Lab 2:
Tissue Processing
Objectives
 What is tissue processing?
 What are the major steps in tissue processing?
 Identify types of fixative.
 The advantage of each fixative.
 The disadvantage of each fixative.
 What chemical or alterations that can be done to
improve tissue processing?
Overview
 Those who are unfamiliar with histotechnology often ask,
“How does a pathologist look at a biopsy under the
microscope and diagnose a disease ? ” The process is long and
involved, and each sequential step is completely dependent
on the previous one.
 The majority of surgical tissue is treated with various
reagents used to preserve tissue elements (such as nuclei,
cytoplasm, and tissue morphology) and prepare the specimen
for paraffin embedding and microtomy.
Cont.
 Preparing fresh tissue samples for microscopic use has many
steps, such as fixation and processing, embedding and
microtomy, de-paraffinization and staining, and finally cover
slipping.
Tissue Processing
 The term tissue processing refers to treatment of the tissue
necessary to impregnate it into a solid medium, so that the
tissue is rendered sufficiently firm yet elastic for the tissue
sections of desirable thickness to be cut on microtome.
Histopathological Technique
 Histopathological technique deals with the preparation of
tissue for microscopic examination.
 The aim of good histological technique to preserve
microscopic anatomy of tissue and make them hard, so that
very thin section (4 to 5 micron) can be made.
 After staining, the section should represent the anatomy of
the tissue as close to as possible to their structure in life.
These processes are
Fixative
Staining
Dehydration
Cutting
Clearing
Embedding
Fixation
Cover slipping
Staining
Hydration
Dehydration
Tissue
Processing
Deparaffinized
Clearing
Impregnating
in paraffin wax
Embidding
Cutting
1. Fixative
 This is the process by which the constituents of cells and
tissue are fixed in a physical and partly also in a chemical
state so that they will withstand subsequent treatment with
various reagents with minimum loss of architecture.
 This is achieved by exposing the tissue to chemical
compounds, call fixatives.
Mechanism of action of fixatives
 Most fixatives act by denaturing or precipitating proteins
which then form a sponge or meshwork, tending to hold the
other constituents.
 Good fixative is most important factors in the production of
satisfactory results in histopathology.
 No fixative will penetrate a piece of tissue thicker than 1 cm.
Properties of an Ideal Fixative:
 1. Prevents autolysis and bacterial decomposition.
 2. Preserves tissue in their natural state and fix all components.
 3. Make the cellular components insoluble to reagent used in
tissue processing.
 4. Preserves tissue volume.
 5. Avoid excessive hardness of tissue.
 6. Allows enhanced staining of tissue.
 7. Should be non-toxic and non-allergic for user.
 8. Should not be very expensive.
Factor affecting fixation:
 1. Size and thickness of piece of tissue.
 2.Tissue covered by large amount of mucous fix slowly.
 3. The same applies to tissue covered by blood or organ
containing very large amount of blood.
 4. Fatty and lipomatous tissue fix slowly.
 5.Correct choice of fixatives.
 6. Fixation is accelerated by maintaining temperature around
60Co.
Formaldehyde
 The most commonly used fixative is formalin.
 It is prepared by mixing 40% formaldehyde gas in 100w/v of
distilled water.
 Routinely, 10% formalin is used.
Mechanism of action
 It forms cross links between amino acids of protein thereby
making them insoluble.
Cont.
Advantages:
1. Rapid penetration.
2. Easy availability & cheap.
3. Does not over harden the
tissue.
4. Fixes lipids for frozen
sections
Disadvantages:
1. Irritant to the nose, the
eyes
and
mucus
membranes.
2. Formation of precipitate of
paraformaldehyde which
can be prevented by adding
11-165 methanol.
3. Formation of black
formalin pigment, Acid
formaldehyde hematin.
Alcohol (Ethyl Alcohol)
 Absolute alcohol alone has very little place in routine
fixation for histopathology.
 It acts as a reducing agents, become oxidized to acetaldehyde
and then to acetic acid.
Cont.
Advantages:
-Alcohol penetrates rapidly in presence of other fixative hence in
combination e.g. Carnoy's fixative is used to increase the speed of tissue
processing.
-Ethanol preserves some proteins in relatively undenatured state so that it
can be used for immunofluorescence or some histochemical methods to
detect certain enzymes.
-Methyl alcohol is used for fixing blood and bone marrow smears.
Cont.
Disadvantages:
-It is slow to penetrate, hardens and shrinks the
tissue.
Acetone
 Cold acetone is sometimes used as a fixative for the
histochemical demonstration of some tissue enzymes like
phosphatases and lipases.
 Its mode of action as fixative is similar to that of alcohol.
Mercuric Chloride (HgCl2)
Advantages:
 -It penetrates rapidly without destroying lipids.
Disadvantages:
 -It causes shrinkage of the tissue if it used alone.
 -It brings about precipitation of the proteins.
Note: Treatment of the tissue with mercuric chloride brings
out more brilliant staining with most of the dyes.
Picric acid
Advantages:
 -It penetrates well and fixes rapidly.
Disadvantages:
 -It produces marked cells shrinkage hence it is not used
alone.
 -It precipitates proteins and combines with them to form
picrate's some of the picrate's are water-soluble so must be
treated with alcohol before further processing where the
tissue comes into contact with water.
Cont.
Note: All the tissues fixed in picric acid containing fixatives
should be thoroughly washed to remove the yellow
discoloration to ensure proper staining of tissue sections.
 If the fixative is not removed by washing thoroughly with
time even the embedded tissue loses its staining quality.
Potassium dichromate
 It fixes the cytoplasm without precipitation. Valuable in
mixtures for the fixation of lipids especially phospholipids.
 Used for fixing phosphatides and mitochondria.
 Note: Thorough washing of the tissue fixed in dichromate is
required to avoid forming an oxide in alcohol which cannot
be removed later.
2. Dehydration
 Tissues are dehydrated by using increasing strength of
alcohol; e.g. 50%, 70%, 90% and 100%.
 The duration for which tissues are kept in each strength of
alcohol depends upon the size of tissue, fixative used and type
of tissue; e.g. after fixation in aqueous fixative delicate tissue
need to be dehydrated slowly starting in 50% ethyl alcohol
directly whereas most tissue specimens may be put into 70%
alcohol.
 Delicate tissue will get high degree of shrinkage by two great
concentration of alcohol.
Cont.
 Note : If the water is one of the fixative components must be
the work of dehydration step after the fixation process while
the water was not a contents do not need to perform this
step.
3. Clearing
 During dehydration water in tissue has been replaced by
alcohol. The next step alcohol should be replaced by paraffin
wax.
 As paraffin wax is not alcohol soluble, we replace alcohol
with a substance in which wax is soluble.
 This step is call clearing.
Impregnating in Paraffin Wax?
Types ofWax employed for Impregnation:
 1. Paraffin wax
 2.Water soluble wax
 3. Other material, like colloidin, gelatin, paraplast etc.
Paraffin wax is used routinely. It has hard consistency, so
section of 7 micron thickness can be cut.
4. Blocking
 Impregnated tissues are placed in a mould with their labels
and then fresh melted wax is poured in it and allowed to
settle and solidify.
 Once the block has cooled sufficiently to form a surface skin
it should be immersed in cold water to cool it rapidly.
 After the block has completely cooled it is cut into individual
blocks and each is trimmed.
5. Decalcification
 Loss of calcium salts from a bone or tooth OR the process of
removing calcareaous matter.
Methods of decalcification:
 Acid decalcifying agents
 Iron exchange resins with acid and decalcifying fluids
 Electrolytic decalcification
 Chelating agents
Cont.
Acid decalcifying agents
 The commonest method of decalcification is dissolving
calcium salts in an acid solution.
 Some of the acid decalcifying agents are (Nitric acid, Formic
acid and Trichloroacetic acid)
Ion exchange with acid and decalcifying fluids
 The removal of calcium ions from the decalcifying fluid by
the resins leads to quicker and more efficient decalcification.
Cont.
Electrolytic decalcification
 It is the speedier decalcification without damage to cytological
features and staining.
Chelating agents
 EDTA is a chelating agent, it is a white crystalline powder soluble
in distilled water to about 20%.
 As a decalcifying agent it combines with calcium ions to form
soluble, non-ionized compound.
 The volume of solution for decalcifying should be 150 times that
of the tissue.
 The solution should be renewed every 5to7 days during
decalcification.
Cont.