ANA 231:

INTRODUCTION TO TISSUE
PROCESSING
BY
DR MRS OGBA .A.
 STUDY OBJECTIVES;
At the end of this module, students should be
able to
• Define histology and tissues
• Name the 4 primary tissues in the body
• Explain the reasons for tissue processing
• Enumerate the steps in tissue processing
and give reasons for each
• Enumerate the steps in block making,
sectioning and staining
• Describe the principle of H & E staining and
section interpretation
• Identify common artifacts seen in sections.
 Introduction
• Histology is the study of the microscopic
structure of the tissue and how these tissues are
organized to form organs. It is also called
microscopic anatomy.
• Histology involves all aspects of tissue biology,
to correlate the tissue structure with its function.
• Tissues are a group of similar cells working
together in functionally related groups. Cells are
able to form tissues by attachment (like
physically touching each other and
communication.
 Types of tissue;
• Epithelial tissue
• Muscle tissue
• Connective tissue
• Nervous tissue.
 Tissue processing
• To be able to study the microscopic
features of the tissue, it should undergo
several steps. Each step is done for a
certain reason.
• Tissues are exposed to a series of reagents
that fix, dehydrate, clear, infiltrate and lastly
they are embedded in a medium that
provides support for the tissues.
• Each step In tissue processing is very
important from procurement of sample to
determination of reagent and staining.
• Tissue processing is a technique by which
tissues are made suitable for embedding
within a supportive medium.
• The principle of Tissue processing is
therefore designed to remove all
extractable water from the tissue and
replacing it with a support medium that
provides sufficient rigidity to enable
sectioning of a tissue without damage or
distortion.
Steps in tissue processing.
o Collection of samples and grossing: once
tissues are removed from the body, they
begin to undergo autolysis. Autolysis is
independent of any bacteria.
o Samples collected must be labeled and
identified with a unique number generated
in the lab.
o Samples collected should not be more
than 4mm thick.
 FIXATION
• This is the most important step in tissue
processing. Fixation is a process by which
constituents of a cell or tissue are fixed in a
physical or chemical state so that they can
withstand subsequent treatment with
various reagents with minimum loss,
distortion or decomposition. Fixation is
done using substances known as fixatives.
• Fixatives are fluids often a mixture of
several reactive chemicals into which
histological specimens are placed to
prevent autolysis. Specimens are also
hardened to withstand further processing.
Fixation can be done using different fixatives such as formalin, Boulins fluid,
cold acetone, cornoy’s fluid, Helly’s fluid. The most common fixative used in
the laboratory is 10% formalin.
TYPES OF FIXATION
Physical method: this includes heating, microwaving, freeze drying methods.
Chemical method: coagulant fixations, cross linking and compound fixation.
 Importance of fixation
• Prevent tissue damage
• Maintain tissue structure
• Maintain cellular structure
• Harden the tissue
• Protect it from pathogen
• Prevent autolysis.
 DEHYDRATION
• This involved the removal of water from the
tissue.
• Many dehyrdrating agents are hydrophilic
thus possessing strong polar groups that
interact with water in the tissue. Dehydration
is done slowly using different grade
concentration of the dehydrant as excessive
dehydration can cause shrinking of tissue
and loss of tissue component.
• If the dehydrant is ethanol, the tissue is first
placed in 70% ethanol, followed by 90% and
then absolute ethanol.
• Other dehydrants are ethanol acetone,
methanol, isopropyl and glycol.
• For really delicate tissues it is
recommended to start the process from
30% ethanol.
• Ethanol is a choice dehydrant because it
ensures total dehydration.
 CLEARING
• The clearing agent acts as an intermediary
between the dehydration and impregnation
solution. A good clearing agent should be
miscible with both solutions.
• When the dehydrating agent has been
entirely replaced by most of the clearing
solvent, the tissue has a translucent
appearance hence the term ‘clearing”.
• Most clearing agent are flammable which
warrants caution while in use.
• Prolonged exposure to clearing agent can
make the tissue brittle.
• The most suitable clearing agent for routine
use is xylene. Others include, toluene,
chloroform, methyl benzoate, methyl
salicylate.
Xylene: is the most commonly used clearing
agent in histology. It is a flammable,
colourless liquid with a xtic aromatic smell.
It is miscible with most organic solvent and
paraffin. Suitable for tissue blocks that are
less than 5mm in thickness.
Toluene: has similar properties with xylene. It
is less damaging with prolonged immersion.
More flammable and volatile than xylene.
 Criteria for choosing a clearing
agent
• Rapid removal of dehydrating agent
• Ease of removal by melted paraffin
• Minimal tissue damage
• Flammability
• Toxicity
• Cost.
 INFILTRATION/IMPREGNATION
• This is the process whereby the clearing agent is
completely removed from the tissue and replaced
with a medium that will completely fill all the
tissue cavities thereby giving the tissue a firm
consistency.
• After clearing, tissues are transferred to molten
paraffin wax for infiltration and impregnation.
During this process, xylene diffuses out and
molten paraffin wax is infiltrated.
• The wax which has infiltrated the tissue gets
deposited and this process is called impregnation.
• Impregnation allows easier handling and cutting
of tissue into sections without damage.
• Routinely two changes are given in the
wax to get proper impregnation.
• The duration and number of changes
required for thorough impregnation
depends on size and type of tissues, the
clearing agent used.
 EMBEDDING
• This step involves the enclosing properly
processed tissue in a medium that provides
external support during microscopy.
• The embedding medium must fill the spaces
within the tissue and supporting cellular
components.
• The tissue is impregnated with wax which forms
a matrix thus preventing distortion during
microtomy. The means of getting fixed tissue
into paraffin is known as tissue processing.
• For times when paraffin wax is unsuitable for
the type of sectioning required, other options
such as resin, Agar gel, gelatin, celloidin can be
used.
 MICROTOMY/SECTIONING
• This is the means by which the tissue is
sectioned and attached to a surface for
further microscopic examination.
• The instrument used for microtomy is a
microtome.
• Tissues embedded in paraffin can be
sectioned from 3-10 microns but usually
6-8microns
• There are several microtome designed for
various specific purposes although many
are multifunctional.
 Types of microtome:
• Rotary microtome: can cut thin sections
2-3mm and easy to adapt to all kinds of
tissues.
• Base sledge microtome: used for larger
tissue blocks, hard tissues or whole blocks
• Sliding microtome.
 STAINING
• This process involves clearing, hydration and
staining proper. The clearing process involves
reversing the embedding process, getting
wax or resin out of the tissue and allowing
water soluble dyes to penetrate it. This
process is done using a clearing agent.
(histoclear)
• Staining cannot be done on tissues
containing paraffin.
• In paraffin method, the slide is hydrated using
descending grades of alcohol (100,95,70%).
The slide is then immersed in a preferable
stain and rinsed in running water.
Dyes are used differently.
Progressive dye: this is the simplest with the
dyes applied to the section until the desired
density of colour is reached.
Regressive dye: involves over staining the
tissue so it I darker than needed then
removing the excess to bring the colour down
to the required level. This method is mostly
used as dyes will not only stain the structures
been demonstrated but will also colour the
background.
Stains help to confer a contrast to cells
structures as most unstained cells are
essentially transparent including RBC.
The most common type of stain used in
histology is the H & E stain.
• Hematoxylin stains the acidic regions of a
cytoplasm, nucleus, cartilage matrix blue
• Eosin stains the basic region of the
cytoplasm and collagen fibres pink. .
• Other histological stains include, Geimsa,
weigert’s stain, Orcein’s elastic stain.
 Factors that influence the rate of
tissue processing
When tissue is immersed in fluid, interchange
occurs between the fluid within the tissue
and the surrounding fluid. The following
factor influences the rate at which the
interchange occurs.
• Agitation: This increases the flow of fresh
solution around the tissue. Efficient agitation
can reduce processing time by 30%
• Heat: heat increases the rate of penetration
and fluid exchange. It must be used sparingly
to reduce the possibility of shrinkage and
hardening of tissue. Temp must be limited to
45 deg as higher temp can affect staining.
• Viscosity: it is the property of resistance
to the flow of the fluid. The smaller the
size of the molecule in the solution, the
faster the rate of fluid penetration (low
viscosity). If the molecules are large,
penetration is slower.
 ARTEFACTS
These are things in a biological specimen
that are not present naturally but
introduced or produced during the
procedure.
Types of artefacts:
• Fixation artefacts
• Embedding artefacts
• Sectioning artefacts
• Mounting artefacts.