor
such as Leica Biosystems 6. Orientate Specimens Carefully
PELORIS).
- Guidelines for the placement - Specimens are carefully
of processing reagents are orientated. Competent
ignored, meaning that grossing ensures flat
ineffective, contaminated, or surfaces on most specimens.
diluted reagents are used Staff performing embedding
(e.g. “out-of-threshold” has ready access to each
warnings from the PELORIS specimen description and is
reagent management system appropriately trained.
are ignored). This can cause - Orientation is incorrect. This
poor processing quality. can result in loss of tissue as
re-embedding is required.
4. Use High-Quality Wax Some poorly prepared
specimens require extensive
- High-quality wax is used for trimming on the microtome to
infiltration and especially for obtain a full-face section
embedding (blocking out) to
7. Choose an Appropriate Mold